U.S. patent application number 17/591033 was filed with the patent office on 2022-08-04 for terpene production in plants.
The applicant listed for this patent is ALTRIA CLIENT SERVICES LLC, UNIVERSITY OF VIRGINIA PATENT FOUNDATION. Invention is credited to Chengalrayan KUDITHIPUDI, Roel RABARA, Yanxin SHEN, Michael Paul TIMKO, Dongmei XU.
Application Number | 20220243215 17/591033 |
Document ID | / |
Family ID | 1000006302277 |
Filed Date | 2022-08-04 |
United States Patent
Application |
20220243215 |
Kind Code |
A1 |
KUDITHIPUDI; Chengalrayan ;
et al. |
August 4, 2022 |
TERPENE PRODUCTION IN PLANTS
Abstract
Composition and methods for the modification of the secondary
metabolic functions of glandular trichomes in plants, such as
tobacco or Cannabis, that control the formation of terpenes that
impart specific flavor and aroma characteristics to the plant
leaves are provided. Enhanced terpene production and composition
are achieved through targeted modification in the biochemical
synthesis pathways for menthol and cis-abienol. This application
provides novel nucleotide sequences encoding the enzymology for
production of these terpenes in tobacco and Cannabis application to
their use plants.
Inventors: |
KUDITHIPUDI; Chengalrayan;
(Midlothian, VA) ; SHEN; Yanxin; (Henrico, VA)
; XU; Dongmei; (Glen Allen, VA) ; TIMKO; Michael
Paul; (Charlottesville, VA) ; RABARA; Roel;
(Charlottesville, VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALTRIA CLIENT SERVICES LLC
UNIVERSITY OF VIRGINIA PATENT FOUNDATION |
Richmond
Charlottesville |
VA
VA |
US
US |
|
|
Family ID: |
1000006302277 |
Appl. No.: |
17/591033 |
Filed: |
February 2, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
63145259 |
Feb 3, 2021 |
|
|
|
63145262 |
Feb 3, 2021 |
|
|
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63145263 |
Feb 3, 2021 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/8243 20130101;
C12N 15/8223 20130101; C07K 14/415 20130101; C12P 5/007
20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C07K 14/415 20060101 C07K014/415; C12P 5/00 20060101
C12P005/00 |
Claims
1. A modified tobacco or Cannabis plant, tobacco or Cannabis seed,
or part thereof comprising a recombinant nucleic acid molecule
comprising a nucleic acid sequence encoding a polypeptide involved
in the biosynthesis of at least one terpene operably linked to a
heterologous promoter.
2. A modified tobacco or Cannabis plant, tobacco or Cannabis seed,
or part thereof comprising at least one non-natural mutation in an
endogenous gene encoding a polypeptide involved in the biosynthesis
of at least one terpene, wherein the modified tobacco or Cannabis
plant exhibits increased mRNA expression of the endogenous gene or
increased accumulation of the polypeptide as compared to a control
tobacco or Cannabis plant lacking the at least one non-natural
mutation when grown under comparable conditions.
3. A modified tobacco or Cannabis plant, tobacco or Cannabis seed,
or part thereof, comprising a recombinant DNA construct comprising
a heterologous promoter operably linked to a nucleic acid molecule
comprising a nucleic acid sequence encoding at least one small RNA
molecule capable of binding to and reducing the expression of a
nucleic acid sequence encoding a polypeptide involved in the
biosynthesis of at least one terpene.
4. The modified tobacco or Cannabis plant, tobacco or Cannabis
seed, or part thereof of claim 1, wherein the polypeptide is
selected from the group consisting of geranyl diphosphate synthase
(GDP), limonene synthase (LS), limonene 3-hydroxylase (L30H),
isopiperitenol dehydrogenase (IPD), pulegone reductase, menthofuran
synthase (MFS), geranylgeranyl diphosphate synthase (GGPPS),
neomenthol dehydrogenase (NtNMD), phylloplanin, premnaspirodiene
oxygenase (PSO), kolavenyl diphosphate synthase (KPS), solanesyl
phosphate synthase 3, terpene synthase 10-like, germacrene D
synthase, geranylgeranyl diphosphate synthase (GGPPS2),
8-hydroxy-copalyl diphosphate synthase, cis-abienol synthase,
cembratrienol synthase 2a, levopimaradiene synthetase,
2-isopropylmalate synthetase (IPS), 2-oxoisovalerate dehydrogenase,
and 2-alkenal reductase.
5. The modified tobacco or Cannabis plant, tobacco or Cannabis
seed, or part thereof of claim 1, wherein the at least one terpene
is selected from the group consisting of menthol or a related
compound, a labdanoid, neophytadiene, cembratrienediol, levopimaric
acid, and L-leucine.
6. (canceled)
7. The modified tobacco or Cannabis plant, tobacco or Cannabis
seed, or part thereof of claim 4, wherein the cis-abienol synthase
is selected from the group consisting of cis-abienol synthase
ISOFORM1 and cis-abienol synthase ISOFORM2.
8. (canceled)
9. (canceled)
10. The modified tobacco or Cannabis plant, tobacco or Cannabis
seed, or part thereof of claim 5, wherein the labdanoid is
cis-abienol.
11.-16. (canceled)
17. The modified tobacco or Cannabis plant, tobacco or Cannabis
seed, or part thereof of claim 1, wherein the nucleic acid sequence
comprises a sequence at least 70% identical to a nucleic acid
sequence selected from the group consisting of SEQ ID NOs: 1-21,
112-125, 155, 158, and 176-190.
18. The modified tobacco or Cannabis plant, tobacco or Cannabis
seed, or part thereof of claim 2, wherein the endogenous gene
comprises a sequence at least 70% identical to a nucleic acid
sequence selected from the group consisting of SEQ ID NOs: 22-42,
126-139, 156, 159, and 161-175.
19. The modified tobacco or Cannabis plant, tobacco or Cannabis
seed, or part thereof of claim 1, wherein the polypeptide comprises
an amino acid sequence at least 70% identical or similar to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 43-63, 140-153, 157, 160, and 191-205.
20. The modified tobacco or Cannabis plant, tobacco or Cannabis
seed, or part thereof of claim 1, wherein the heterologous promoter
comprises a constitutive promoter.
21. The modified tobacco or Cannabis plant, tobacco or Cannabis
seed, or part thereof of claim 1, wherein the heterologous promoter
comprises a tissue-preferred promoter or tissue-specific
promoter.
22. The modified tobacco or Cannabis plant, tobacco or Cannabis
seed, or part thereof of claim 21, wherein the tissue-preferred
promoter or tissue-specific promoter comprises a trichome-preferred
promoter or a trichome-specific promoter.
23. The modified tobacco or Cannabis plant, tobacco or Cannabis
seed, or part thereof of claim 1, wherein the heterologous promoter
comprises a nucleic acid sequence at least 90% identical to SEQ ID
NOs: 104-111, 206-208, or a functional fragment thereof.
24. The modified tobacco or Cannabis plant, tobacco or Cannabis
seed, or part thereof of claim 1, wherein the modified tobacco or
Cannabis plant comprises an increased amount of the at least one
terpene as compared to a control tobacco or Cannabis plant lacking
the recombinant nucleic acid molecule when grown under comparable
conditions.
25. (canceled)
26. The modified tobacco or Cannabis plant, tobacco or Cannabis
seed, or part thereof of claim 24, wherein the increased amount of
the at least one terpene comprises an increase of at least 5% as
compared to the control tobacco or Cannabis plant.
27. The modified tobacco plant, tobacco seed, or part thereof of
claim 1, wherein the tobacco plant, tobacco seed, or part thereof
is of a tobacco variety selected from the group consisting of a
flue-cured variety, a bright variety, a Burley variety, a Virginia
variety, a Maryland variety, a dark variety, a Galpao variety, an
Oriental variety, and a Turkish variety.
28. The modified tobacco or Cannabis plant, tobacco or Cannabis
seed, or part thereof of claim 1, wherein the tobacco or Cannabis
plant is male sterile or cytoplasmically male sterile.
29.-35. (canceled)
36. Cured tobacco material or Cannabis material from the modified
tobacco or Cannabis plant, tobacco or Cannabis seed, or part
thereof of claim 1.
37. The cured tobacco material of claim 36, wherein the cured
tobacco material is made by a curing process selected from the
group consisting of flue curing, air curing, fire curing, and sun
curing.
38.-63. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF
SEQUENCE LISTING
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 63/145,259, filed Feb. 3, 2021; U.S.
Provisional Patent Application No. 63/145,262, filed Feb. 3, 2021;
and U.S. Provisional Patent Application No. 63/145,263, filed Feb.
3, 2021, all of which are incorporated by reference herein in their
entireties. This application also contains a Sequence Listing,
which has been submitted electronically in ASCII format and is
hereby incorporated by reference in its entirety. Said ASCII copy,
created on Jan. 25, 2022, is named P34829US01_SL.txt and is 743,305
bytes in size as measured in Microsoft Windows.RTM..
FIELD
[0002] The present disclosure relates to terpenoid biosynthesis
genes and genetic manipulation thereof in plants, including tobacco
and Cannabis.
Sequences
[0003] Table 1 provides nucleic acid sequences and amino acid
sequences used in this disclosure.
TABLE-US-00001 TABLE 1 Sequences used in this disclosure. SEQ
Annotation ID NO Gene ID (NT3.1/NCBI) Sequence Type 1 g49326
Geranylgeranyl diphosphate synthase/diphosphate synthase Nucleic
acid, coding (GGPPS2)GQ911584 nucleotide coding sequence 2 g49765
Geranylgeranyl pyrophosphate synthase/geranyl diphosphate synthase
Nucleic acid, coding nucleotide coding sequence 3 g20844 Copal-8-ol
diphosphate hydratase, chloroplastic/Nicotiana tabacum cps2 Nucleic
acid, coding gene for 8-hydroxy-copalyl diphosphate synthase
nucleotide coding sequence 4 g2330 Cis-abienol synthase,
chloroplastic/(cis-abienol synthase/ABS) Isoform 1 Nucleic acid,
coding (NtaABS) nucleotide coding sequence 5 g2330 Cis-abienol
synthase, chloroplastic/(cis-abienol synthase/ABS) Isoform 2
Nucleic acid, coding (NtLIBS) nucleotide coding sequence 6 g59058
Ent-kaurene synthase/Nicotiana tabacum cis-abienol synthase,
Nucleic acid, coding chloroplastic-like (LOC107805659), transcript
variant X1, mRNA 7 g20765 Geranyl diphosphate synthase Nucleic
acid, coding 8 g29598 (-)-camphenetricyclene synthase,
chloroplastic/(-)-limonene synthase (LS) Nucleic acid, coding 9
g15165 Terpene synthase/limonene synthase X1_citrus_sinensis
Nucleic acid, coding 10 g47974 Cytochrome
P450/(-)-limonene-3-hydroxylase Nucleic acid, coding 11 g19518
Cytochrome P450/limonene-3-hydroxylase_mentha_spicata Nucleic acid,
coding 12 g28322 Cytochrome
P450/limonene-3-hydroxylase_mentha_spicata Nucleic acid, coding 13
g18898 Short-chain dehydrogenase reductase 4/ Nucleic acid, coding
isopiperitenol_dehydrogenase_mentha 14 g7706 NADP-dependent
dehydrogenase/pulegone_reductase_tobacco Nucleic acid, coding 15
g29837 (+)-neomenthol dehydrogenase/menthone_reductase_capsicum
Nucleic acid, coding 16 g96188 Cytochrome P450/menthofuran synthase
Nucleic acid, coding 17 g58533 Cembratrienol synthase 2a/ Nucleic
acid, coding Nicotiana sylvestris cembratrienol synthase 2a mRNA,
complete cds 18 g33184 Copalyl diphosphate synthase/Levopimaradiene
synthetase asparagus Nucleic acid, coding 19 g36718
2-isopropylmalate synthase 2/2-isopropylmalate synthetase
arabidopsis Nucleic acid, coding 20 g50844 2-oxoisovalerate
dehydrogenase subunit alpha/ Nucleic acid, coding 2-oxoisovalerate
dehydrogenase subunit alpha 21 g63865 2-oxoisovalerate
dehydrogenase subunit alpha/ Nucleic acid, coding 2-oxoisovalerate
dehydrogenase subunit alpha 22 g49326 Geranylgeranyl diphosphate
synthase/diphosphate synthase (GGPPS2) Nucleic acid, GQ911584
genomic 23 g49765 Geranylgeranyl pyrophosphate synthase/geranyl
diphosphate synthase Nucleic acid, genomic 24 g20844 Copal-8-ol
diphosphate hydratase,chloroplastic/Nicotiana tabacum cps2 Nucleic
acid, gene for 8-hydroxy-copalyl diphosphate synthase genomic 25
g2330 Cis-abienol synthase, chloroplastic/(cis-abienol
synthase/ABS) Isoform 1 Nucleic acid, (NtaABS) genomic 26 g2330
Cis-abienol synthase, chloroplastic/(cis-abienol synthase/ABS)
Isoform 2 Nucleic acid, (NtABS) genomic 27 g59058 Ent-kaurene
synthase/Nicotiana tabacum cis-abienol synthase, Nucleic acid,
chloroplastic-like (LOC107805659), transcript variant X1, mRNA
genomic 28 g20765 Geranyl diphosphate synthase Nucleic acid,
genomic 29 g29598 (-)-camphenetricyclene synthase,
chloroplastic/(-)-limonene synthase (LS) Nucleic acid, genomic 30
g15165 Terpene synthase/limonene synthase_X1_citrus_sinensis
Nucleic acid, genomic 31 g47974 Cytochrome
P450/(-)-limonene-3-hydroxylase Nucleic acid, genomic 32 g19518
Cytochrome P450/limonene-3-hydroxylase_mentha_spicata Nucleic acid,
genomic 33 g28322 Cytochrome
P450/limonene-3-hydroxylase_mentha_spicata Nucleic acid, genomic 34
g18898 Short-chain dehydrogenase reductase 4/ Nucleic acid,
isopiperitenol_dehydrogenase_mentha genomic 35 g7706 NADP-dependent
dehydrogenase/pulegone_reductase_tobacco Nucleic acid, genomic 36
g29837 (+)-neomenthol dehydrogenase/menthone_reductase_capsicum
Nucleic acid, genomic 37 g96188 Cytochrome P450/menthofuran
synthase Nucleic acid, genomic 38 g58533 Cembratrienol synthase 2a/
Nucleic acid, Nicotiana sylvestris cembratrienol synthase 2a mRNA,
complete cds genomic 39 g33184 Copalyl diphosphate
synthase/Levopimaradiene_synthetase_asparagus Nucleic acid, genomic
40 g36718 2-isopropylmalate synthase 2/ Nucleic acid,
2-isopropylmalate synthetase_arabidopsis genomic 41 g50844
2-oxoisovalerate dehydrogenase subunit alpha/ Nucleic acid,
2-oxoisovalerate dehydrogenase subunit alpha genomic 42 g63865
2-oxoisovalerate dehydrogenase subunit alpha/ Nucleic acid,
2-oxoisovalerate dehydrogenase subunit alpha genomic 43 g49326
Geranylgeranyl diphosphate synthase/diphosphate synthase Amino acid
(GGPPS2)GQ911584 44 g49765 Geranylgeranyl pyrophosphate
synthase/geranyl diphosphate synthase Amino acid 45 g20844
Copal-8-ol diphosphate hydratase,chloroplastic/Nicotiana tabacum
cps2 Amino acid gene for 8-hydroxy-copalyl diphosphate synthase 46
g2330 Cis-abienol synthase, chloroplastic/(cis-abienol
synthase/ABS) Isoform 1 Amino acid 47 g2330 Cis-abienol synthase,
chloroplastic/(cis-abienol synthase/ABS) Isoform 2 Amino acid 48
g59058 Ent-kaurene synthase/Nicotiana tabacum cis-abienol synthase,
Amino acid chloroplastic-like (LOC107805659), transcript variant
X1, mRNA 49 g20765 Geranyl diphosphate synthase Amino acid 50
g29598 (-)-camphenetricyclene synthase, chloroplastic/(-)-limonene
synthase (LS) Amino acid 51 g15165 Terpene synthase/limonene
synthase_X1_citrus_sinensis Amino acid 52 g47974 Cytochrome
P450/(-)-limonene-3-hydroxylase Amino acid 53 g19518 Cytochrome
P450/limonene-3-hydroxylase mentha_spicata Amino acid 54 g28322
Cytochrome P450/limonene-3-hydroxylase mentha_spicata Amino acid 55
g18898 Short-chain dehydrogenase reductase 4/ Amino acid
isopiperitenol dehydrogenase mentha 56 g7706 NADP-dependent
dehydrogenase/pulegone_reductase_tobacco Amino acid 57 g29837
(+)-neomenthol dehydrogenase/menthone_reductase_capsicum Amino acid
58 g96188 Cytochrome P450/menthofuran synthase Amino acid 59 g58533
Cembratrienol synthase 2a/ Amino acid Nicotiana sylvestris
cembratrienol synthase 2a mRNA, complete cds 60 g33184 Copalyl
diphosphate synthase/Levopimaradiene_synthetase_asparagus Amino
acid 61 g36718 2-isopropylmalate synthase 2/ Amino acid
2-isopropylmalate synthetase_arabidopsis 62 g50844 2-oxoisovalerate
dehydrogenase subunit alpha/ Amino acid 2-oxoisovalerate
dehydrogenase subunit alpha 63 g63865 2-oxoisovalerate
dehydrogenase subunit alpha/ Amino acid 2-oxoisovalerate
dehydrogenase subunit alpha 64 g49765 - Geranylgeranyl
pyrophosphate synthase/geranyl diphosphate synthase Nucleic acid
FWD forward primer primer 65 g49765 - Geranylgeranyl pyrophosphate
synthase/geranyl diphosphate synthase Nucleic acid REV reverse
primer primer 66 g29598 - (-)-camphenetricyclene synthase,
chloroplastic/(-)-limonene synthase (LS) Nucleic acid FWD forward
primer primer 67 g29598 - (-)-camphenetricyclene synthase,
chloroplastic/(-)-limonene synthase (LS) Nucleic acid REV reverse
primer primer 68 g2330 - Cis-abienol synthase,
chloroplastic/(cis-abienol synthase/ABS) Nucleic acid FWD forward
primer primer 69 g2330 - Cis-abienol synthase,
chloroplastic/(cis-abienol synthase/ABS) Nucleic acid REV reverse
primer primer 70 g47974 - Cytochrome
P450/(-)-limonene-3-hydroxylase forward primer Nucleic acid FWD
primer 71 g47974 - Cytochrome P450/(-)-limonene-3-hydroxylase
reverse primer Nucleic acid REV primer 72 g96188 - Cytochrome
P450/menthofuransynthase forward primer Nucleic acid FWD primer 73
g96188 - Cytochrome P450/menthofuran synthase reverse primer
Nucleic acid REV primer 74 g20844 - Copal-8-ol diphosphate
hydratase, chloroplastic/Nicotiana tabacum cps2 Nucleic acid FWD
gene for 8-hydroxy-copalyl diphosphate synthase forward primer
primer 75 g20844 - Copal-8-ol diphosphate hydratase,
chloroplastic/Nicotiana tabacum cps2 Nucleic acid REV gene for
8-hydroxy-copalyl diphosphate synthase reverse primer primer 76
g36718 - 2-isopropylmalate synthase 2/ Nucleic acid FWD
2-isopropylmalate synthetase_arabidopsis forward primer primer 77
g36718 - 2-isopropylmalate synthase 2/ Nucleic acid REV
2-isopropylmalate synthetase_arabidopsis reverse primer primer 78
g50844 - 2-oxoisovalerate dehydrogenase subunit alpha/ Nucleic acid
FWD 2-oxoisovalerate dehydrogenase subunit alpha forward primer
primer 79 g50844 - 2-oxoisovalerate dehydrogenase subunit alpha/
Nucleic acid REV 2-oxoisovalerate dehydrogenase subunit alpha
reverse primer primer 80 g20765 - Geranyl diphosphate synthase
forward primer Nucleic acid FWD primer 81 g20765 - Geranyl
diphosphate synthase reverse primer Nucleic acid REV primer 82
g18898 - Short-chain dehydrogenase reductase 4/ Nucleic acid FWD
isopiperitenol_dehydrogenase_mentha forward primer primer 83 g18898
- Short-chain dehydrogenase reductase 4/ Nucleic acid REV
isopiperitenol_dehydrogenase_mentha reverse primer primer 84 g33184
- Copalyl diphosphate synthase/Levopimaradiene_synthetase_asparagus
Nucleic acid FWD forward primer primer 85 g33184 - Copalyl
diphosphate synthase/Levopimaradiene_synthetase_asparagus Nucleic
acid REV reverse primer primer 86 g15165 - Terpene
synthase/limonene_synthase_X1_citrus_sinensis forward primer
Nucleic acid FWD primer 87 g15165 - Terpene
synthase/limonene_synthase_X1_citrus_sinensis reverse primer
Nucleic acid REV primer 88 g19518 - Cytochrome
P450/limonene-3-hydroxylase_mentha_spicata Nucleic acid FWD primer
primer 89 g19518 - Cytochrome
P450/limonene-3-hydroxylase_mentha_spicata reverse primer Nucleic
acid REV primer 90 g29837 - (+)-neomenthol
dehydrogenase/menthone_reductase_capsicum forward Nucleic acid
FWD primer primer 91 g29837 - (+)-neomenthol
dehydrogenase/menthone_reductase_capsicum reverse Nucleic acid REV
primer primer 92 g7706 - NADP-dependent
dehydrogenase/pulegone_reductase_tobacco forward Nucleic acid FWD
primer primer 93 g7706 - NADP-dependent
dehydrogenase/pulegone_reductase_tobacco reverse Nucleic acid REV
primer primer 94 g63865 - 2-oxoisovalerate dehydrogenase subunit
alpha/ Nucleic acid FWD 2-oxoisovalerate dehydrogenase subunit
alpha forward primer primer 95 g63865 - 2-oxoisovalerate
dehydrogenase subunit alpha/ Nucleic acid REV 2-oxoisovalerate
dehydrogenase subunit alpha reverse primer primer 96 g28322 -
Cytochrome P450/limonene-3-hydroxylase_mentha_spicata forward
Nucleic acid FWD primer primer 97 g28322 - Cytochrome
P450/limonene-3-hydroxylase_mentha_spicata reverse primer Nucleic
acid REV primer 98 g59058 - Ent-kaurene synthase/Nicotiana tabacum
cis-abienol synthase, Nucleic acid FWD chloroplastic-like
(LOC107805659), transcript variant X1 forward primer primer 99
g59058 - Ent-kaurene synthase/Nicotiana tabacum cis-abienol
synthase, Nucleic acid REV chloroplastic-like (LOC107805659),
transcript variant X1 reverse primer primer 100 g49326 -
Geranylgeranyl diphosphate synthase/diphosphate synthase Nucleic
acid FWD (GGPPS2)GQ911584 forward primer primer 101 g49326 -
Geranylgeranyl diphosphate synthase/diphosphate synthase Nucleic
acid REV (GGPPS2)GQ911584 reverse primer primer 102 g58533 -
Cembratrienol synthase 2a/ Nucleic acid FWD Nicotiana sylvestris
cembratrienol synthase 2a forward primer primer 103 g58533 -
Cembratrienol synthase 2a/ REV Nicotiana sylvestris cembratrienol
synthase 2a reverse primer Nucleic acid primer 104 RbcsT promoter
Nucleic acid (1243 nt) 105 RbcsT promoter (436 nt) Nucleic acid 106
NtPSO 1.5 kb promoter Nucleic acid 107 NtPSO 1.0 kb promoter
Nucleic acid 108 NtPHY 1.5 kb promoter Nucleic acid 109 NtPHY 0.5
kb promoter Nucleic acid 110 NtCYC 1.5 kb promoter Nucleic acid 111
NtCYC 0.5 kb promoter Nucleic acid 112 g29597 (-)-limonene synthase
(LS) Nucleic acid, coding 113 g69975 Geranylgeranyl diphosphate
synthase Nucleic acid, coding 114 g56096 Cembratrienol synthase 2a
Nucleic acid, coding 115 g59063 8-hydroxy-copalyl-diphosphate
synthase Nucleic acid, coding 116 g59492 2-isopropylmalate
synthetase Nucleic acid, coding 117 g76515 (-)-limonene synthase
(LS) Nucleic acid, coding 118 g19519 Limonene-3-hydroxylase Nucleic
acid, coding 119 g20478 Cis-abienol synthase Nucleic acid, coding
120 g80474 Menthofuran synthase Nucleic acid, coding 121 g27500
Isopiperitenol dehydrogenase Nucleic acid, coding 122 g68795
Limonene synthase Nucleic acid, coding 123 g53620
(-)-limonene-3-hydroxylase Nucleic acid, coding 124 g86048
2-isopropylmalate synthetase Nucleic acid, coding 125 g59293
Geranyl diphosphate synthase Nucleic acid, coding 126 g 29597
(-)-limonene synthase(LS), genomic sequence Nucleic acid, genomic
127 g 69975 Geranylgeranyl diphosphate synthase, genomic sequence
Nucleic acid, genomic 128 g 56096 Cembratrienolsynthase 2a, genomic
sequence Nucleic acid, genomic 129 g 59063
8-hydroxy-copalyl-diphosphate synthase, genomic sequence Nucleic
acid, genomic 130 g 59492 2-isopropylmalate synthetase, genomic
sequence Nucleic acid, genomic 131 g 76515 (-)-limonene synthase
(LS), genomic sequence Nucleic acid, genomic 132 g 19519
Limonene-3-hydroxylase, genomic sequence Nucleic acid, genomic 133
g 20478 Cis-abienol synthase, genomic sequence Nucleic acid,
genomic 134 g 80474 Menthofuran synthase, genomic sequence Nucleic
acid, genomic 135 g 27500 Isopiperitenol dehydrogenase, genomic
sequence Nucleic acid, genomic 136 g 68795 Limonene synthase,
genomic sequence Nucleic acid, genomic 137 g 53620
(-)-limonene-3-hydroxylase, genomic sequence Nucleic acid, genomic
138 g 86048 2-isopropylmalate synthetase, genomic sequence Nucleic
acid, genomic 139 g 59293 Geranyl diphosphate synthase, genomic
sequence Nucleic acid, genomic 140 g29597 (-)-limonene
synthase(LS), genomic sequence Amino acid 141 g69975 Geranylgeranyl
diphosphate synthase, genomic sequence Amino acid 142 g56096
Cembratrienol synthase 2a, genomic sequence Amino acid 143 g59063
8-hydroxy-copalyldiphosphate synthase, genomic sequence Amino acid
144 g59492 2-isopropylmalate synthetase, genomic sequence Amino
acid 145 g76515 (-)-limonene synthase(LS) , genomic sequence Amino
acid 146 g19519 Limonene-3-hydroxylase, genomic sequence Amino acid
147 g20478 Cis-abienol synthase, genomic sequence Amino acid 148
g80474 Menthofuran synthase, genomic sequence Amino acid 149 g27500
Isopiperitenol dehydrogenase, genomic sequence Amino acid 150
g68795 Limonene synthase, genomic sequence Amino acid 151 g53620
(-)-limonene-3-hydroxylase, genomic sequence Amino acid 152 g86048
2-isopropylmalate synthetase, genomic sequence Amino acid 153
g59293 Geranyl diphosphate synthase, genomic sequence Amino acid
154 pGWB55 plasmid Nucleic acid 1, 12, 723 bp 155 g62191
Phylloplanin Nucleic acid, coding 156 g62191 Phylloplanin Nucleic
acid, genomic 157 g62191 Phylloplanin Amino acid 158 g89817
Cytochrome P450/premnaspirodiene oxygenase (PSO) Nucleic acid,
coding 159 g89817 Cytochrome P450/premnaspirodiene oxygenase (PSO)
Nucleic acid, genomic 160 g89817 Cytochrome P450/premnaspirodiene
oxygenase (PSO) Amino acid 161 geranylgeranyl pyrophosphate
synthase chloroplastic [Cannabis sativa] Nucleic acid, genomic 162
(-)-kolavenyl diphosphate synthase TPS28. chloroplastic [Cannabis
sativa] Nucleic acid, genomic 163 solanesyl diphosphate synthase 3
chloroplastic/mitochondrial [Cannabis Nucleic acid, sativa] genomic
164 terpene synthase 10-like isoform X1 [Cannabis sativa] Nucleic
acid, genomic 165 (-)-limonene synthase chloroplastic isoform X1
[Cannabis sativa] Nucleic acid, genomic 166 cytochrome P450 71D11
(XP_030510234.1) [Cannabis sativa] Nucleic acid, genomic 167
cytochrome P450 71D9 [Cannabis sativa] Nucleic acid, genomic 168
cytochrome P450 71D11 (XP_030502273.1) [Cannabis sativa] Nucleic
acid, genomic 169 (-)-isopiperitenol/(-)-carveol dehydrogenase.
mitochondrial-like [Cannabis Nucleic acid, sativa] genomic 170
2-alkenal reductase (NADP(+)-dependent) [Cannabis sativa] Nucleic
acid, genomic 171 uncharacterized protein LOC115704491 [Cannabis
sativa] Nucleic acid, genomic 172 cytochrome P450 71A22-like
[Cannabis sativa] Nucleic acid, genomic 173 (-)-germacrene D
synthase-like [Cannabis sativa] Nucleic acid, genomic 174
2-isopropylmalate synthase 2. chloroplastic [Cannabis sativa]
Nucleic acid, genomic 175 transcription factor MYB61 isoform X1
[Cannabis sativa] Nucleic acid, genomic 176 geranylgeranyl
pyrophosphate synthase chloroplastic [Cannabis sativa] Nucleic
acid, coding 177 (-)-kolavenyl diphosphate synthase TPS28.
chloroplastic [Cannabis sativa] Nucleic acid, coding 178 solanesyl
diphosphate synthase 3 chloroplastic/mitochondrial [Cannabis
Nucleic acid, coding sativa] 179 terpene synthase 10-like isoform
X1 [Cannabis sativa] Nucleic acid, coding 180 (-)-limonene synthase
chloroplastic isoform X1 [Cannabis sativa] Nucleic acid, coding 181
cytochrome P450 71D11 (XP_030510234.1) [Cannabis sativa] Nucleic
acid, coding 182 cytochrome P450 71D9 [Cannabis sativa] Nucleic
acid, coding 183 cytochrome P450 71D11 (XP_030502273.1) [Cannabis
sativa] Nucleic acid, coding 184 (-)-isopiperitenol/(-)-carveol
dehydrogenase. mitochondrial-like [Cannabis sativa] Nucleic acid,
coding 185 2-alkenal reductase (NADP(+)-dependent) [Cannabis
sativa] Nucleic acid, coding 186 uncharacterized protein
LOC115704491 [Cannabis sativa] Nucleic acid, coding 187 cytochrome
P450 71A22-like [Cannabis sativa] Nucleic acid, coding 188
(-)-germacrene D synthase-like [Cannabis sativa] Nucleic acid,
coding 189 2-isopropylmalate synthase 2. chloroplastic [Cannabis
sativa] Nucleic acid, coding 190 transcription factor MYB61 isoform
X1 [Cannabis sativa] Nucleic acid, coding 191 geranylgeranyl
pyrophosphate synthase chloroplastic [Cannabis sativa] Amino acid
192 (-)-kolavenyl diphosphate synthase TPS28. chloroplastic
[Cannabis sativa] Amino acid 193 solanesyl diphosphate synthase 3
chloroplastic/mitochondrial [Cannabis Amino acid sativa] 194
terpene synthase 10-like isoform X1 [Cannabis sativa] Amino acid
195 (-)-limonene synthase chloroplastic isoform X1 [Cannabis
sativa] Amino acid 196 cytochrome P450 71D11 (XP_030510234.1)
[Cannabis sativa] Amino acid 197 cytochrome P450 71D9 [Cannabis
sativa] Amino acid 198 cytochrome P450 71D11 (XP 030502273.1)
[Cannabis sativa] Amino acid 199 (-)-isopiperitenol/(-)-carveol
dehydrogenase. mitochondrial-like [Cannabis Amino acid sativa] 200
2-alkenal reductase (NADP(+)-dependent) [Cannabis sativa] Amino
acid 201 uncharacterized protein LOC115704491 [Cannabis sativa]
Amino acid 202 cytochrome P450 71A22-like [Cannabis sativa] Amino
acid 203 (-)-germacrene D synthase-like [Cannabis sativa] Amino
acid 204 2-isopropylmalate synthase 2. chloroplastic [Cannabis
sativa] Amino acid 205 transcription factor MYB61 isoform X1
[Cannabis sativa] Amino acid 206 Cyclase promoter Nucleic acid 207
Premnaspirodiene oxygenase (PSO) promoter Nucleic acid 208 Ribulose
bisphosphate carboxylase small chain clone 512 promoter Nucleic
acid
BACKGROUND
[0004] Plants are capable of producing a wide variety of secondary
metabolites, and many secondary metabolites are produced in
above-ground structures called glandular trichomes. Terpenes,
produced by a variety of plants and by some insects, represent a
large and diverse class of organic compounds consisting of
isoprene, a five carbon building block. Terpenoids are modified
terpenes that contain additional functional groups.
[0005] Terpenoids comprise the largest and most diverse family of
secondary metabolites present in plants. Terpenoids, which can be
cyclic or acyclic, vary in size from five-carbon hemiterpenes to
long complex molecules containing thousands of isoprene units. The
olefinic backbone of terpenoids is made of multiples of the
five-carbon (C) isoprene unit, with the major groups being
monoterpenes (10C), sesquiterpenes (15C), and diterpenes (20C).
These terpenoids are produced through the condensation of 5-C
isoprene units (dimethylallyl diphosphate [DMAPP] and isopentenyl
diphosphate [IPP]) most often by the sequential head-to-tail
addition of DMAPP to IPP.
[0006] Terpenoid levels in plants such as tobacco and Cannabis can
be enhanced and modified by targeted manipulation of gene
expression of genes in terpene biosynthetic pathways in order to
improve flavor and aroma characteristics of downstream plant-based
products.
SUMMARY
[0007] In one aspect, this disclosure provides a modified tobacco
plant, tobacco seed, or part thereof, or a modified Cannabis plant,
Cannabis seed, or part thereof, comprising a recombinant nucleic
acid molecule comprising a nucleic acid sequence encoding a
polypeptide involved in the biosynthesis of at least one terpene
operably linked to a heterologous promoter.
[0008] In one aspect, this disclosure provides a modified tobacco
plant, tobacco seed, or part thereof, or a modified Cannabis plant,
Cannabis seed, or part thereof, comprising at least one non-natural
mutation in an endogenous gene encoding a polypeptide involved in
the biosynthesis of at least one terpene, where the modified
tobacco plant or the modified Cannabis plant exhibits increased
mRNA expression of the endogenous gene or increased accumulation of
the polypeptide as compared to a control tobacco plant or a control
Cannabis plant lacking the at least one non-natural mutation when
grown under comparable conditions.
[0009] In one aspect, this disclosure provides a modified tobacco
plant, tobacco seed, or part thereof, or a modified Cannabis plant,
Cannabis seed, or part thereof, comprising a recombinant DNA
construct comprising a heterologous promoter operably linked to a
nucleic acid encoding at least one small RNA molecule capable of
binding to and reducing the expression of a nucleic acid sequence
encoding a polypeptide involved in the biosynthesis of at least one
terpene.
[0010] In one aspect, this disclosure provides a recombinant
nucleic acid molecule comprising a nucleic acid sequence encoding a
polypeptide involved in the biosynthesis of at least one terpene
operably linked to a heterologous promoter.
[0011] In one aspect, this disclosure provides a method of
producing a modified tobacco plant or a modified Cannabis plant
comprising: (a) introducing a recombinant nucleic acid molecule to
at least one tobacco cell or Cannabis cell, where the recombinant
nucleic acid molecule comprises a nucleic acid sequence encoding a
polypeptide involved in terpene biosynthesis operably linked to a
heterologous promoter; (b) selecting at least one tobacco cell or
Cannabis cell comprising the recombinant nucleic acid molecule; and
(c) regenerating a modified tobacco or Cannabis plant from the at
least one tobacco or Cannabis cell selected in step (b) where the
modified tobacco or Cannabis plant comprises an increased amount of
at least one terpene in at least one tissue as compared to a
control tobacco plant lacking the recombinant nucleic acid molecule
when grown under comparable conditions.
[0012] In one aspect, this disclosure provides a method of
producing a modified tobacco plant or a modified Cannabis plant
comprising: (a) inducing at least one non-natural mutation in at
least one tobacco or Cannabis cell in an endogenous gene encoding a
polypeptide involved in terpene biosynthesis; (b) selecting at
least one tobacco or Cannabis cell comprising the at least one
non-natural mutation from step (a); and (c) regenerating a modified
tobacco or Cannabis plant from the at least one tobacco or Cannabis
cell selected in step (b), where the modified tobacco or Cannabis
plant comprises an increased amount of at least one terpene in at
least one tissue as compared to a control tobacco or Cannabis plant
lacking the at least one non-natural mutation when grown under
comparable conditions.
[0013] In one aspect, this disclosure provides a method of
producing a modified tobacco plant or a modified Cannabis plant
comprising: (a) introducing a recombinant DNA construct to at least
one tobacco or Cannabis cell, where said recombinant DNA construct
comprises a heterologous promoter operably linked to a nucleic acid
encoding at least one small RNA molecule capable of binding to and
reducing the expression of a nucleic acid sequence encoding a
polypeptide involved in the biosynthesis of at least one terpene;
(b) selecting at least one tobacco or Cannabis cell comprising the
recombinant DNA construct; and (c) regenerating at least one
modified tobacco or Cannabis plant from the at least one tobacco or
Cannabis cell selected in step (b), where the modified tobacco or
Cannabis plant comprises a reduced amount of at least one terpene
in at least one tissue as compared to a control tobacco or Cannabis
plant lacking the recombinant DNA construct when grown under
comparable conditions.
[0014] In one aspect, this disclosure provides a method comprising
preparing a tobacco product or a Cannabis product using cured
tobacco or Cannabis material from a modified tobacco or Cannabis
plant, where the modified tobacco or Cannabis plant comprises a
recombinant nucleic acid molecule comprising a nucleic acid
sequence encoding a polypeptide involved in the biosynthesis of at
least one terpene operably linked to a heterologous promoter.
[0015] In one aspect, this disclosure provides a method comprising
preparing a tobacco product or a Cannabis product using cured
tobacco or Cannabis material from a modified tobacco or Cannabis
plant, where the modified tobacco or Cannabis plant comprises at
least one non-natural mutation in an endogenous gene encoding a
polypeptide involved in the biosynthesis of at least one terpene,
where the modified tobacco or Cannabis plant exhibits increased
mRNA expression of the endogenous gene or increased accumulation of
the polypeptide as compared to a control tobacco or Cannabis plant
lacking the at least one non-natural mutation when grown under
comparable conditions.
[0016] In one aspect, this disclosure provides a method comprising
preparing a tobacco or Cannabis product using cured tobacco or
Cannabis material from a modified tobacco or Cannabis plant, where
the modified tobacco or Cannabis plant comprises a recombinant DNA
construct comprising a heterologous promoter operably linked to a
nucleic acid molecule encoding at least one small RNA molecule
capable of binding to and reducing the expression of a nucleic acid
sequence encoding a polypeptide involved in the biosynthesis of at
least one terpene.
[0017] In one aspect, this disclosure provides a method comprising
transforming a tobacco or Cannabis cell with a recombinant nucleic
acid molecule, where the recombinant nucleic acid molecule
comprises a nucleic acid sequence encoding a polypeptide involved
in the biosynthesis of at least one terpene operably linked to a
heterologous promoter.
[0018] In one aspect, this disclosure provides a method comprising
transforming a tobacco or Cannabis cell with a recombinant nucleic
acid molecule, where the recombinant nucleic acid molecule
comprises a heterologous promoter operably linked to a nucleic acid
encoding at least one small RNA molecule capable of binding to and
reducing the expression of a nucleic acid sequence encoding a
polypeptide involved in the biosynthesis of at least one
terpene.
[0019] In one aspect, this disclosure provides a method for
producing a tobacco plant or a Cannabis plant, the method
comprising: (a) crossing at least one tobacco or Cannabis plant of
a first tobacco or Cannabis variety with at least one tobacco or
Cannabis plant of a second tobacco or Cannabis variety to produce
at least one progeny tobacco or Cannabis seed, where the at least
one tobacco or Cannabis plant of the first tobacco or Cannabis
variety comprises a recombinant nucleic acid molecule comprising a
nucleic acid sequence encoding a polypeptide involved in the
biosynthesis of at least one terpene operably linked to a
heterologous promoter; and (b) selecting for at least one progeny
tobacco or Cannabis seed, or a plant germinated therefrom, where
the at least one progeny tobacco or Cannabis seed or plant
germinated therefrom comprises the recombinant nucleic acid
molecule.
[0020] In one aspect, this disclosure provides a method for
producing a tobacco plant or a Cannabis plant, the method
comprising: (a) crossing at least one tobacco or Cannabis plant of
a first tobacco or Cannabis variety with at least one tobacco or
Cannabis plant of a second tobacco or Cannabis variety to produce
at least one progeny tobacco or Cannabis seed, where the at least
one tobacco or Cannabis plant of the first tobacco or Cannabis
variety comprises at least one non-natural mutation in an
endogenous gene encoding a polypeptide involved in the biosynthesis
of at least one terpene, where the at least one tobacco or Cannabis
plant of the first tobacco or Cannabis variety exhibits increased
mRNA expression of the endogenous gene or increased accumulation of
the polypeptide as compared to a control tobacco or Cannabis plant
of the first tobacco or Cannabis variety lacking the at least one
non-natural mutation when grown under comparable conditions; and
(b) selecting for at least one progeny tobacco or Cannabis seed, or
a plant germinated therefrom, where the at least one progeny
tobacco or Cannabis seed or plant germinated therefrom comprises
the at least one non-natural mutation.
[0021] In one aspect, this disclosure provides for a method for
producing a tobacco plant or a Cannabis plat, the method
comprising: (a) crossing at least one tobacco or Cannabis plant of
a first tobacco or Cannabis variety with at least one tobacco or
Cannabis plant of a second tobacco or Cannabis variety to produce
at least one progeny tobacco or Cannabis seed, where the at least
one tobacco or Cannabis plant of the first tobacco or Cannabis
variety comprises a recombinant nucleic acid molecule comprising a
heterologous promoter operably linked to a nucleic acid molecule
encoding at least one small RNA molecule capable of binding to and
reducing the expression of a nucleic acid sequence encoding a
polypeptide involved in the biosynthesis of at least one terpene;
and (b) selecting for at least one progeny tobacco or Cannabis
seed, or a plant germinated therefrom, where the at least one
progeny tobacco or Cannabis seed or plant germinated therefrom
comprises the recombinant nucleic acid molecule.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 depicts phenylpropanoid, cytoplasmic mevalonate
(MVA), plastidic methylerythritol phosphate (MEP), and polyketide
biosynthetic pathways in plants. Transport of precursors is
represented by dashed arrows, while direct catalytic reactions are
depicted by bold arrows. Abbreviations used: IPP, isopentenyl
diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl
diphosphate; FPP, farnesyl diphosphate; MVA, mevalonate; MEP,
methylerythritol phosphate.
[0023] FIG. 2 depicts the menthol biosynthetic pathway in
plants.
[0024] FIG. 3 depicts the cis-abienol biosynthetic pathway in
plants.
[0025] FIG. 4 depicts the cembratrienediol biosynthesis pathway in
plants.
[0026] FIG. 5 depicts the levopimaric acid biosynthesis pathway in
plants.
[0027] FIG. 6 depicts the L-leucine biosynthesis pathway in
plants.
[0028] FIG. 7 depicts gene expression in trichomes of terpene
biosynthesis genes.
[0029] FIG. 8 depicts the abundance of transcripts of NtNMD
generated by RNA-sequencing in the flower and leaves of tobacco
cultivars TN90 and K326 at different stages of plant development
(d: day; w: week).
[0030] FIG. 9 depicts the abundance of transcripts of NABS
generated by RNA-sequencing in the flower and leaves of tobacco
cultivars TN90 and K326 at different stages of plant development
(d: day; w: week).
[0031] FIG. 10 depicts a representative vector construct for
overexpression of candidate genes in tobacco. Each promoter is
operably linked to a candidate gene and a sequence encoding green
fluorescent protein (G3GFP).
[0032] FIG. 11 depicts a map of a representative vector, pGWB551,
that can be used for overexpression and knockdown of candidate
terpene biosynthesis genes in tobacco.
[0033] FIGS. 12 to 18 depict green fluorescent protein (GFP)
expression (driven by the CaMV 35S promoter) in calli in transgenic
tobacco plant lines. FIG. 12 shows geranylgeranyl diphosphate
synthase-TN90; FIG. 13 shows geranylgeranyl diphosphate
synthase-Nb; FIG. 14 shows cembratrienol synthase 2a-Nb; FIG. 15
shows neomenthol dehydrogenase-Nb; FIG. 16 shows cis-abienol
synthase-TN90; FIG. 17 shows menthofuran synthase-Nb; and FIG. 18
shows GFP-TN90. For FIGS. 12 to 18, Nb=N. benthamiana; and
TN90=tobacco cultivar `TN90.`
[0034] FIG. 19 depicts relative gene expression of NtaABS, and FIG.
20 depicts relative gene expression of NABS in T.sub.0 transgenic
tobacco plants transformed with cis-abienol synthase overexpression
constructs. NtaABS and NtABS are each operably linked to a CaMV 35S
promoter.
[0035] FIG. 21 depicts relative gene expression of NtNMD operably
linked to a CaMV 35S promoter in T.sub.0 transgenic tobacco plants
transformed with menthone reductase overexpression constructs.
[0036] FIG. 22 depicts relative gene expression of neophytadiene in
NtNMD overexpression T.sub.0 transgenic tobacco plants compared to
a T90 control plant.
[0037] FIG. 23 depicts relative gene expression in NtABS/NtaABS
overexpression T.sub.0 transgenic tobacco plants compared to a TN90
control plant.
[0038] FIG. 24 depicts the relative gene expression levels of NtNMD
(SEQ ID NO: 15) in T.sub.1 transgenic plants of the tobacco line
TN90 overexpressing NtNMD as compared to a wildtype TN90 plant (not
shown).
[0039] FIG. 25 depicts the relative gene expression levels of
NtaABS (SEQ ID NO: 4) in T.sub.1 transgenic plants of the tobacco
line TN90 overexpressing NtaABS as compared to a wildtype TN90
plant (not shown).
DETAILED DESCRIPTION
[0040] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. One skilled in the art will recognize
many methods can be used in the practice of the present disclosure.
Indeed, the present disclosure is in no way limited to the methods
and materials described. Where a term is provided in the singular,
the inventors also contemplate aspects of the disclosure described
by the plural of that term, and vice versa. Where there are
discrepancies in terms and definitions used in references that are
incorporated by reference, the terms used in this application shall
have the definitions given herein. Other technical terms used have
their ordinary meaning in the art in which they are used, as
exemplified by various art-specific dictionaries, for example, "The
American Heritage.RTM. Science Dictionary" (Editors of the American
Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and
New York), the "McGraw-Hill Dictionary of Scientific and Technical
Terms" (6th edition, 2002, McGraw-Hill, New York), or the "Oxford
Dictionary of Biology" (6th edition, 2008, Oxford University Press,
Oxford and New York).
[0041] Any references cited herein, including, e.g., all patents,
published patent applications, and non-patent publications, are
incorporated herein by reference in their entirety.
[0042] When a grouping of alternatives is presented, any and all
combinations of the members that make up that grouping of
alternatives is specifically envisioned. For example, if an item is
selected from a group consisting of A, B, C, and D, the inventors
specifically envision each alternative individually (e.g., A alone,
B alone, etc.), as well as combinations such as A, B, and D; A and
C; B and C; etc. The term "and/or" when used in a list of two or
more items means any one of the listed items by itself or in
combination with any one or more of the other listed items. For
example, the expression "A and/or B" is intended to mean either or
both of A and B--i.e., A alone, B alone, or A and B in combination.
The expression "A, B and/or C" is intended to mean A alone, B
alone, C alone, A and B in combination, A and C in combination, B
and C in combination, or A, B, and C in combination.
[0043] When a range of numbers is provided herein, the range is
understood to be inclusive of the edges of the range as well as any
number between the defined edges of the range. For example,
"between 1 and 10" includes any number between 1 and 10, as well as
the number 1 and the number 10
[0044] The term "about" is used herein to mean approximately,
roughly, around, or in the region of. When the term "about" is used
in conjunction with a numerical range, it modifies that range by
extending the boundaries above and below the numerical values set
forth and is understood to mean plus or minus 10%. For example,
"about 100" would include from 90 to 110.
[0045] As used herein, the singular form "a," "an," and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a compound" or "at least one
compound" may include a plurality of compounds, including mixtures
thereof.
[0046] As used herein, terms or phrases such as "about," "at
least," "at least about," "at most," "less than," "greater than,"
"within," or "alike," when followed by a series of list of numbers
of percentages, such terms or phrases are deemed to modify each and
every number of percentage in the series or list, regardless
whether the adverb, preposition, or other modifier phrase is
reproduced prior to each and every member.
[0047] Any tobacco plant, or part thereof, provided herein is
specifically envisioned for use with any method provided herein.
Similarly, any modified tobacco plant, or part thereof, is
specifically envisioned for use with any method provided herein.
Any Cannabis plant, or part thereof, provided herein is
specifically envisioned for use with any method provided herein.
Similarly, any modified Cannabis plant, or part thereof, is
specifically envisioned for use with any method provided
herein.
[0048] Any nucleic acid sequence, amino acid sequence, or other
composition provided herein is specifically envisioned for use with
any method provided herein.
[0049] Terpenes represent a large and diverse class of organic
compounds consisting of isoprene, a five carbon building block.
Terpenes are produced by a variety of plants and by some insects.
Terpenes often have a strong odor, and may protect the plants that
produce them by deterring herbivores. Terpenoids are modified
terpenes that contain additional functional groups.
[0050] The largest and most diverse family of secondary metabolite
in plants is the terpenoids. Terpenoids are synthesized through the
condensation of 5-C isoprene units (dimethylallyl diphosphate
[DMAPP] and isopentenyl diphosphate [IPP]) most often by the
sequential head-to-tail addition of DMAPP to IPP. The plastidic
methylerythritol phosphate (MEP) pathway consists of seven steps
that convert pyruvate and glyceraldehyde-3-phosphate into IPP and
DMAPP. See FIG. 1. The initial cyclization processes are catalyzed
by different terpene synthases (TPS) and enzyme variation leads to
variation in monoterpene structure. Terpenes can range in size from
the five-carbon hemiterpenes to long complex molecules containing
thousands of isoprene units. Some monoterpene synthases can produce
more than one product.
[0051] In addition to terpene synthases, trans-isoprenyl
diphosphate synthases (IDSs) are among the core enzymes involved in
the generation of diverse terpenoids. TPSs and IDSs share a
conserved alpha-terpenoid synthase fold and a trinuclear metal
cluster for catalysis.
[0052] A monoterpene is a cyclic molecule composed of two isoprene
units. Monoterpenes are C.sub.10 colorless, lipophilic, and
structurally diverse (cyclic and acyclic) volatile molecules that
impart characteristic aromas and flavors on plants. These molecules
also play an important role as chemoattractant between plants and
their pollinator species as well as deterrents in plant-plant and
plant-herbivore communication and are key components of plant
defense. Monoterpene production and accumulation are generally
restricted to glandular trichomes, with plants lacking such
specialized structures typically accumulating only trace quantities
of monoterpenes.
[0053] Terpene synthase (TPS) genes can be grouped into seven
clades (TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, TPS-g, and TPS-h)
where TPS-d and TPS-h are specific to gymnosperms and the lycopod
Selaginalla moellendorffii and TPS-a, TPS-b, and TPS-g are specific
to angiosperm. TPS-a contains mostly sesquiterpene synthases and
diterpene synthases. Neophytadiene is an example of an abundant
sesquiterpenoid in tobacco. The TPS-b and TPS-g clades comprise
mostly monoterpene synthases. The genome of cultivated tomato (S.
lycopersicum), a relative of tobacco, contains 44 TPS genes of
which 29 appear to be functional or potentially functional. The
expression patterns of the tomato TPS genes in leaf and stem
trichomes are quantitatively and qualitatively different,
consistent with the distinct volatile profiles of these two organs.
The tissue-specific differences in gene expression in Solanaceae
species is in part responsible for the observed distinct
intraspecific and interspecific chemical variability. Many terpene
synthases form multiple products from the same substrate.
[0054] The aromatic (smell) and organoleptic (taste/flavor)
properties of tobacco leaf tissues can be enhanced by targeted
genetic manipulation of the biosynthetic pathways that control
terpenoid formation. Similarly, Cannabis resin contains a variety
of monoterpenes and sesquiterpenes. Two major biosynthetic pathways
control terpenoid formation. One pathway controls the formation of
menthol and related compounds (see FIG. 2) and the other controls
the formation of labdanoids, such as cis-abienol (see FIG. 3).
[0055] Targeted overexpression of genes in the menthol biosynthetic
pathway can enhance the accumulation of menthol in tobacco plants.
The C10 monoterpene (-)-menthol is a characteristic constituent of
Mentha x piperita (peppermint), a plant of economic importance for
the production of essential oil used various industrial
manufacturing and medicinal purposes. In Mentha species, menthol
biosynthesis and storage are restricted to the peltate glandular
trichomes (oil glands) on the aerial surfaces of the plant.
[0056] The biosynthesis of (-)-menthol involves eight enzymatic
steps. The formation and subsequent cyclization of the universal
monoterpene precursor geranyl diphosphate to the parent olefin
(-)-(4S)-limonene is the first committed enzymatic reaction in the
menthol biosynthetic pathway.
[0057] Genes encoding enzymes of the menthol biosynthetic pathway
include geranyl diphosphate synthase (GDP) (SEQ ID NOs: 7, 28, and
49), Limonene synthase (LS) (SEQ ID NOs: 8, 29, and 50), Limonene
3-hydroxylase (L30H) (SEQ ID NOs: 10, 31, and 52), Limonene
Synthase chloroplastic isoform X1 (SEQ ID NOs: 165, 181, and 197),
Isopiperitenol dehydrogenase (IPD) (SEQ ID NOs: 13, 34, 55, 169,
185, and 201), pulegone reductase (SEQ ID NOs: 14, 35, and 56),
menthofuran synthase (MFS) (SEQ ID NOs: 16, 37, and 58) and
Menthone reductase, which is alternatively called neomenthol
dehydrogenase (NtNMD) (SEQ ID NOs: 15, 36, and 57). See FIG. 2.
[0058] The second major terpenoid biosynthetic pathway is involved
in the formation of labdanoids, such as cis-abienol (see FIG. 3).
The major labdanoids found in some aromatic plants, including some
forms of tobacco, are Cis-abienol and labdenediol. Cis-abienol is a
diterpenoid abundant in some Oriental tobacco varieties, but not
common in commercial flue and Burley type tobaccos such as
TN90.
[0059] The biosynthesis of Cis-abienol proceeds in two steps (see
FIG. 3). A class-II terpene synthase (8-hydroxy-copalyl diphosphate
synthase) (SEQ ID NOs: 3, 24, 45, 115, 129, and 143) synthesizes
8-hydroxy-copalyl diphosphate from geranylgeranyl diphosphate, and
a kaurene synthase-like (KSL) enzyme (cis-abienol synthase) (SEQ ID
NOs: 4, 5, 6, 25, 26, 27, 46, 47, 48, 119, 133, and 147) converts
8-hydroxy-copalyl diphosphate to Cis-abienol.
[0060] Additional biosynthetic pathways used for targeted aroma
enhancement include the cembratrienediol biosynthesis pathway, see
FIG. 4, the levopimaric acid biosynthesis pathway, see FIG. 5, and
the L-leucine biosynthesis pathway, see FIG. 6. Cembratrienol
synthase 2a (SEQ ID NOs: 17, 38, 59, 114, 128, and 142) is a key
enzyme in the cembratrienediol biosynthesis pathway (see FIG. 4).
The diterpenes cembratrieneols (CBTols) and cembratrienediols
(CBTdiols), are considered important defense compounds, and are
produced in the trichomes. Levopimaradiene synthase (SEQ ID NOs:
18, 39, and 60) is a key enzyme in the levopimaric acid
biosynthesis pathway (see FIG. 5). Levopimaric acid is reported to
be involved in diterpene resin acid production in conifers.
2-isopropylmalate synthetase (IPS) (SEQ ID NOs: 19, 40, 61, 116,
124, 130, 138, 144, 152, 175, 191, and 207) and 2-oxoisovalerate
dehydrogenase (SEQ ID NOs: 20, 21, 41, 42, 62, and 63) are key
enzymes in the L-leucine biosynthesis pathway (see FIG. 6). Leucine
is one of the branched-chain amino acids that contributes to aroma
characteristics in oriental tobacco.
[0061] In an aspect this disclosure provides a modified tobacco or
Cannabis plant, tobacco or Cannabis seed, or part thereof
comprising a recombinant nucleic acid molecule comprising a nucleic
acid sequence encoding a polypeptide involved in the biosynthesis
of at least one terpene operably linked to a heterologous
promoter.
[0062] In a further aspect, this disclosure provides a modified
tobacco or Cannabis plant, tobacco or Cannabis seed, or part
thereof comprising at least one non-natural mutation in an
endogenous gene encoding a polypeptide involved in the biosynthesis
of at least one terpene, where the modified tobacco or Cannabis
plant exhibits increased mRNA expression of the endogenous gene or
increased accumulation of the polypeptide as compared to a control
tobacco or Cannabis plant lacking the at least one non-natural
mutation when grown under comparable conditions.
Terpenes
[0063] Terpenes are a class of aromatic organic compound produced
by plants and some insects. Terpenes are members of a group of
volatile unsaturated hydrocarbons found in the essential oils of
plants, based on a cyclic molecule having the formula
C.sub.10H.sub.16; and related structures such as, but not limited
to, compounds such as sesquiterpene with the formula
C.sub.15H.sub.24, or a simple derivative of such a compound.
Terpenes are hydrocarbon molecules that are often used by plants to
either directly deter herbivory or to attract predators or
parasites of plant herbivores. Non-limiting examples of terpenes
include citral, menthol, camphor, salvinorin A, cannabinoids, and
curcuminoids.
[0064] Several classes of terpenes have been identified in
Cannabis, including, but not limited to, myrcene, pinene, limonene,
caryophyllene, linalool, terpinolene, camphene, terpineol,
phellandrene, carene, humulene, pulegone, and geraniol. Myrcene,
specifically beta-myrcene, is a monoterpene and the most common
terpene produced by Cannabis. Pinene is a bicyclic monoterpenoid.
Two naturally occurring structural isomers of pinene are known:
alpha-pinene and beta-pinene. Limonene is a monocyclic
monoterpenoid and one of the two major compounds formed from
pinene. Caryophyllene, specifically beta caryophyllene, is a
sesquiterpene common to many plants besides Cannabis, including
cloves, cinnamon leaves, and black pepper. Caryophyllene is the
only terpene known to interact with the endocannabinoid system, as
beta-caryophyllene is a functional cannabinoid-2 receptor (CB2)
agonist. Linalool is a non-cyclic monoterpenoid, a critical
precursor in the formation of Vitamin E, and has been isolated from
many different plants. Delta-3-carene is a bicyclic monoterpene.
Alpha-Terpineol, terpinen-4-ol, and 4-terpineol are three closely
related monoterpenoids. Humulene is a sesquiterpene also known as
alpha-humulene. Pulegone, a monocyclic monoterpenoid, is a minor
component of Cannabis.
[0065] In an aspect, a terpene is a terpenoid. Terpenoids (also
referred to as isoprenoids) are modified terpenes that contain
additional functional groups, which often include oxygen.
Terpenoids, which can be cyclic or acyclic, vary in size from
five-carbon hemiterpenes to long complex molecules containing
thousands of isoprene units. Terpenoids are produced through the
condensation of five-carbon isoprene units (e.g., dimethylallyl
diphosphate (DMAPP) and isopentenyl diphosphate (IPP)), most often
by the sequential head-to-tail addition of DMAPP to IPP. The
initial cyclization processes are catalyzed by different terpene
synthases and enzyme variation leads to variation in monoterpene
structure.
[0066] Terpenoids are classified according to the number of
isoprene units that comprise the parent terpene. A hemiterpenoid
comprises one isoprene unit. A monoterpenoid comprises two isoprene
units. A sesquiterpenoid comprises three isoprene units. A
diterpenoid comprises four isoprene units. A sesterterpenoid
comprises five isoprene units. A triterpenoid comprises six
isoprene units. A tetraterpenoid comprises eight isoprene units. A
polyterpenoid comprises more than eight isoprene units.
[0067] In an aspect, a polypeptide is involved in the biosynthesis
of at least one terpene. In an aspect, a polypeptide is involved in
the biosynthesis of at least one terpenoid. In an aspect, a
polypeptide is involved in the biosynthesis of at least one
terpenoid selected from the group consisting of a hemiterpenoid, a
monoterpenoid, a sesquiterpenoid, a diterpenoid, a sesterterpenoid,
a triterpenoid, a tetraterpenoid, and a polyterpenoid. In an
aspect, a polypeptide is involved in the biosynthesis of at least
one terpene selected from the group consisting of a hemiterpene, a
monoterpene, a sesquiterpene, a diterpene, a sesterterpene, a
triterpene, a tetraterpene, and a polyterpene.
[0068] As used herein, the term "biosynthesis" refers to the
production of a complex molecule (e.g., without being limiting, a
terpene or terpenoid) within a plant or plant cell. To be
"involved" with the biosynthesis of a compound, a polypeptide can
directly interact with a substrate during the biosynthesis of the
compound, or the polypeptide can affect the expression (positively
or negatively) of a polypeptide that directly interacts with a
substrate (e.g., a transcription factor that promotes the
expression of an enzyme that converts a substrate to a new form or
a repressor that inhibits expression of an enzyme that converts a
substrate to a new form). Examples of biosynthetic pathways can be
found in FIGS. 1-6. In an aspect, a transcription factor is MYB61
isoform X1. As a non-limiting example, SEQ ID NOs: 208 and 176 are
amino acid and nucleic acid sequences, respectively, for
transcription factor MYB61 isoform X1.
[0069] In an aspect, a polypeptide is involved in the biosynthesis
of a hemiterpene. In an aspect, a polypeptide is involved in the
biosynthesis of a hemiterpenoid. In an aspect, a polypeptide is
involved in the biosynthesis of a monoterpene. In an aspect, a
polypeptide is involved in the biosynthesis of a monoterpenoid. In
an aspect, a polypeptide is involved in the biosynthesis of a
sesquiterpene. In an aspect, a polypeptide is involved in the
biosynthesis of a sesquiterpenoid. In an aspect, a polypeptide is
involved in the biosynthesis of a diterpene. In an aspect, a
polypeptide is involved in the biosynthesis of a diterpenoid. In an
aspect, a polypeptide is involved in the biosynthesis of a
sesterterpene. In an aspect, a polypeptide is involved in the
biosynthesis of a sesterterpenoid. In an aspect, a polypeptide is
involved in the biosynthesis of a triterpene. In an aspect, a
polypeptide is involved in the biosynthesis of a triterpenoid. In
an aspect, a polypeptide is involved in the biosynthesis of a
tetraterpene. In an aspect, a polypeptide is involved in the
biosynthesis of a polyterpenoid. In an aspect, a polypeptide is
involved in the biosynthesis of a monoterpene. In an aspect, a
polypeptide is involved in the biosynthesis of a polyterpenoid.
[0070] Terpene synthase (TPS) genes can be grouped into seven
clades: TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, TPS-g, and TPS-h.
TPS-a, TPS-b, and TPS-g are restricted to angiosperms, and TPS-d
and TPS-h are specific to gymnosperms and the lycopod Selaginalla
moellendorffii. The TPS-a clade comprises mostly sesquiterpene
synthases and diterpene synthases, while the TPS-b and TPS-g clades
comprise mostly monoterpene synthases.
Terpene Synthase-Like Genes
[0071] In an aspect, a polypeptide involved in the biosynthesis of
at least one terpenoid is a TPS-a clade member. In an aspect, a
polypeptide involved in the biosynthesis of at least one terpenoid
is a TPS-b clade member. In an aspect, a polypeptide involved in
the biosynthesis of at least one terpenoid is a TPS-c clade member.
In an aspect, a polypeptide involved in the biosynthesis of at
least one terpenoid is a TPS-e/f clade member. In an aspect, a
polypeptide involved in the biosynthesis of at least one terpenoid
is a TPS-g clade member. In an aspect, a polypeptide involved in
the biosynthesis of at least one terpenoid is a member of a clade
selected from the group consisting of TPS-a, TPS-b, TPS-c, TPS-e/f,
and TPS-g.
[0072] In an aspect, a terpene is menthol. In an aspect, a terpene
is menthol or a related compound. In an aspect, a terpene is a
labdanoid. In an aspect, a terpene is cembratrienediol. In an
aspect, a terpene is levopimaric acid. In an aspect, a terpene is
L-leucine. In an aspect, a terpene is neophytadiene. In an aspect,
a labdanoid is cis-abienol. In an aspect, a labdanoid is
labdane-diol. In an aspect, a terpene is selected from the group
consisting of menthol or a related compound, a labdanoid,
cembratrienediol, levopimaric acid, and L-leucine. In an aspect, a
terpene is selected from the group consisting of menthol or a
related compound, a labdanoid, cembratrienediol, levopimaric acid,
L-leucine, and neophytadiene. In an aspect, a terpene is selected
from the group consisting of menthol, a labdanoid,
cembratrienediol, levopimaric acid, and L-leucine. In an aspect, a
terpene is selected from the group consisting of menthol, a
labdanoid, cembratrienediol, levopimaric acid, L-leucine, and
neophytadiene.
[0073] In an aspect, a terpene is myrcene. In an aspect, a terpene
is pinene. In an aspect, a terpene is limonene. In an aspect, a
terpene is caryophyllene. In an aspect, a terpene is linalool. In
an aspect, a terpene is terpinolene. In an aspect, a terpene is
camphene. In an aspect, a terpene is terpineol. In an aspect, a
terpene is phellandrene. In an aspect, a terpene is carene. In an
aspect, a terpene is humulene. In an aspect, a terpene is pulegone.
In an aspect, a terpene is geraniol.
[0074] As used herein, "menthol" refers to the organic compound
having a chemical formula of C.sub.10H.sub.20O and the
International Union of Pure and Applied Chemistry (IUPAC) name
5-Methyl-2-(propan-2-yl)cyclohexan-1-ol. Menthol is also referred
to as "(-)-Menthol." Related compounds of menthol include, but are
not limited to, (+)-Menthol, (+)-Isomenthol, (+)-Neomenthol,
(+)-Neoisomenthol, (-)-Isomenthol, (-)-Neomethol, and
(-)-Neoisomenthol. In an aspect, a related compound of menthol is
selected from the group consisting of (+)-Menthol, (+)-Isomenthol,
(+)-Neomenthol, (+)-Neoisomenthol, (-)-Isomenthol, (-)-Neomethol,
and (-)-Neoisomenthol.
[0075] As used herein, "neophytadiene" refers to the organic
compound having a chemical formula of C.sub.20H.sub.38 and the
IUPAC name of 7,11,15-trimethyl-3-methylidenehexadec-1-ene.
[0076] As used herein, "cembratrienediol" refers to the organic
compound having a chemical formula of C.sub.20H.sub.34O.sub.2 and
the IUPAC name
(1R,3R,4Z,8Z,12S,13Z)-1,5,9-trimethyl-12-propan-2-ylcyclotetradeca-4,8,13-
-triene-1,3-diol. Cembratrienediol is also referred to as
"beta-Cembrenediol."
[0077] As used herein, "levopimaric acid" refers to the organic
compound having a chemical formula of C.sub.20H.sub.30O.sub.2 and
the IUPAC name
(1R,4aR,4bS,10aR)-1,4a-dimethyl-7-propan-2-yl-2,3,4,4b,5,9,10,10a-octahyd-
rophenanthrene-1-carboxylic acid. Levopimaric acid is also referred
to as "L-Pimaric acid." In an aspect, levopimaric acid is involved
in diterpene resin acid production.
[0078] As used herein, "L-leucine" refers to the amino acid having
the chemical formula C.sub.6H.sub.12NO.sub.2 and the IUPAC name
(2S)-2-amino-4-methylpentanoic acid.
[0079] As used herein, a "labdanoid" refers to a terpenoid
derivative of the fundamental parent labdane, a diterpene. A
labdane has the chemical formula C.sub.20H.sub.38 and the IUPAC
name
(1S,2S,4aS,8aR)-2,5,5,8a-tetramethyl-1-[(3R)-3-methylpentyl]-1,2,3,4,4a,6-
,7,8-octahydronaphthalene.
[0080] A non-limiting example of a labdanoid is cis-abienol. As
used herein, "cis-abienol" refers to the organic compound having a
chemical formula of C.sub.20H.sub.34O and the IUPAC name
(1R,2R,4aS,8aS)-2,5,5,8a-tetramethyl-1-[(2Z)-3-methylpenta-2,4,-dienyl]-3-
,4,4a,6,7,8-hexahydro-1H-naphthalen-2-ol.
[0081] As used herein, "myrcene" refers to the compound having a
chemical formula of C.sub.10H.sub.16 and the IUPAC name
7-Methyl-3-methylene-octa-1,6-diene. Myrcene, also known as
beta-myrcene or alpha-myrcene, belongs to the class of organic
compounds known as acyclic monoterpenoids. Thus, myrcene is
considered to be an isoprenoid lipid molecule.
[0082] As used herein, "pinene" refers to the compound having a
chemical formula of C.sub.10H.sub.16 and the IUPAC name
(1S,5S)-2,6,6-Trimethylbicyclo[3.1.1]hept-2-ene
((-)-.alpha.-Pinene). (+)-a-Pinene, also known as 2-pinene or
acintene a, belongs to the class of organic compounds known as
bicyclic monoterpenoids. These are monoterpenoids containing
exactly 2 rings, which are fused to each other. Alpha-pinene is a
pinene that is bicyclo[3.1.1]hept-2-ene substituted by methyl
groups at positions 2, 6 and 6 respectively. It has a role as a
plant metabolite.
[0083] As used herein, "limonene" refers to the compound having a
chemical formula of C.sub.10H.sub.16 and the IUPAC name
1-methyl-4-(1-methylethenyl)-cyclohexene. Limonene, (+/-)- is a
racemic mixture of limonene, a natural cyclic monoterpene.
[0084] As used herein, "caryophyllene" or "beta caryophyllene"
refers to the organic compound having a chemical formula of
C.sub.15H.sub.24 and the IUPAC name
(1R,4E,9S)-4,11,11-trimethyl-8-methylidenebicyclo[7.2.0]undec-4-ene.
Beta-Caryophyllene is classified as a sesquiterpenoid.
(-)-beta-caryophyllene is a beta-caryophyllene in which the
stereocenter adjacent to the exocyclic double bond has S
configuration, while the remaining stereocenter has R
configuration. It is the most commonly occurring form of
beta-caryophyllene, occurring in many essential oils.
[0085] As used herein, "linalool" refers to the compound having a
chemical formula of C.sub.10H.sub.18O and the IUPAC name
3,7-dimethylocta-1,6-dien-3-ol. Beta-linalool, also known as
3,7-Dimethyl-1,6-octadien-3-ol or 2,6-dimethylocta-2,7-dien-6-ol,
is classified as an acyclic monoterpenoid.
[0086] As used herein, "terpinolene" refers to the organic compound
having a chemical formula of C.sub.10H.sub.16 and the IUPAC name
1-methyl-4-propan-2-ylidenecyclohexene. Terpinolene, also known as
alpha-terpinolene or isoterpinene, is classified as a menthane
monoterpenoid. These are monoterpenoids with a structure based on
an o-, m-, or p-menthane backbone.
[0087] As used herein, "camphene" refers to the organic compound
having a chemical formula of C.sub.10H.sub.16 and the IUPAC name
2,2-dimethyl-3-methylidenebicyclo[2.2.1]heptane. Camphene is
classified as a bicyclic monoterpenoid, containing two rings which
are fused to each other. Camphene is a monoterpene with a bicyclic
skeleton that is bicyclo[2.2.1]heptane substituted by geminal
methyl groups at position 2 and a methylidene group at position
3.
[0088] As used herein, "terpineol" refers to the monoterpene
alcohol of which there are four isomers: alpha-terpineol,
beta-terpineol, gamma-terpineol, and terpinen-4-ol. Beta-terpineol
and gamma-terpineol differ only by the location of the double bond.
Terpineol is usually a mixture of these isomers with
alpha-terpineol as the major constituent. As used herein, "alpha
terpineol" refers to the organic compound having a chemical formula
of C.sub.10H.sub.18O and the IUPAC name
2-(4-methylcyclohex-3-en-1-yl)propan-2-ol.
[0089] As used herein, "phellandrene" refers to the cyclic
monoterpenes "alpha-phellandrene" or "beta-phellandrene." As used
herein, "alpha-phellandrene" refers to the organic compound having
a chemical formula of C.sub.10H.sub.16 and the IUPAC name
2-methyl-5-propan-2-ylcyclohexa-1,3-diene. Alpha-phellandrene is
also known as alpha-phellandren or dihydro-p-cymene. As used
herein, "beta-phellandrene" refers to the organic compound having a
chemical formula of C.sub.10H.sub.16 and the IUPAC name
3-methylidene-6-propan-2-ylcyclohexene. Beta-phellandrene is also
known as beta-phellandren or 2-p-menthadiene.
[0090] As used herein, "carene" or "alpha carene" refers to the
organic compound having a chemical formula of C.sub.10H.sub.16 and
the IUPAC name 3,7,7-trimethylbicyclo[4.1.0]hept-3-ene.
Alpha-Carene, also known as 3-carene or delta-car-3-ene, is
classified as a bicyclic monoterpenoid.
[0091] As used herein, "humulene" refers to the organic compound
having a chemical formula of C.sub.15H.sub.24 and the IUPAC name
(1E,4E,8E)-2,6,6,9-tetramethylcycloundeca-1,4,8-triene.
[0092] As used herein, "pulegone" refers to the organic compound
having a chemical formula of C.sub.10H.sub.16O and the IUPAC name
(5R)-5-methyl-2-propan-2-ylidenecyclohexan-1-one. (+)-Pulegone,
also known as D-pulegone or cis-isopulegone, is classified as a
menthane monoterpenoid.
[0093] As used herein, "geraniol" refers to the organic compound
having a chemical formula of C.sub.10H.sub.18O and the IUPAC name
(2E)-3,7-dimethylocta-2,6-dien-1-ol. Geraniol is an acyclic
monoterpenoid, and is also referred to as 2E-Geraniol,
(e)-geraniol, or geranyl alcohol.
[0094] In an aspect, a polypeptide is geranylgeranyl diphosphate
synthase. In an aspect, a polypeptide is 8-hydroxy-copalyl
diphosphate synthase. In an aspect, a polypeptide is cis-abienol
synthase. In an aspect, a polypeptide is cembratrienol synthase 2a.
In an aspect, a polypeptide is levopimaradiene synthase. In an
aspect, a polypeptide is 2-isopropylmalate synthetase. In an
aspect, a polypeptide is 2-oxoisovalerate dehydrogenase. In an
aspect, a polypeptide is geranyl diphosphate synthase. In an
aspect, a polypeptide is limonene synthase. In an aspect, a
polypeptide is limonene 3-hydroxylase. In an aspect, a polypeptide
is isopiperitenol dehydrogenase. In an aspect, a polypeptide is
pulegone reductase. In an aspect, a polypeptide is menthofuran
synthase. In an aspect, a polypeptide is neomenthol dehydrogenase.
In an aspect, a polypeptide is geranylgeranyl pyrophosphate
synthase. In an aspect, a polypeptide is kolavenyl diphosphate
synthase. In an aspect, a polypeptide is solanesyl diphosphate
synthase. In an aspect, a polypeptide is terpene synthase. In an
aspect, a polypeptide is 2-alkenal reductase. In an aspect, a
polypeptide is germacrene synthase. In an aspect, a polypeptide is
cytochrome P450. In an aspect, a polypeptide is a carveol
dehydrogenase. In an aspect, a polypeptide is a MYB61 transcription
factor.
[0095] In an aspect, a polypeptide is selected from the group
consisting of geranyl diphosphate synthase (GDP), limonene synthase
(LS), limonene 3-hydroxylase (L30H), isopiperitenol dehydrogenase
(IPD), pulegone reductase, menthofuran synthase, and GGPPS
neomenthol dehydrogenase (NtNMD).
[0096] In an aspect, a polypeptide is selected from the group
consisting of geranylgeranyl diphosphate synthase (GGPPS2),
8-hydroxy-copalyl diphosphate synthase, and cis-abienol
synthase.
[0097] In an aspect, a polypeptide is selected from the group
consisting of geranylgeranyl diphosphate synthase,
8-hydroxy-copalyl diphosphate synthase, cis-abienol synthase,
cembratrienol synthase 2a, levopimaradiene synthetase,
2-isopropylmalate synthetase, 2-oxoisovalerate dehydrogenase,
geranyl diphosphate synthase, limonene synthase, limonene
3-hydroxylase, isopiperitenol dehydrogenase, pulegone reductase,
menthofuran synthase, neomenthol dehydrogenase, phylloplanin, and
premnaspirodiene oxygenase.
[0098] In an aspect, a polypeptide is selected from the group
consisting of geranylgeranyl pyrophosphate synthase, kolavenyl
diphosphate synthase, solanesyl diphosphate synthase, terpene
synthase, limonene synthase, isopiperitenol dehydrogenase,
2-alkenal reductase, germacrene synthase, 2-isopropylmalate
synthase, and MYB61.
[0099] In an aspect, a polypeptide is selected from the group
consisting of geranylgeranyl diphosphate synthase,
8-hydroxy-copalyl diphosphate synthase, cis-abienol synthase,
cembratrienol synthase 2a, levopimaradiene synthetase,
2-isopropylmalate synthetase, 2-oxoisovalerate dehydrogenase,
geranyl diphosphate synthase, limonene synthase, limonene
3-hydroxylase, isopiperitenol dehydrogenase, pulegone reductase,
menthofuran synthase, neomenthol dehydrogenase, phylloplanin,
premnaspirodiene oxygenase, geranylgeranyl pyrophosphate synthase,
kolavenyl diphosphate synthase, solanesyl diphosphate synthase,
terpene synthase, 2-alkenal reductase, germacrene synthase, and
MYB61.
[0100] As a non-limiting example, SEQ ID NOs: 45 and 3 are
representative examples of amino acid and nucleic acid sequences,
respectively, for 8-hydroxy-copalyl diphosphate synthase. As a
non-limiting example, SEQ ID NOs: 59 and 17 are representative
examples of amino acid and nucleic acid sequences, respectively,
for cembratrienol synthase 2a. As a non-limiting example, SEQ ID
NOs: 60 and 18 are representative examples of amino acid and
nucleic acid sequences, respectively, for levopimaradiene synthase.
As a non-limiting example, SEQ ID NOs: 61 and 207 are
representative examples of amino acid sequences for
2-isopropylmalate synthase. As a non-limiting example, SEQ ID NOs:
61 and 174 are representative examples of nucleic acid sequences
for 2-isopropylmalate synthase.
[0101] As a non-limiting example, SEQ ID NOs: 160 and 158 are
representative examples of amino acid and nucleic acid sequences,
respectively, for premnaspirodiene oxygenase (Gene id g89817). As a
non-limiting example, SEQ ID NOs: 157 and 155 are representative
examples of amino acid and nucleic acid sequences, respectively,
for phylloplanin (Gene id g62191). As a non-limiting example, SEQ
ID NOs: 194 and 164 are representative amino acid and nucleic acid
sequences, respectively, for a terpene synthase.
[0102] In an aspect, geranylgeranyl diphosphate synthase is a
component of the cis-abienol biosynthesis pathway. As a
non-limiting example, SEQ ID NOs: 43 and 1 are representative
examples of amino acid and nucleic acid sequences, respectively,
for geranylgeranyl diphosphate synthase. In an aspect,
geranylgeranyl pyrophosphate synthase is a component of the
cis-abienol biosynthesis pathway. As a non-limiting example, SEQ ID
NOs: 44 and 191 are non-limiting examples of amino acid sequences
for geranylgeranyl pyrophosphate synthase. As a non-limiting
example, SEQ ID NOs: 2 and 161 are representative examples of
nucleic acid sequences for geranylgeranyl pyrophosphate
synthase.
[0103] In an aspect, 2-oxoisovalerate dehydrogenase is a component
of the L-leucine biosynthesis pathway in plants. As a non-limiting
example, SEQ ID NOs: 62 and 20 are representative examples of amino
acid and nucleic acid sequences, respectively, for dehydrogenase
subunit alpha (Gene id g50844). As a non-limiting example, SEQ ID
NOs: 63 and 21 are representative examples of amino acid and
nucleic acid sequences, respectively, for 2-oxoisovalerate
dehydrogenase subunit alpha (Gene id g63865).
[0104] In an aspect, cis-abienol synthase is a component of the
cis-abienol biosynthesis pathway. As a non-limiting example, SEQ ID
NOs: 147 and 119 are representative examples of amino acid and
nucleic acid sequences, respectively, for cis-abienol synthase. In
an aspect, a cis-abienol synthase is selected from the group
consisting of cis-abienol synthase ISOFORM1 and cis-abienol
synthase ISOFORM 2. In an aspect, a cis-abienol synthase is
selected from the group consisting of cis-abienol synthase
ISOFORM1, cis-abienol synthase ISOFORM 2, and Ent-kaurene synthase.
In an aspect, a cis-abienol synthase is cis-abienol synthase
ISOFORM 1. In an aspect, a cis-abienol synthase is cis-abienol
synthase ISOFORM 2. In an aspect, cis-abienol synthase is
ent-kaurene synthase. As a non-limiting example, SEQ ID NOs: 46 and
4 are representative examples of amino acid and nucleic acid
sequences, respectively, for cis-abienol synthase ISOFORM 1. As a
non-limiting example, SEQ ID NOs: 47 and 5 are representative
examples of amino acid and nucleic acid sequences, respectively,
for cis-abienol synthase ISOFORM 2. As a non-limiting example, SEQ
ID NOs: 48 and 6 are representative examples of amino acid and
nucleic acid sequences, respectively, for ent-kaurene synthase.
[0105] I an aspect, kolavenyl diphosphate synthase catalyzes the
first reaction of salvinorin A biosynthesis, which is the formation
of (-)-kolavenyl diphosphate ((-)-KPP). (-)-KPP is subsequently
dephosphorylated to result in (-)-kolavenol. As a non-limiting
example, SEQ ID NOs: 192 and 162 are representative examples of
amino acid and nucleic acid sequences, respectively, for kolavenyl
diphosphate synthase.
[0106] In an aspect, NAD(P)-dependent oxidoreductases catalyze
NADPH-dependent reduction of a wide range of substrates. In an
aspect, an oxidoreductase protein contains an oxidoreductase
domain. In an aspect, SDR family oxidoreductase shares a conserved
oxidoreductase domain with identified [mammalian] carbonyl
reductases. In an aspect, an example of a protein containing a SDR
family oxidoreductase conserved domain is uncharacterized protein
LOC115704491. As a non-limiting example, SEQ ID NOs: 201 and 171
are representative examples of amino acid and nucleic acid
sequences, respectively, of uncharacterized protein
LOC115704491.
[0107] In an aspect, another example of an enzyme in the
oxidoreductase family is 2-alkenal reductase. Substrates of
2-alkenal reductase are n-alkanal, NAD+, and NADP+. 2-alkenal
reductase is also called NADPH-dependent alkenal/one
oxidoreductase. As a non-limiting example, SEQ ID NOs: 200 and 170
are representative examples of amino acid and nucleic acid
sequences, respectively, for 2-alkenal reductase.
[0108] In an aspect, cytochromes P450 (CYP) are a large enzyme
family that catalyze the hydroxylation of diverse terpene
substrates. In an aspect, premnaspirodiene oxygenase (PSO) (SEQ ID
NOs: 158 and 160) catalyzes the hydroxylation of diverse
sesquiterpene substrates. Hydroxylation reactions catalyzed by
various cytochrome P450s are regio-specific and stereo-specific. As
a non-limiting example, SEQ ID NOs: 31-33, 37, 159, 166-168, and
172 are representative examples of nucleic acid sequences for
cytochrome P450. As a non-limiting example, SEQ ID NOs: 52-54, 58,
160, 196-198, and 202 are representative examples of amino acid
sequences for cytochrome P450.
[0109] In an aspect, germacrene D synthase is a sesquiterpene
synthase that catalyzes the formation of germacrene D. In an
aspect, a germacrene D synthase is (-)-germacrene D synthase. As a
non-limiting example, SEQ ID NOS: 203 and 173 are representative
examples of amino acid and nucleic acid sequences, respectively,
for (-)-germacrene D synthase.
[0110] In an aspect, a modified plant, seed, or plant part
comprising a recombinant nucleic acid provided herein comprises an
increased amount of at least one terpene as compared to a control
plant, seed, or plant part lacking the recombinant nucleic acid
molecule when grown under comparable conditions. In an aspect, a
modified tobacco or Cannabis plant, tobacco or Cannabis seed, or
tobacco plant part comprising a recombinant nucleic acid provided
herein comprises an increased amount of at least one terpene as
compared to a control tobacco or Cannabis plant, tobacco or
Cannabis seed, or tobacco or Cannabis plant part lacking the
recombinant nucleic acid molecule when grown under comparable
conditions.
[0111] In an aspect, an increased amount of at least one terpene
comprises an increase of at least 0.5%. In an aspect, an increased
amount of at least one terpene comprises an increase of at least
1%. In an aspect, an increased amount of at least one terpene
comprises an increase of at least 2%. In an aspect, an increased
amount of at least one terpene comprises an increase of at least
3%. In an aspect, an increased amount of at least one terpene
comprises an increase of at least 4%. In an aspect, an increased
amount of at least one terpene comprises an increase of at least
5%. In an aspect, an increased amount of at least one terpene
comprises an increase of at least 10%. In an aspect, an increased
amount of at least one terpene comprises an increase of at least
12.5%. In an aspect, an increased amount of at least one terpene
comprises an increase of at least 15%. In an aspect, an increased
amount of at least one terpene comprises an increase of at least
17.5%. In an aspect, an increased amount of at least one terpene
comprises an increase of at least 20%. In an aspect, an increased
amount of at least one terpene comprises an increase of at least
25%. In an aspect, an increased amount of at least one terpene
comprises an increase of at least 30%. In an aspect, an increased
amount of at least one terpene comprises an increase of at least
40%. In an aspect, an increased amount of at least one terpene
comprises an increase of at least 50%. In an aspect, an increased
amount of at least one terpene comprises an increase of at least
60%. In an aspect, an increased amount of at least one terpene
comprises an increase of at least 70%. In an aspect, an increased
amount of at least one terpene comprises an increase of at least
80%. In an aspect, an increased amount of at least one terpene
comprises an increase of at least 90%. In an aspect, an increased
amount of at least one terpene comprises an increase of at least
100%. In an aspect, an increased amount of at least one terpene
comprises an increase of at least 150%. In an aspect, an increased
amount of at least one terpene comprises an increase of at least
200%. In an aspect, an increased amount of at least one terpene
comprises an increase of at least 250%. In an aspect, an increased
amount of at least one terpene comprises an increase of at least
500%.
[0112] In an aspect, an increased amount of at least one terpene
comprises an increase of between 0.5% and 500%. In an aspect, an
increased amount of at least one terpene comprises an increase of
between 0.5% and 250%. In an aspect, an increased amount of at
least one terpene comprises an increase of between 0.5% and 100%.
In an aspect, an increased amount of at least one terpene comprises
an increase of between 0.5% and 75%. In an aspect, an increased
amount of at least one terpene comprises an increase of between
0.5% and 50%. In an aspect, an increased amount of at least one
terpene comprises an increase of between 0.5% and 25%. In an
aspect, an increased amount of at least one terpene comprises an
increase of between 0.5% and 10%. In an aspect, an increased amount
of at least one terpene comprises an increase of between 0.5% and
5%. In an aspect, an increased amount of at least one terpene
comprises an increase of between 0.5% and 500%. In an aspect, an
increased amount of at least one terpene comprises an increase of
between 5% and 250%. In an aspect, an increased amount of at least
one terpene comprises an increase of between 5% and 100%. In an
aspect, an increased amount of at least one terpene comprises an
increase of between 5% and 50%. In an aspect, an increased amount
of at least one terpene comprises an increase of between 25% and
500%. In an aspect, an increased amount of at least one terpene
comprises an increase of between 25% and 250%. In an aspect, an
increased amount of at least one terpene comprises an increase of
between 50% and 100%. In an aspect, an increased amount of at least
one terpene comprises an increase of between 100% and 500%.
[0113] Terpenes may be extracted using any method known in the art,
for example, solvent extraction. See, for example, Jiang et al.,
Curr Protoc Plant Biol., 1:345-358 (2016). Solventless extraction
methods are also known in the art. See, for example, Yang and Xie,
Science Direct, 162: 135-139 (2006).
[0114] The amount of terpenes in a plant can be measured using any
method known in the art, including, without being limiting, gas
chromatography mass spectrometry (GC-MS), Nuclear Magnetic
Resonance Spectroscopy, and liquid chromatography-linked mass
spectrometry. See The Handbook of Plant Metabolomics, edited by
Weckwerth and Kahl, (Wiley-Blackwell) (May 28, 2013). In an aspect,
an amount of at least one terpene refers to the concentration of
that terpene in the tissue sampled.
[0115] In an aspect, a modified tobacco plant or Cannabis plant
provided herein further comprises a recombinant nucleic acid
molecule comprising a nucleic acid sequence encoding a polypeptide
operably linked to a heterologous promoter, where the polypeptide
causes an increase in average trichome density as compared to a
control tobacco plant or Cannabis plant lacking the transgene or
modification when grown under comparable conditions. Non-limiting
examples of genes that can be overexpressed to increase average
trichome density include NtGIS, NtMYB86, NbGIS, Cannabis MYB61,
Cannabis GIS3, and Cannabis non-specific lipid-transfer protein
1-like.
Cannabinoids
[0116] Cannabinoids are naturally occurring compounds found in
Cannabis plants. Many cannabinoids are concentrated in a resin
produced in glandular trichomes, and at least 113 cannabinoids are
known. Two main cannabinoids are tetrahydrocannabinol (THC) and
cannabidiol (CBD), though over 100 additional cannabinoids have
been identified. CBD composes approximately 40% of the plant resin
extract.
[0117] Cannabinoids exert their effects by interacting with
specific cannabinoid receptors. The two main types of cannabinoid
receptors in humans are cannabinoid receptor type 1 (CB1) and
cannabinoid receptor type 2 (CB2).
[0118] In an aspect, a heterologous polynucleotide is involved in
the biosynthesis of a cannabinoid. There are several main classes
of natural cannabinoids. Cannabis plant-derived cannabinoids are
termed phytocannabinoids. All classes derive from cannabigerol-type
(CBG) compounds and differ mainly in the way this precursor is
cyclized.
[0119] In an aspect, a cannabinoid is selected from the group
consisting of a cannabigerol-type (CBG) cannabinoid, a
cannabichromene-type (CBC) cannabinoid, a cannabidiol-type (CBD)
cannabinoid, a tetrahydrocannabinol-type (THC) cannabinoid, a
cannabinol-type (CBN) cannabinoid, a cannabinodiol-type (CBDL)
cannabinoid, a cannabielsoin-type (CBE) cannabinoid, an
iso-tetrahydrocannabinol-type (iso-THC) cannabinoid, a
cannabicyclol-type (CBL) cannabinoid, and a cannabitriol-type (CBT)
cannabinoid.
[0120] Cannabigerol (CBG) is one of the major phytocannabinoids
present in Cannabis sativa. CBG is the non-acidic form of
cannabigerolic acid, the parent molecule from which other
cannabinoids are synthesized. Cannabichromene (CBC) is a
non-psychoactive cannabinoid. CBC is also called cannabichrome,
cannanbichromene, pentylcannabichromene, or cannabinochromene.
[0121] Cannabidiol (CBD) is a naturally occurring compound found in
the resinous flower of Cannabis. CBD is a major phytocannabinoid,
accounting for up to 40% of the Cannabis plant's extract, and binds
to a wide variety of physiological targets of the endocannabinoid
system. Tetrahydrocannabinol (THC) is also known as
Delta-9-tetrahydrocannabinol. Some Cannabis plants contain very
little THC, and are considered "industrial hemp."
[0122] Cannabinol (CBN) is a mildly psychoactive cannabinoid found
only in trace amounts in Cannabis, and is mostly found in aged
Cannabis. THC breaks down into CBN after prolonged periods of time.
The degradation of THC to CBN can be accelerated by exposing dried
plant matter to oxygen and heat. Cannabinodiol (CBDL) is a
fully-aromatized extract of CBD. CBDL is a psychoactive
cannabinoid, present in Cannabis sativa at low concentrations.
[0123] Cannabielsoin (CBE) is a non-psychoactive cannabinoid
metabolite derived from CBD. Cannabicyclol (CBL) is a
non-psychoactive cannabinoid found in Cannabis. Cannabichromene
degrades into CBL through natural irradiation or under acidic
conditions. Cannabitriol CBT is a cannabinoid that is not always
detectable in Cannabis, but when detectable, CBT is often present
in low concentrations. CBT is structurally similar to THC. There
are currently nine known types of CBT.
[0124] In an aspect, a Cannabis-derived cannabinoid, or a
phytocannabinoid, is selected from the group consisting of
tetrahydrocannabinol, tetrahydrocannabinolic acid, cannabidiol,
cannabidiolic acid, cannabinol, cannabigerol, cannabichromene,
cannabicyclol, cannabivarin, tetrahydrocannabivarin,
cannabidivarin, cannabichromevarin, cannabigerovarin, cannabigerol
monomethyl ether, cannabielsoin, and cannabicitran.
[0125] As used herein, "tetrahydrocannabinol" ("THC") refers to the
organic compound having a chemical formula of
C.sub.21H.sub.30O.sub.2 and the IUPAC name of
(-)-(6aR,10aR)-6,6,9-trimethyl-3-pentyl-6a,7,8,10a-tetrahydro-6H-benzo[c]-
chromen-1-ol.
[0126] As used herein, "tetrahydrocannabinolic acid" ("THCA" or
"2-COOH-THC") refers to the organic compound having a chemical
formula of C.sub.22H.sub.30O.sub.4.
[0127] As used herein, "cannabidiol" ("CBD") refers to the organic
compound having a chemical formula of C.sub.21H.sub.30O.sub.2, a
molecular weight of 314.469 g/mol, and the IUPAC name of
2-[(1R,6R)-3-methyl-6-(prop-1-en-2-yl)cyclohex-2-en-1-yl]-5-pentylbenzene-
-1,3-diol. It is a 21-carbon terpenophenolic compound, which is
formed following decarboxylation from a cannabidiolic acid
precursor, although it can also be produced synthetically. CBD is
normally taken to refer to the naturally occurring (-)-enantiomer.
(+) CBD has been synthesized.
[0128] As used herein, "cannabidiolic acid" ("CBDA") refers to the
organic compound having a chemical formula of
C.sub.22H.sub.30O.sub.4 and the IUPAC name of
2,4-dihydroxy-3-[(1R,6R)-3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]-6-p-
entylbenzoic acid.
[0129] In plants, THC and CBD are derived from their acidic
precursors THCA and CBDA, respectively, which are both derived from
cannabigerolic acid (CBGA). The final biosynthesis step differs,
with THCA synthase producing THCA and CBDA synthase producing CBDA,
respectively, from CBGA. Subsequent decarboxylation of THCA and
CBDA via light exposure, heating, or aging, results in the
biosynthesis of THC or CBD. THCA synthase catalyzes oxidative
cyclization of the monoterpene moiety of CBGA to form THCA. CBDA
synthase catalyze oxidative cyclization of the monoterpene moiety
of CBGA to form CBDA. THC is generated from THCA by non-enzymatic
decarboxylation. CBD is generated from CBDA by non-enzymatic
decarboxylation.
[0130] As used herein, "cannabinol" refers to the organic compound
having a chemical formula of C.sub.21H.sub.26O.sub.2 and the IUPAC
name of 6,6,9-trimethyl-3-pentylbenzo[c]chromen-1-ol.
[0131] As used herein, "cannabigerol" refers to the organic
compound having a chemical formula of C.sub.21H.sub.32O.sub.2 and
the IUPAC name of
2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-pentylbenzene-1,3-diol.
[0132] As used herein, "cannabichromene" refers to the organic
compound having a chemical formula of C.sub.21H.sub.30O.sub.2 and
the IUPAC name of
2-methyl-2-(4-methylpent-3-enyl)-7-pentylchromen-5-ol.
[0133] As used herein, "cannabicyclol" refers to the organic
compound having a chemical formula of C.sub.21H.sub.30O.sub.2 and
the IUPAC name of
9,13,13-trimethyl-5-pentyl-8-oxatetracyclo[7.4.1.0.0]tetradeca-2,4,6-t-
rien-3-ol.
[0134] As used herein, "cannabivarin" refers to the organic
compound having a chemical formula of C.sub.19H.sub.22O.sub.2 and
the IUPAC name of 6,6,9-trimethyl-3-propylbenzo[c]chromen-1-ol.
[0135] As used herein, "tetrahydrocannabivarin" refers to the
organic compound having a chemical formula of
C.sub.19H.sub.26O.sub.2 and the IUPAC name of
(6aR,10aR)-6,6,9-trimethyl-3-propyl-6a,7,8,10a-tetrahydrobenzo[c]chromen--
1-ol.
[0136] As used herein, "cannabidivarin" refers to the organic
compound having a chemical formula of C.sub.19H.sub.26O.sub.2 and
the IUPAC name of
2-[(1R,6R)-3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]-5-propylbenzen-
e-1,3-diol.
[0137] As used herein, "cannabichromevarin" refers to the organic
compound having a chemical formula of C.sub.19H.sub.26O.sub.2 and
the IUPAC name of
2-methyl-2-(4-methylpent-3-enyl)-7-propylchromen-5-ol.
[0138] As used herein, "cannabigerovarin" refers to the organic
compound having a chemical formula of C.sub.19H.sub.28O.sub.2 and
the IUPAC name of
2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-propylbenzene-1,3-diol.
[0139] As used herein, "cannabigerol monomethyl ether" refers to
the organic compound having a chemical formula of
C.sub.22H.sub.28O.sub.2 and the IUPAC name of
1-methoxy-6,6,9-trimethyl-3-pentylbenzo[c]chromene.
[0140] As used herein, "cannabielsoin" refers to the organic
compound having a chemical formula of C.sub.21H.sub.30O.sub.3 and
the IUPAC name of
(5aS,6S,9R,9aR)-6-methyl-3-pentyl-9-prop-1-en-2-yl-7,8,9,9a-tetrahydro-
-5aH-dibenzofuran-1,6-diol.
[0141] As used herein, "cannabicitran" refers to the organic
compound having a chemical formula of C.sub.21H.sub.30O.sub.2 and
the IUPAC name of
1,5,5-trimethyl-9-pentyl-6,15-dioxatetracyclo[9.3.1.0.0]pentadeca-7(12-
),8,10-triene.
Promoters
[0142] As commonly understood in the art, the term "promoter"
refers to a DNA sequence that contains an RNA polymerase binding
site, a transcription start site, and/or a TATA box and assists or
promotes the transcription and expression of an associated
transcribable polynucleotide sequence and/or gene (or transgene). A
promoter can be synthetically produced, varied, or derived from a
known or naturally occurring promoter sequence or other promoter
sequence. A promoter can also include a chimeric promoter
comprising a combination of two or more heterologous sequences. A
promoter of the present application can thus include variants of
promoter sequences that are similar in composition, but not
identical to, other promoter sequence(s) known or provided
herein.
[0143] In one aspect, this disclosure provides a recombinant
nucleic acid molecule comprising a nucleic acid sequence encoding a
polypeptide involved in the biosynthesis of at least one terpene
operably linked to a heterologous promoter. In one aspect, this
disclosure provides a recombinant nucleic acid molecule comprising
a nucleic acid sequence encoding a polypeptide involved in the
biosynthesis of at least one terpene operably linked to a
heterologous promoter. In one aspect, this disclosure provides a
recombinant nucleic acid molecule comprising a nucleic acid
sequence encoding a polypeptide involved in the biosynthesis of at
least one terpene operably linked to a heterologous promoter.
[0144] It is appreciated in the art that a fragment of a promoter
sequence can function to drive transcription of an operably linked
nucleic acid molecule. For example, without being limiting, if a
1000 bp promoter is truncated to 500 bp, and the 500 bp fragment is
capable of driving transcription, the 500 bp fragment is referred
to as a "functional fragment."
[0145] In an aspect, a heterologous promoter comprises a nucleic
acid sequence at least 90% identical to SEQ ID NOs: 104-111,
206-208, or a functional fragment thereof. In an aspect, a
heterologous promoter comprises a nucleic acid sequence at least
92.5% identical to SEQ ID NOs: 104-111, 206-208, or a functional
fragment thereof. In an aspect, a heterologous promoter comprises a
nucleic acid sequence at least 95% identical to SEQ ID NOs:
104-111, 206-208, or a functional fragment thereof. In an aspect, a
heterologous promoter comprises a nucleic acid sequence at least
96% identical to SEQ ID NOs: 104-111, 206-208, or a functional
fragment thereof. In an aspect, a heterologous promoter comprises a
nucleic acid sequence at least 97% identical to SEQ ID NOs:
104-111, 206-208, or a functional fragment thereof. In an aspect, a
heterologous promoter comprises a nucleic acid sequence at least
98% identical to SEQ ID NOs: 104-111, 206-208, or a functional
fragment thereof. In an aspect, a heterologous promoter comprises a
nucleic acid sequence at least 99% identical to SEQ ID NOs:
104-111, 206-208, or a functional fragment thereof. In an aspect, a
heterologous promoter comprises a nucleic acid sequence at least
99.9% identical to SEQ ID NOs: 104-111, 206-208, or a functional
fragment thereof. In an aspect, a heterologous promoter comprises a
nucleic acid sequence 100% identical to SEQ ID NOs: 104-111,
206-208, or a functional fragment thereof.
[0146] Promoters that drive expression in all or most tissues of
the plant are referred to as "constitutive" promoters. In an
aspect, a constitutive promoter is selected from the group
consisting of a Cauliflower Mosaic Virus 35S promoter, a ubiquitin
promoter, an actin promoter, an opine promoter, glyceraldehyde
3-phosphate dehydrogenase (GAPDH), and an alcohol dehydrogenase
promoter.
[0147] Promoters that drive expression during certain periods or
stages of development are referred to as "developmental"
promoters.
[0148] Promoters that drive enhanced expression in certain tissues
of an organism relative to other tissues of the organism are
referred to as "tissue-preferred" promoters. Thus, a
"tissue-preferred" promoter causes relatively higher or
preferential expression in a specific tissue(s) of a plant, but
with lower levels of expression in other tissue(s) of the plant. As
a non-limiting example, a trichome tissue-preferred promoter
exhibits higher activity in trichomes, but may also exhibit
activity, albeit at lower levels, in additional tissues such as
stem, leaves, and floral tissues. A "tissue-specific" promoter
causes expression only in a specific tissue. As a non-limiting
example, a trichome tissue-specific promoter drives expression only
in trichomes. In an aspect, a tissue-specific promoter is a
trichome tissue-specific promoter. In another aspect, a
tissue-preferred promoter is a trichome tissue-preferred
promoter.
[0149] In an aspect, a promoter is selected from the group
consisting of a cyclase promoter, a premnaspirodiene oxygenase
(PSO) promoter, and a ribulose biphosphate carboxylase
promoter.
[0150] In an aspect, this disclosure provides a modified tobacco
plant, tobacco seed, or part thereof, where a tissue-preferred
promoter comprises a trichome-preferred promoter. In an aspect,
this disclosure provides a modified tobacco plant, tobacco seed, or
part thereof, where a tissue-specific promoter comprises a
trichome-specific promoter. In an aspect, this disclosure provides
a modified tobacco plant, tobacco seed, or part thereof where a
tissue-preferred promoter or tissue-specific promoter comprises a
trichome-preferred promoter or a trichome-specific promoter. In an
aspect, a trichome-specific promoter sequence is selected from the
group consisting of SEQ ID NOs: 104-111 and 206-208. In an aspect,
a trichome-preferred promoter sequence is selected from the group
consisting of SEQ ID NOs: 104-111 and 206-208.
[0151] An "inducible" promoter is a promoter that initiates
transcription in response to an environmental stimulus such as
heat, cold, drought, light, or other stimuli, such as wounding or
chemical application.
[0152] In an aspect, a promoter provided herein is a constitutive
promoter. In another aspect, a promoter provided herein is an
inducible promoter. In a further aspect, a promoter provided herein
is a developmental promoter. In another aspect, a promoter is a
tissue-preferred or tissue-specific promoter. In a further aspect,
a promoter is selected from the group consisting of a constitutive
promoter, a tissue-preferred promoter, a tissue-specific promoter,
and an inducible promoter.
[0153] In an aspect, this disclosure provides a heterologous
promoter. In another aspect, this disclosure provides a promoter
that is operably linked to a heterologous polynucleotide. In
another aspect, this disclosure provides a polynucleotide sequence
that is operably linked to a heterologous promoter.
[0154] As used herein, "operably linked" refers to a functional
linkage between two or more elements. For example, an operable
linkage between a polynucleotide of interest and a regulatory
sequence (e.g., a promoter) is a functional link that allows for
expression of the polynucleotide of interest. Operably linked
elements may be contiguous or non-contiguous. In an aspect, a
promoter provided herein is operably linked to a heterologous
nucleic acid molecule.
Plants
[0155] In an aspect, this disclosure provides a modified tobacco or
Cannabis plant, tobacco or Cannabis seed, or part thereof
comprising a recombinant nucleic acid molecule comprising a nucleic
acid sequence encoding a polypeptide involved in the biosynthesis
of at least one terpene operably linked to a heterologous
promoter.
[0156] In an aspect, this disclosure provides a modified tobacco or
Cannabis plant, tobacco or Cannabis seed, or part thereof
comprising at least one non-natural mutation in an endogenous gene
encoding a polypeptide involved in the biosynthesis of at least one
terpene, where the modified tobacco or Cannabis plant exhibits
increased mRNA expression of the endogenous gene or increased
accumulation of the polypeptide as compared to a control tobacco or
Cannabis plant lacking the at least one non-natural mutation when
grown under comparable conditions.
[0157] In an aspect, a plant provided herein is a modified plant.
In an aspect, a seed provided herein is a modified seed. In an
aspect, a plant part provided herein is a modified plant part. As
used herein, "modified," in the context of a plant, seed, or plant
part, refers to a plant, seed, or plant part, comprising a genetic
alteration introduced for certain purposes and beyond natural
polymorphisms. Without being limiting, a modified plant, seed, or
plant part comprises a recombinant nucleic acid molecule. In
another aspect, a modified plant, seed, or plant part comprises a
genetic modification. In an aspect, a modified plant, seed, or
plant part is transgenic.
[0158] Cannabis cultivars range from plants grown to produce
Cannabis for recreational purposes to plants produced to use hemp
fiber derived from the stems of the plant (e.g., industrial hemp).
In cultivars utilized for recreational purposes, THC quantity
exceeds CBD quantity in the dried female inflorescences used for
smoking and oral administration. Industrial hemp cultivars produce
substantially lower levels of THC and higher levels of CBD.
[0159] The plant genus Cannabis encompasses the species C. sativa,
C. indica, and C. ruderalis. Another classification scheme applied
to Cannabis is a chemotaxonomic classification system. There are
five chemotaxonomic types of Cannabis: one with high levels of THC,
one with high levels of CBD, one that has similar levels of THC and
CBD, one with high levels of CBG, and one with low to no detectable
cannabinoids.
[0160] In addition to genetic characteristics, cultivated Cannabis
plants are influenced by environmental conditions and production
technology during their life cycle. A study evaluating the effects
of ambient temperature and humidity, soil temperature and
precipitation on the content of THC and CBD in industrial hemp
noted that these agroclimatic conditions have differing effects on
THC and CBD. For example, CBD content is positively affected by
soil temperature and ambient temperature, but negatively influenced
by precipitation. See, for example, Sikora, V., et al., 2011,
Genetika, 43(3):449-456.
[0161] In an aspect, a plant is a tobacco plant. In an aspect, a
plant is a Nicotiana plant. In an aspect, a tobacco plant is a
Nicotiana tabacum plant.
[0162] In an aspect, a Nicotiana plant, seed, or plant part is
selected from the group consisting of Nicotiana tabacum, Nicotiana
amplexicaulis PI 271989; Nicotiana benthamiana PI 555478; Nicotiana
bigelovii PI 555485; Nicotiana debneyi; Nicotiana excelsior PI
224063; Nicotiana glutinosa PI 555507; Nicotiana goodspeedii PI
241012; Nicotiana gossei PI 230953; Nicotiana hesperis PI 271991;
Nicotiana knightiana PI 555527; Nicotiana maritima PI 555535;
Nicotiana megalosiphon PI 555536; Nicotiana nudicaulis PI 555540;
Nicotiana paniculata PI 555545; Nicotiana plumbaginfolia PI 555548;
Nicotiana repanda PI 555552; Nicotiana rustica; Nicotiana
suaveolens PI 230960; Nicotiana sylvestris PI 555569; Nicotiana
tomentosa PI 266379; Nicotiana tomentosiformis; and Nicotiana
trigonophylla PI 555572.
[0163] In an aspect, a plant is a Cannabis plant. In an aspect, a
plant is a Cannabis plant. In an aspect, a Cannabis plant is a
Cannabis sativa plant. In an aspect, a Cannabis plant is a Cannabis
indica plant. In an aspect, a Cannabis plant is a Cannabis
ruderalis plant. In an aspect, a Cannabis plant is selected from
the group consisting of Cannabis sativa, Cannabis indica, and
Cannabis ruderalis.
[0164] In an aspect, a Cannabis plant may be placed into chemotype
or chemotaxonomic grouping based on ratios of THC/CBD. In an
aspect, a ratio of THC/CBD is based on relative levels of THC and
CBD within a given Cannabis plant. A Cannabis plant with a THC/CBD
ratio of greater than 1.0 has a high level of THC. A Cannabis plant
with a THC/CBD ratio of approximately 1.0 has similar levels of THC
and CBD. A Cannabis plant with a THC/CBD ratio of less than 1.0 has
a high level of CBD.
[0165] In an aspect, a Cannabis plant is placed within a
chemotaxonomic type comprising high levels of THC, relative to
other assessed cannabinoids. In an aspect, a Cannabis plant is
placed within a chemotaxonomic type comprising high levels of CBD,
relative to other assessed cannabinoids. In an aspect, a Cannabis
plant is placed within a chemotaxonomic type comprising similar
levels of THC and CBD. In an aspect, a Cannabis plant is placed
within a chemotaxonomic type comprising high levels of CBG,
relative to other assessed cannabinoids. In an aspect, a Cannabis
plant is placed within a chemotaxonomic type comprising low to no
detectable cannabinoids.
[0166] In an aspect, a Cannabis plant, plant part, or seed
comprises low to no detectable levels of THC. In an aspect, a
Cannabis plant, plant part, or seed comprises low to no detectable
levels of CBD. In an aspect, a Cannabis plant, plant part, or seed
comprises low to no detectable levels of CBG.
[0167] In an aspect, a seed is a tobacco seed. In an aspect, a seed
is a Nicotiana seed. In an aspect, a tobacco seed is a Nicotiana
tabacum seed.
[0168] In an aspect, a seed is a Cannabis seed. In an aspect, a
seed is a Cannabis seed. In an aspect, a Cannabis seed is a
Cannabis sativa seed. In an aspect, a Cannabis seed is a Cannabis
indica seed. In an aspect, a Cannabis seed is a Cannabis ruderalis
seed. In an aspect, a Cannabis seed is selected from the group
consisting of Cannabis sativa, Cannabis indica, and Cannabis
ruderalis.
[0169] In an aspect, a Cannabis seed is placed within a
chemotaxonomic type comprising high levels of THC, relative to
other assessed cannabinoids. In an aspect, a Cannabis seed is
placed within a chemotaxonomic type comprising high levels of CBD,
relative to other assessed cannabinoids. In an aspect, a Cannabis
seed is placed within a chemotaxonomic type comprising similar
levels of THC and CBD. In an aspect, a Cannabis seed is placed
within a chemotaxonomic type comprising high levels of CBG,
relative to other assessed cannabinoids. In an aspect, a Cannabis
seed is placed within a chemotaxonomic type comprising low to no
detectable cannabinoids.
[0170] In an aspect, a plant part is a tobacco plant part. In an
aspect, a plant part is a Nicotiana plant part. In an aspect, a
tobacco plant part is a Nicotiana tabacum plant part.
[0171] In an aspect, a plant part is a Cannabis plant part. In an
aspect, a plant part is a Cannabis plant part. In an aspect, a
Cannabis plant part is a Cannabis sativa plant part. In an aspect,
a Cannabis plant part is a Cannabis indica plant part. In an
aspect, a Cannabis plant part is a Cannabis ruderalis plant part.
In an aspect, a Cannabis plant part is selected from the group
consisting of Cannabis sativa, Cannabis indica, and Cannabis
ruderalis.
[0172] In an aspect, a Cannabis plant part is placed within a
chemotaxonomic type comprising high levels of THC, relative to
other assessed cannabinoids. In an aspect, a Cannabis plant part is
placed within a chemotaxonomic type comprising high levels of CBD,
relative to other assessed cannabinoids. In an aspect, a Cannabis
plant part is placed within a chemotaxonomic type comprising
similar levels of THC and CBD. In an aspect, a Cannabis plant part
is placed within a chemotaxonomic type comprising high levels of
CBG, relative to other assessed cannabinoids. In an aspect, a
Cannabis plant part is placed within a chemotaxonomic type
comprising low to no detectable cannabinoids.
[0173] In an aspect, a plant part provided includes, but is not
limited to, a leaf, a stem, a root, a trichome, a seed, a flower,
pollen, an anther, an ovule, a pedicel, a fruit, a meristem, a
cotyledon, a hypocotyl, a pod, an embryo, endosperm, an explant, a
callus, a tissue culture, a shoot, a cell, and a protoplast. In an
aspect, a plant part does not include a seed. In an aspect, this
disclosure provides plant cells, tissues, and organs that are not
reproductive material and do not mediate the natural reproduction
of the plant. In another aspect, this disclosure also provides
plant cells, tissues, and organs that are reproductive material and
mediate the natural reproduction of the plant. In another aspect,
this disclosure provides plant cells, tissues, and organs that
cannot maintain themselves via photosynthesis. In another aspect,
this disclosure provides somatic plant cells. Somatic cells,
contrary to germline cells, do not mediate plant reproduction.
[0174] Cells, tissues and organs can be from seed, fruit, leaf,
cotyledon, hypocotyl, meristem, embryos, endosperm, root, shoot,
stem, trichome, pod, flower, inflorescence, stalk, pedicel, style,
stigma, receptacle, petal, sepal, pollen, anther, filament, ovary,
ovule, pericarp, phloem, vascular tissue. In another aspect, this
disclosure provides a plant chloroplast. In a further aspect, this
disclosure provides epidermal cells, stomata cell, leaf or root
hairs, a storage root, or a tuber. In another aspect, this
disclosure provides a tobacco protoplast. In another aspect, this
disclosure provides a Cannabis protoplast.
[0175] Skilled artisans understand that tobacco and Cannabis plants
naturally reproduce via seeds, not via asexual reproduction or
vegetative propagation. In an aspect, this disclosure provides
plant endosperm.
[0176] This disclosure provides cells from plants provided
herein.
[0177] As used herein, a "progeny plant" or "progeny seed" can be
from any filial generation, e.g., F.sub.1, F.sub.2, F.sub.3,
F.sub.4, F.sub.5, F.sub.6, F.sub.7, etc.
[0178] In an aspect, a tobacco plant, seed, or plant part, is of a
tobacco variety selected from the group consisting of a flue-cured
variety, a bright variety, a Burley variety, a Virginia variety, a
Maryland variety, a dark variety, a Galpao variety, an Oriental
variety, and a Turkish variety.
[0179] In an aspect, a tobacco cell is of a tobacco variety
selected from the group consisting of a flue cured variety, a
bright variety, a Burley variety, a Virginia variety, a Maryland
variety, a dark variety, a Galpao variety, an Oriental variety, and
a Turkish variety.
[0180] In an aspect, a tobacco leaf is of a tobacco variety
selected from the group consisting of a flue cured variety, a
bright variety, a Burley variety, a Virginia variety, a Maryland
variety, a dark variety, a Galpao variety, an Oriental variety, and
a Turkish variety.
[0181] In an aspect, a cured tobacco leaf or plant part is of a
tobacco variety selected from the group consisting of a flue cured
variety, a bright variety, a Burley variety, a Virginia variety, a
Maryland variety, a dark variety, a Galpao variety, an Oriental
variety, and a Turkish variety. Skilled artisans further understand
that cured tobacco does not constitute a living organism and is not
capable of growth or reproduction
[0182] Flue-cured tobaccos (also called "Virginia" or "bright"
tobaccos) amount to approximately 40% of world tobacco production.
Flue-cured tobaccos are often also referred to as "bright tobacco"
because of the golden-yellow to deep-orange color it reaches during
curing. Flue-cured tobaccos have a light, bright aroma and taste.
Flue-cured tobaccos are generally high in sugar and low in oils.
Major flue-cured tobacco growing countries are Argentina, Brazil,
China, India, Tanzania and the United States of America. In one
aspect, tobacco plants, seeds, or plant parts provided herein are
of a flue-cured tobacco variety selected from the group consisting
of the varieties listed in Table 2, and any variety essentially
derived from any one of the foregoing varieties. See WO 2004/041006
A1. In a further aspect, tobacco plants, seeds, or plant parts
provided herein are in a flue-cured variety selected from the group
consisting of K326, K346, and NC196.
TABLE-US-00002 TABLE 2 Flue-cured Tobacco Varieties 400 (TC 225)
401 (TC 226) 401 Cherry Red (TC 227) 401 Cherry Red Free (TC 228)
Cash (TC 250) Cash (TI 278) CC 101 CC 1063 CC 13 CC 143 CC 200 CC
27 CC 301 CC 33 CC 35 CC 37 CC 400 CC 500 CC 600 CC 65 CC 67 CC 700
CC 800 CC 900 Coker 139 (TC 259) Coker 139 ybl, yb2 Coker 140 (TC
260) Coker 176 (TC 262) Coker 187 (TC 263) Coker 187-Hicks (TC 265)
Coker 209 (TC 267) Coker 258 (TC 270) Coker 298 (TC 272) Coker 316
(TC 273) Coker 319 (TC 274) Coker 347 (TC 275) Coker 371-Gold (TC
276) Coker 411 (TC 277) Coker 48 (TC 253) Coker 51 (TC 254) Coker
86 (TC 256) CU 263 (TC 619) CU 561 DH95-1562-1 Dixie Bright 101 (TC
290) Dixie Bright 102 (TC 291) Dixie Bright 244 (TC 292) Dixie
Bright 27 (TC 288) Dixie Bright 28 (TC 289) GF 157 GF 318 GL 26H GL
338 GL 350 GL 368 GL 395 GL 600 GL 737 GL 939 GL 939 (TC 628) Hicks
(TC 310) Hicks Broadleaf (TC 311) K 149 (TC 568) K 317 K 326 K 326
(TC 319) K 340 (TC 320) K 346 K 346 (TC 569) K 358 K 394 (TC 321) K
399 K 399 (TC 322) K 730 Lonibow (TI 1573) Lonibow (TI 1613) McNair
10 (TC 330) McNair 135 (TC 337) McNair 30 (TC 334) McNair 373 (TC
338) McNair 944 (TC 339) MK94 (TI 1512) MS K 326 MS NC 71 MS NC 72
NC 100 NC 102 NC 1071 (TC 364) NC 1125-2 NC 12 (TC 346) NC 1226 NC
196 NC 2326 (TC 365) NC 27 NF (TC 349) NC 291 NC 297 NC 299 NC 37
NF (TC 350) NC 471 NC 55 NC 567 (TC 362) NC 60 (TC 352) NC 606 NC
6140 NC 71 NC 72 NC 729 (TC 557) NC 810 (TC 659) NC 82 (TC 356) NC
8640 NC 89 (TC 359) NC 92 NC 925 NC 95 (TC 360) NC 98 (TC 361) NC
EX 24 NC PY 10 (TC 367) NC TG 61 Oxford 1 (TC 369) Oxford 1-181 (TC
370) Oxford 2 (TC 371) Oxford 207 (TC 632) Oxford 26 (TC 373)
Oxford 3 (TC 372) Oxford 414 NF PD 611 (TC 387) PVH 03 PVH 09 PVH
1118 PVH 1452 PVH 1600 PVH 2110 PVH 2275 R 83 (Line 256-1) (TI
1400) Reams 134 Reams 158 Reams 713 Reams 744 Reams M1 RG 11 (TC
600) RG 13 (TC 601) RG 17 (TC 627) RG 22 (TC 584) RG 8 (TC 585) RG
81 (TC 618) RG H51 RG4H 217 RGH 12 RGH 4 RGH 51 RGH 61 SC 58 (TC
400) SC 72 (TC 403) Sp. G-168 SPEIGHT 168 Speight 168 (TC 633)
Speight 172 (TC 634) Speight 178 Speight 179 Speight 190 Speight
196 SPEIGHT 220 SPEIGHT 225 SPEIGHT 227 SPEIGHT 236 Speight G-10
(TC 416) Speight G-102 Speight G-108 Speight G-111 Speight G-117
Speight G-126 Speight G-15 (TC 418) Speight G-23 Speight G-28 (TC
420) Speight G-33 Speight G-41 Speight G-5 Speight G-52 Speight
G-58 Speight G-70 Speight G-70 (TC 426) Speight G-80 (TC 427)
Speight NF3 (TC 629) STNCB VA 182 VA 45 (TC 559) Vesta 30 (TC 439)
Vesta 33 (TC 440) Vesta 5 (TC 438) Vesta 62 (TC 441) Virginia (TI
220) Virginia (TI 273) Virginia (TI 877) Virginia 115 (TC 444)
Virginia 21 (TC 443) Virginia Bright (TI 964) Virginia Bright Leaf
(TC 446) Virginia Gold (TC 447) White Stem Orinoco (TC 451)
[0183] Air-cured tobaccos include "Burley," Maryland," and "dark"
tobaccos. The common factor linking air-cured tobaccos is that
curing occurs primarily without artificial sources of heat and
humidity. Burley tobaccos are light to dark brown in color, high in
oil, and low in sugar. Burley tobaccos are typically air-cured in
barns. Major Burley growing countries include Argentina, Brazil,
Italy, Malawi, and the United States of America.
[0184] Maryland tobaccos are extremely fluffy, have good burning
properties, low nicotine and a neutral aroma. Major Maryland
growing countries include the United States of America and
Italy.
[0185] In one aspect, tobacco plants, seeds, or plant parts
provided herein are of a Burley tobacco variety selected from the
group consisting of the tobacco varieties listed in Table 3, and
any variety essentially derived from any one of the foregoing
varieties. In a further aspect, tobacco plants, seeds, or plant
parts provided herein are in a Burley variety selected from the
group consisting of TN 90, KT 209, KT 206, KT212, and 89 4488.
TABLE-US-00003 TABLE 3 Burley Tobacco Varieties 4407 LC AA-37-1
Burley 21 (TC 7) Burley 49 (TC 10) Burley 64 (TC 11) Burley Mammoth
KY 16 (TC 12) Clay 402 Clay 403 Clay 502 Clays 403 GR 10 (TC 19) GR
10 (TC 19) GR10A (TC 20) GR 13 (TC 21) GR 14 (TC 22) GR 149 LC GR
153 GR 17 (TC 23) GR 17B (TC 24) GR 18 (TC 25) GR 19 (TC 26) GR 2
(TC 15) GR 24 (TC 27) GR 36 (TC 28) GR 38 (TC 29) GR 38A (TC 30) GR
40 (TC 31) GR 42 (TC 32) GR 42C (TC 33) GR 43 (TC 34) GR 44 (TC 35)
GR 45 (TC 36) GR 46 (TC 37) GR 48 (TC 38) GR 5 (TC 16) GR 53 (TC
39) GR 6 (TC 17) GR 9 (TC 18) GR139 NS GR139 S HB 04P HB 04P LC HB
3307P LC HB 4108P HB 4151P HB 4192P HB 4194P HB 4196 HB 4488 HB
4488P HB04P HB 4488 LC HIB 21 HPB 21 HY 403 Hybrid 403 LC Hybrid
404 LC Hybrid 501 LC KDH-959 (TC 576) KDH-960 (TC 577) KT 200 LC KT
204 LC KT 206 LC KT 209 LC KT 210 LC KT 212 LC KT 215 LC KY 1 (TC
52) KY 10 (TC 55) KY 12 (TC 56) KY 14 (TC 57) KY 14 x L8 LC KY 15
(TC 58) KY 16 (TC 59) KY 17 (TC 60) KY 19 (TC 61) KY 21 (TC 62) KY
22 (TC 63) KY 24 (TC 64) KY 26 (TC 65) KY 33 (TC 66) KY 34 (TC 67)
KY 35 (TC 68) KY 41A (TC 69) KY 5 (TC 53) KY 52 (TC 70) KY 54 (TC
71) KY 56 (TC 72) KY 56 (TC 72) KY 57 (TC 73) KY 58 (TC 74) KY 8654
(TC 77) KY 8959 KY 9 (TC 54) KY 907 LC KY 908 (TC 630) NBH 98
(Screened) NC 1206 NC 129 NC 2000 LC NC 2002 LC NC 3 LC NC 5 LC NC
6 LC NC 7 LC NC BH 129 LC NC03-42-2 Newton 98 R 610 LC R 630 LC R
7-11 R 7-12 LC RG 17 TKF 1801 LC TKF 2002 LC TKF 4024 LC TKF 4028
LC TKF 6400 LC TKF 7002 LC TKS 2002 LC TN 86 (TC 82) TN 90 LC TN 97
Hybrid LC TN 97 LC VA 116 VA 119 Virgin A Mutante (TI 1406)
Virginia 509 (TC 84)
[0186] In another aspect, tobacco plants, seeds, or plant parts
provided herein are of a Maryland tobacco variety selected from the
group consisting of the tobacco varieties listed in Table 4, and
any variety essentially derived from any one of the foregoing
varieties.
TABLE-US-00004 TABLE 4 Maryland Tobacco Varieties Maryland 10 (TC
498) Maryland 14 D2 (TC 499) Maryland 201 (TC 503) Maryland 21 (TC
500) Maryland 341 (TC 504) Maryland 40 Maryland 402 Maryland 59 (TC
501) Maryland 601 Maryland 609 (TC 505) Maryland 64 (TC 502)
Maryland 872 (TC 506) Maryland Mammoth (TC 507)
[0187] Dark air-cured tobaccos are distinguished from other tobacco
types primarily by its curing process, which gives dark air-cured
tobacco its medium-brown to dark-brown color and a distinct aroma.
Dark air-cured tobaccos are mainly used in the production of
chewing tobacco and snuff. In one aspect, tobacco plants, seeds, or
plant parts provided herein are of a dark air-cured tobacco variety
selected from the group consisting of Sumatra, Jatim, Dominican
Cubano, Besuki, One sucker, Green River, Virginia sun-cured, and
Paraguan Passado, and any variety essentially derived from any one
of the foregoing varieties.
[0188] Dark fire-cured tobaccos are generally cured with
low-burning wood fires on the floors of closed curing barns. Dark
fire-cured tobaccos are typically used for making pipe blends,
cigarettes, chewing tobacco, snuff, and strong-tasting cigars.
Major growing regions for dark fire-cured tobaccos are Tennessee,
Kentucky, and Virginia in the United States of America. In one
aspect, tobacco plants, seeds, or plant parts provided herein are
of a dark fire-cured tobacco variety selected from the group
consisting of the tobacco varieties listed in Table 5, and any
variety essentially derived from any one of the foregoing
varieties.
TABLE-US-00005 TABLE 5 Dark Fire-Cured Tobacco Varieties Black
Mammoth (TC 461) Black Mammoth Small Stalk (TC 641) Certified
Madole (TC 463) D-534-A-1 (TC 464) DAC ULT 302 DAC ULT 303 DAC ULT
306 DAC ULT 308 DAC ULT 312 DF 300 (TC 465) DF 485 (TC 466) DF 516
(TC 467) DF 911 (TC 468) DT 508 DT 518 (Screened) DT 538 LC DT 592
Improved Madole (TC 471) Jernigan's Madole (TC 472) KT 14LC KT
D17LC KT D4 LC KT D6 LC KT D8 LC KY 153 (TC 216) KY 157 (TC 217) KY
160 KY 160 (TC 218) KY 163 (TC 219) KY 165 (TC 220) KY 170 (TC 474)
KY 171 (PhPh) KY 171 (TC 475) KY 171 LC KY 171 NS KY 180 (TC 573)
KY 190 (TC 574) Little Crittenden Little Crittenden (TC 476) Little
Crittenden LC (certified) Little Crittenden PhPh Lizard Tail Turtle
Foot TN D94 Madole (TC 478) Madole (TC 479) MS KY 171 MS NL Madole
LC MS TN D950 LC Nance (TC 616) Narrow Leaf Madole LC (certified)
Neal Smith Madole (TC 646) Newtons VH Madole NL Madole NL Madole
(PhPh) NL Madole (TC 484) NL Madole LC NL Madole LC (PhPh) NL
Madole NS One Sucker (TC 224) OS 400 PD 302H PD 312H PD 318H PD
7302 LC PD 7305 PD 7309 LC PD 7312 LC PD 7318 LC PD 7319 LC Petico
M PG04 PY KY 160 (TC 612) PY KY 171 (TC 613) Shirey TI 1372 TN D94
(TC 621) TN D950 TN D950 (PhPh) TN D950 TN D950 (TC 622) TR Madole
(TC 486) VA 309 VA 309 (TC 560) VA 309 LC (certified) VA 310 (TC
487) VA 331 (TC 592) VA 355 (TC 638) VA 359 VA 359 (Screened) VA
359 (TC 639) VA 359 LC (certified) VA 403 (TC 580) VA 405 (TC 581)
VA 409 (TC 562) VA 510 (TC 572)
[0189] Oriental tobaccos are also referred to as Greek, aroma and
Turkish tobaccos due to the fact that they are typically grown in
eastern Mediterranean regions such as Turkey, Greece, Bulgaria,
Macedonia, Syria, Lebanon, Italy, and Romania. The small plant
size, small leaf size, and unique aroma properties of Oriental
tobacco varieties are a result of their adaptation to the poor soil
and stressful climatic conditions in which they have been
developed. In one aspect, tobacco plants, seeds, or plant parts
provided herein are of an Oriental tobacco variety selected from
the group consisting of the tobacco varieties listed in Table 6,
and any variety essentially derived from any one of the foregoing
varieties.
TABLE-US-00006 TABLE 6 Oriental Tobacco Varieties Bafra (TI 1641)
Bahce (TI 1730) Bahia (TI 1416) Bahia (TI 1455) Baiano (TI 128)
Basma Basma (TI 1666) Basma Drama Basma Hybrid (PhPh) Basma Zihna I
Bitlis (TI 1667) Bitlis (TI 1725) Bubalovac (TI 1282) Bursa (TI
1650) Bursa (TI 1668) Canik (TI 1644) Djebel 174 (TI 1492) Djebel
359 (TI 1493) Djebel 81 Dubec 566 (TI 1409) Dubec 7 (TI 1410) Dubek
566 (TI 1567) Duzce (TI 1670) Edirne (TI 1671) Ege (TI 1642) Ege-64
(TI 1672) Izmir (Akhisar) (TI 1729) Izmir (Gavurkoy) (TI 1727)
Izmir Ege 64 Izmir-Incekara (TI 1674) Izmir-Ozbas (TI 1675) Jaka
Dzebel (TI 1326) Kaba-Kulak Kagoshima Maruba (TI 158) Katerini
Katerini S53 Krumovgrad 58 MS Basma MS Katerini S53 Nevrokop 1146
Ozbas (TI 1645) Perustitza (TI 980) Prilep (TI 1291) Prilep (TI
1325) Prilep 12-2/1 Prilep 23 Samsun (TC 536) Samsun 959 (TI 1570)
Samsun Evkaf (TI 1723) Samsun Holmes NN (TC 540) Samsun Maden (TI
1647) Samsun NO 15 (TC 541) Samsun-BLK SHK Tol (TC 542)
Samsun-Canik (TI 1678) Samsun-Maden (TI 1679) Saribaptar 407 -
Izmir Region Smyrna (TC 543) Smyrna No. 23 (TC 545) Smyrna No. 9
(TC 544) Smyrna-Blk Shk Tol (TC 546) Trabzon (TI 1649) Trabzon (TI
1682) Trapezund 161 (TI 1407) Turkish (TC 548) Turkish Angshit (TI
90) Turkish Samsum (TI 92) Turkish Tropizoid (TI 93) Turkish
Varotic (TI 89) Xanthi (TI 1662)
[0190] In an aspect, tobacco plants, seeds, or plant parts provided
herein are of a cigar tobacco variety selected from the group
consisting of the tobacco varieties listed in Table 7, and any
variety essentially derived from any one of the foregoing
varieties.
TABLE-US-00007 TABLE 7 Cigar Tobacco Varieties Bahai (TI 62)
Beinhart 1000 Beinhart 1000 (TI 1562) Beinhart 1000-1 (TI 1561)
Bergerac C Bergerac C (TI 1529) Big Cuban (TI 1565) Castillo Negro,
Blanco, Pina (TI 448) Castillo Negro, Blanco, Pina (TI 448A)
Castillo Negro, Blanco, Pina (TI 449) Caujaro (TI 893) Chocoa (TI
289) Chocoa (TI 313) Connecticut 15 (TC 183) Connecticut Broadleaf
Connecticut Broadleaf (TC 186) Connecticut Shade (TC 188) Criollo,
Colorado (TI 1093) Enshu (TI 1586) Florida 301 Florida 301 (TC 195)
PA Broadleaf (TC 119) Pennsylvania Broadleaf Pennsylvania Broadleaf
(TC 119) Petite Havana SR1 Petite Havana SR1 (TC 105)
[0191] In an aspect, tobacco plants, seeds, or plant parts provided
herein are of a tobacco variety selected from the group consisting
of the tobacco varieties listed in Table 8, and any variety
essentially derived from any one of the foregoing varieties.
TABLE-US-00008 TABLE 8 Other Tobacco Varieties Chocoa (TI 319) Hoja
Parada (TI 1089) Hoj a Parado (Galpoa) (TI 1068) Perique (St. James
Parrish) Perique (TC 556) Perique (TI 1374) Sylvestris (TI 984) TI
179
[0192] In an aspect, a tobacco plant or plant part is from a
variety selected from the group consisting of the tobacco varieties
listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and
Table 8. In another aspect, a tobacco plant or plant part is from a
variety listed in Table 2. In another aspect, a tobacco plant or
plant part is from a variety listed in Table 3. In another aspect,
a tobacco plant or plant part is from a variety listed in Table 4.
In another aspect, a tobacco plant or plant part is from a variety
listed in Table 5. In another aspect, a tobacco plant or plant part
is from a variety listed in Table 6. In another aspect, a tobacco
plant or plant part is from a variety listed in Table 7. In another
aspect, a tobacco plant or plant part is from a variety listed in
Table 8.
[0193] In an aspect, a tobacco seed is from a variety selected from
the group consisting of the tobacco varieties listed in Table 2,
Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8. In
another aspect, a tobacco seed is from a variety listed in Table 2.
In another aspect, a tobacco seed is from a variety listed in Table
3. In another aspect, a tobacco seed is from a variety listed in
Table 4. In another aspect, a tobacco seed is from a variety listed
in Table 5. In another aspect, a tobacco seed is from a variety
listed in Table 6. In another aspect, a tobacco seed is from a
variety listed in Table 7. In another aspect, a tobacco seed is
from a variety listed in Table 8.
[0194] In an aspect, a tobacco cell is from a variety selected from
the group consisting of the tobacco varieties listed in Table 2,
Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8. In
another aspect, a tobacco cell is from a variety listed in Table 2.
In another aspect, a tobacco cell is from a variety listed in Table
3. In another aspect, a tobacco cell is from a variety listed in
Table 4. In another aspect, a tobacco cell is from a variety listed
in Table 5. In another aspect, a tobacco cell is from a variety
listed in Table 6. In another aspect, a tobacco cell is from a
variety listed in Table 7. In another aspect, a tobacco cell is
from a variety listed in Table 8.
[0195] All foregoing mentioned specific varieties of flue-cured,
dark air-cured, Burley, Maryland, dark fire-cured, cigar, or
Oriental type are listed only for exemplary purposes. Any
additional flue-cured, dark air-cured, Burley, Maryland, dark
fire-cured, cigar, or Oriental varieties are also contemplated in
the present application.
[0196] In an aspect, a plant or variety provided herein is an
inbred plant or variety. As used herein, an "inbred" variety is a
variety that has been bred for genetic homogeneity.
[0197] As used herein, a "hybrid" is created by crossing two plants
from different varieties or species, such that the progeny
comprises genetic material from each parent. Skilled artisans
recognize that higher order hybrids can be generated as well. For
example, a first hybrid can be made by crossing Variety C with
Variety D to create a C.times.D hybrid, and a second hybrid can be
made by crossing Variety E with Variety F to create an E.times.F
hybrid. The first and second hybrids can be further crossed to
create the higher order hybrid (C.times.D).times.(E.times.F)
comprising genetic information from all four parent varieties. In
an aspect, a plant or seed provided herein is a hybrid plant or
seed.
[0198] In an aspect, a tobacco plant provided herein is an inbred
tobacco plant. In an aspect, a tobacco seed provided herein is an
inbred tobacco seed. In an aspect, a tobacco plant provided herein
is a hybrid tobacco plant. In another aspect, a tobacco seed
provided herein is a hybrid tobacco seed.
[0199] In an aspect, a Cannabis plant provided herein is an inbred
Cannabis plant. In an aspect, a Cannabis seed provided herein is an
inbred Cannabis seed. In an aspect, a Cannabis plant provided
herein is a hybrid Cannabis plant. In another aspect, a Cannabis
seed provided herein is a hybrid Cannabis seed.
[0200] Unless specified otherwise, all comparisons to control
plants require similar growth conditions or comparable growth
conditions for the two plants being compared. As used herein,
"grown under comparable conditions," "similar growth conditions" or
"comparable growth conditions" refer to similar environmental
conditions and/or agronomic practices for growing and making
meaningful comparisons between two or more plant genotypes so that
neither environmental conditions nor agronomic practices would
contribute to or explain any difference observed between the two or
more plant genotypes. Environmental conditions include, for
example, light, temperature, water (humidity), and nutrition (e.g.,
nitrogen and phosphorus). Agronomic practices include, for example,
seeding, clipping, undercutting, transplanting, topping, and
suckering. See Chapters 4B and 4C of Tobacco, Production, Chemistry
and Technology, Davis & Nielsen, eds., Blackwell Publishing,
Oxford (1999), pp 70-103. See, also, Amaducci et al. Field Crops
Research, 107:161-169 (2008). As used herein, a "control plant"
refers to a plant of identical, or nearly identical, genetic makeup
as the modified plant being compared, except for the non-natural
mutation or recombinant DNA construct provided herein that was
introduced to the modified plant.
[0201] In an aspect, a first plant variety and a second plant
variety are the same variety. In an aspect, a first plant variety
and a second plant variety are two different varieties. In an
aspect, a second plant variety comprises a recombinant nucleic acid
molecule.
[0202] In an aspect, a first plant variety is heterozygous for a
recombinant nucleic acid molecule. In an aspect, a first plant
variety is hemizygous for a recombinant nucleic acid molecule. In
an aspect, a first plant variety is homozygous for a recombinant
nucleic acid molecule. In an aspect, a second plant variety is
heterozygous for a recombinant nucleic acid molecule. In an aspect,
a second plant variety is hemizygous for a recombinant nucleic acid
molecule. In an aspect, a second plant variety is homozygous for a
recombinant nucleic acid molecule. In an aspect, a progeny seed, or
a plant germinated therefrom, is heterozygous for a recombinant
nucleic acid molecule. In an aspect, a progeny seed, or a plant
germinated therefrom, is hemizygous for a recombinant nucleic acid
molecule. In an aspect, a progeny seed, or a plant germinated
therefrom, is homozygous for a recombinant nucleic acid
molecule.
[0203] In an aspect, a first plant variety is a tobacco plant
variety. In an aspect, a second plant variety is a tobacco plant
variety.
[0204] In an aspect, a first plant variety is a Cannabis plant
variety. In an aspect, a second plant variety is a Cannabis plant
variety.
[0205] As used herein, the term "crossing" refers to the deliberate
mating of two plants. In an aspect, crossing comprises pollination
and/or fertilization of a first plant by a second plant. The two
plants being crossed can be distantly related, closely related, or
identical. In an aspect, the two plants being crossed are both
modified plants. In an aspect, the two plants being crossed are of
the same variety. In an aspect, the two plants being crossed are of
two different varieties. In an aspect, one of the two plants being
crossed is male sterile. In an aspect, one of the two plants being
crossed is female sterile. In an aspect, at least one of the two
plants being crossed is a hybrid tobacco plant. In an aspect, at
least one of the two plants being crossed is a hybrid Cannabis
plant. In an aspect, at least one of the two plants being crossed
is a modified plant.
[0206] In an aspect, a plant of a first variety is the male parent
in a crossing step. In an aspect, a plant of a first variety is the
female parent in a crossing step. In an aspect, a plant of a second
variety is the male parent in a crossing step. In an aspect, a
plant of a second variety is the female parent in a crossing
step.
[0207] In an aspect, a plant or variety provided herein is male
sterile. In another aspect, a plant or variety provided herein is
cytoplasmic male sterile (CMS). Male sterile plants can be produced
by any method known in the art. Methods of producing male sterile
tobacco are described in Wernsman, E. A., and Rufty, R. C. 1987.
Chapter Seventeen. Tobacco. Pages 669-698 in: Cultivar Development.
Crop Species. W. H. Fehr (ed.), MacMillan Publishing Go., Inc., New
York, N.Y. 761 pp. See also
kush[dot]com/blog/feminized-Cannabis-seeds-all-you-need-to-know,
which describes processes such as rodelization and spraying female
Cannabis plants with colloidal silver every day after flowering to
reduce ethylene production.
[0208] In another aspect, a plant or variety provided herein is
female sterile. As a non-limiting example, female sterile plants
can be made by mutating the STIGI gene. See, for example, Goldman
et al. 1994, EMBO Journal 13:2976-2984.
[0209] In an aspect, this disclosure provides a method of producing
a modified tobacco plant comprising: (a) introducing a recombinant
nucleic acid molecule to at least one tobacco cell, where the
recombinant nucleic acid molecule comprises a nucleic acid sequence
encoding a polypeptide involved in terpene biosynthesis operably
linked to a heterologous promoter; (b) selecting at least one
tobacco cell comprising the recombinant nucleic acid molecule; and
(c) regenerating a modified tobacco plant from the at least one
tobacco cell selected in step (b) where the modified tobacco plant
comprises an increased amount of at least one terpene in at least
one tissue as compared to a control tobacco plant lacking the
recombinant nucleic acid molecule when grown under comparable
conditions.
[0210] In an aspect, this disclosure provides a method of producing
a modified Cannabis plant comprising: (a) introducing a recombinant
nucleic acid molecule to at least one Cannabis cell, where the
recombinant nucleic acid molecule comprises a nucleic acid sequence
encoding a polypeptide involved in terpene biosynthesis operably
linked to a heterologous promoter; (b) selecting at least one
Cannabis cell comprising the recombinant nucleic acid molecule; and
(c) regenerating a modified Cannabis plant from the at least one
Cannabis cell selected in step (b) where the modified Cannabis
plant comprises an increased amount of at least one terpene in at
least one tissue as compared to a control Cannabis plant lacking
the recombinant nucleic acid molecule when grown under comparable
conditions.
Transformation
[0211] Numerous methods for introducing a recombinant DNA construct
to a plant cell are known in the art, which can be used according
to methods of the present application to produce a transgenic plant
cell and plant. Any suitable method or technique for transformation
of a plant cell known in the art can be used according to present
methods. Effective methods for transformation of plants include
bacterially mediated transformation, such as Agrobacterium-mediated
or Rhizobium-mediated transformation and microprojectile
bombardment-mediated transformation. See, e.g., Feeney and Punja,
In Vitro Cell. and Dev. Biol.--Plant, 39:578-585 (2003). A variety
of methods are known in the art for transforming explants with a
transformation vector via bacterially mediated transformation or
microprojectile bombardment and then subsequently culturing, etc.,
those explants to regenerate or develop transgenic plants. Other
methods for plant transformation, such as microinjection,
electroporation, vacuum infiltration, pressure, sonication, silicon
carbide fiber agitation, polyethylene glycol (PEG)-mediated
transformation, etc., are also known in the art. Transgenic plants
produced by these transformation methods can be chimeric or
non-chimeric for the transformation event depending on the methods
and explants used.
[0212] Methods of transforming plant cells are well known by
persons of ordinary skill in the art. For instance, specific
instructions for transforming plant cells by microprojectile
bombardment with particles coated with recombinant DNA (e.g.,
biolistic transformation) are found in U.S. Pat. Nos. 5,550,318;
5,538,880 6,160,208; 6,399,861; and 6,153,812 and
Agrobacterium-mediated transformation is described in U.S. Pat.
Nos. 5,159,135; 5,824,877; 5,591,616; 6,384,301; 5,750,871;
5,463,174; and 5,188,958, all of which are incorporated herein by
reference. Additional methods for transforming plants can be found
in, for example, Compendium of Transgenic Crop Plants (2009)
Blackwell Publishing. Any appropriate method known to those skilled
in the art can be used to transform a tobacco or Cannabis cell with
any of the nucleic acid molecules provided herein.
[0213] In an aspect, this disclosure provides a method comprising
transforming a tobacco or Cannabis cell with a recombinant nucleic
acid molecule, where the recombinant nucleic acid molecule
comprises a nucleic acid sequence encoding a polypeptide involved
in the biosynthesis of at least one terpene operably linked to a
heterologous promoter.
[0214] In an aspect, this disclosure provides a method for
producing a tobacco or Cannabis plant, the method comprising: (a)
crossing at least one tobacco or Cannabis plant of a first tobacco
or Cannabis variety with at least one tobacco or Cannabis plant of
a second tobacco or Cannabis variety to produce at least one
progeny tobacco or Cannabis seed, where the at least one tobacco or
Cannabis plant of the first tobacco or Cannabis variety comprises a
recombinant nucleic acid molecule comprising a nucleic acid
sequence encoding a polypeptide involved in the biosynthesis of at
least one terpene operably linked to a heterologous promoter; and
(b) selecting for at least one progeny tobacco or Cannabis seed, or
a plant germinated therefrom, where the at least one progeny
tobacco or Cannabis seed or plant germinated therefrom comprises
the recombinant nucleic acid molecule.
[0215] In an aspect, this disclosure provides a method for
producing a tobacco or Cannabis plant, the method comprising: (a)
crossing at least one tobacco or Cannabis plant of a first tobacco
or Cannabis variety with at least one tobacco or Cannabis plant of
a second tobacco or Cannabis variety to produce at least one
progeny tobacco or Cannabis seed, where the at least one tobacco or
Cannabis plant of the first tobacco or Cannabis variety comprises
at least one non-natural mutation in an endogenous gene encoding a
polypeptide involved in the biosynthesis of at least one terpene,
where the at least one tobacco or Cannabis plant of the first
tobacco or Cannabis variety exhibits increased mRNA expression of
the endogenous gene or increased accumulation of the polypeptide as
compared to a control tobacco or Cannabis plant of the first
tobacco or Cannabis variety lacking the at least one non-natural
mutation when grown under comparable conditions; and (b) selecting
for at least one progeny tobacco or Cannabis seed, or a plant
germinated therefrom, where the at least one progeny tobacco or
Cannabis seed or plant germinated therefrom comprises the at least
one non-natural mutation.
[0216] In an aspect, a method of introducing a nucleic acid
molecule to a tobacco or Cannabis cell comprises
Agrobacterium-mediated transformation. In another aspect, a method
of introducing a nucleic acid molecule to a tobacco or Cannabis
cell comprises PEG-mediated transformation. In another aspect, a
method of introducing a nucleic acid molecule to a tobacco or
Cannabis cell comprises biolistic transformation. In another
aspect, a method of introducing a nucleic acid molecule to a
tobacco or Cannabis cell comprises liposome-mediated transfection
(lipofection). In another aspect, a method of introducing a nucleic
acid molecule to a tobacco or Cannabis cell comprises lentiviral
transfection.
[0217] Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386,
4,946,787; and 4,897,355) and lipofection reagents are sold
commercially (e.g., Transfectam.TM. and Lipofectin.TM.). Cationic
and neutral lipids that are suitable for efficient
receptor-recognition lipofection of polynucleotides include those
of International Publication WO 1991/017424 and International
Publication WO 1991/016024. Delivery can be to cells (e.g. in vitro
or ex vivo administration) or target tissues (e.g. in vivo
administration).
[0218] Any tobacco cell from which a fertile tobacco plant can be
regenerated is contemplated as a useful recipient cell for practice
of this disclosure. Similarly, any Cannabis cell from which a
fertile Cannabis plant can be regenerated is contemplated as a
useful recipient cell for practice of this disclosure.
[0219] In an aspect, a recombinant DNA construct is introduced to a
tobacco cell. In an aspect, a recombinant DNA construct is
introduced to a tobacco protoplast cell. In another aspect, a
recombinant DNA construct is introduced to a tobacco callus cell.
In an aspect, a recombinant DNA construct is introduced to a
tobacco cell selected from the group consisting of a seed cell, a
fruit cell, a leaf cell, a cotyledon cell, a hypocotyl cell, a
meristem cell, an embryo cell, an endosperm cell, a root cell, a
shoot cell, a stem cell, a flower cell, an inflorescence cell, a
stalk cell, a pedicel cell, a style cell, a stigma cell, a
receptacle cell, a petal cell, a sepal cell, a pollen cell, an
anther cell, a filament cell, an ovary cell, an ovule cell, a
pericarp cell, and a phloem cell.
[0220] In an aspect, a recombinant DNA construct is introduced to a
Cannabis cell. In an aspect, a recombinant DNA construct is
introduced to a Cannabis protoplast cell. In another aspect, a
recombinant DNA construct is introduced to a Cannabis callus cell.
In an aspect, a recombinant DNA construct is introduced to a
Cannabis cell selected from the group consisting of a seed cell, a
fruit cell, a leaf cell, a cotyledon cell, a hypocotyl cell, a
meristem cell, an embryo cell, an endosperm cell, a root cell, a
shoot cell, a stem cell, a flower cell, an inflorescence cell, a
stalk cell, a pedicel cell, a style cell, a stigma cell, a
receptacle cell, a petal cell, a sepal cell, a pollen cell, an
anther cell, a filament cell, an ovary cell, an ovule cell, a
pericarp cell, and a phloem cell.
[0221] Callus can be initiated from various tissue sources,
including, but not limited to, immature embryos or parts of
embryos, seedling apical meristems, microspores, and the like.
Those cells which are capable of proliferating as callus can serve
as recipient cells for transformation. Practical transformation
methods and materials for making transgenic plants of this
disclosure (e.g., various media and recipient target cells,
transformation of immature embryos, and subsequent regeneration of
fertile transgenic plants) are disclosed, for example, in U.S. Pat.
Nos. 6,194,636 and 6,232,526 and U.S. Patent Application
Publication 2004/0216189, all of which are incorporated herein by
reference.
[0222] In an aspect, this disclosure provides a method of producing
a modified tobacco or Cannabis plant comprising: (a) introducing a
recombinant DNA construct to at least one tobacco or Cannabis cell,
where said recombinant DNA construct comprises a heterologous
promoter operably linked to a nucleic acid encoding at least one
small RNA molecule capable of binding to and reducing the
expression of a nucleic acid sequence encoding a polypeptide
involved in the biosynthesis of at least one terpene; (b) selecting
at least one tobacco or Cannabis cell comprising said recombinant
DNA construct; and (c) regenerating at least one modified tobacco
or Cannabis plant from said at least one tobacco or Cannabis cell
selected in step (b), where the modified tobacco or Cannabis plant
comprises a reduced amount of at least one terpene in at least one
tissue as compared to a control tobacco or Cannabis plant lacking
the recombinant DNA construct when grown under comparable
conditions.
[0223] In an aspect, this disclosure provides a method comprising
transforming a tobacco or Cannabis cell with a recombinant nucleic
acid molecule, where the recombinant nucleic acid molecule
comprises a heterologous promoter operably linked to a nucleic acid
encoding at least one small RNA molecule capable of binding to and
reducing the expression of a nucleic acid sequence encoding a
polypeptide involved in the biosynthesis of at least one
terpene.
[0224] In an aspect, this disclosure provides a method for
producing a tobacco or Cannabis plant, the method comprising: (a)
crossing at least one tobacco or Cannabis plant of a first tobacco
or Cannabis variety with at least one tobacco or Cannabis plant of
a second tobacco or Cannabis variety to produce at least one
progeny tobacco or Cannabis seed, where the at least one tobacco or
Cannabis plant of the first tobacco or Cannabis variety comprises a
recombinant nucleic acid molecule comprising a heterologous
promoter operably linked to a nucleic acid encoding at least one
small RNA molecule capable of binding to and reducing the
expression of a nucleic acid sequence encoding a polypeptide
involved in the biosynthesis of at least one terpene; and selecting
for at least one progeny tobacco or Cannabis seed, or a plant
germinated therefrom, where the at least one progeny tobacco or
Cannabis seed or plant germinated therefrom comprises the
recombinant nucleic acid molecule.
Mutations
[0225] Without being limiting, a modified plant can comprise a
non-natural mutation or a recombinant DNA construct. In an aspect,
a modified tobacco plant comprises a non-natural mutation. In
another aspect, a modified tobacco plant comprises a recombinant
DNA construct. In another aspect, a modified tobacco plant
comprises a genetic modification. In an aspect, a modified Cannabis
plant comprises a non-natural mutation. In another aspect, a
modified Cannabis plant comprises a recombinant DNA construct. In
another aspect, a modified Cannabis plant comprises a genetic
modification.
[0226] In an aspect, this disclosure provides a method of producing
a modified tobacco or Cannabis plant comprising: (a) inducing at
least one non-natural mutation in at least one tobacco or Cannabis
cell in an endogenous gene encoding a polypeptide involved in
terpene biosynthesis; (b) selecting at least one tobacco or
Cannabis plant comprising the at least one non-natural mutation
from step (a); and (c) regenerating a modified tobacco or Cannabis
plant from the at least one tobacco or Cannabis cell selected in
step (b), where the modified tobacco or Cannabis plant comprises an
increased amount of at least one terpene in at least one tissue as
compared to a control tobacco or Cannabis plant lacking the at
least one non-natural mutation when grown under comparable
conditions.
[0227] As used herein, a "mutation" refers to an inheritable
genetic modification introduced into a gene to alter the expression
or activity of a product encoded by a reference sequence of the
gene. A mutation in a certain gene, such as, for example, limonene
synthase (LS), is referred to as an LS mutant. Such a modification
can be in any sequence region of a gene, for example, in a
promoter, 5' untranslated region (UTR), exon, intron, 3'-UTR, or
terminator region. In an aspect, a mutation reduces, inhibits, or
eliminates the expression or activity of a gene product. In another
aspect, a mutation increases, elevates, strengthens, or augments
the expression or activity of a gene product.
[0228] In an aspect, mutations are not natural polymorphisms that
exist in a particular tobacco variety or cultivar. In an aspect, a
mutation is a "non-natural" or "non-naturally occurring" mutation.
As used herein, a "non-natural" or "non-naturally occurring"
mutation refers to a non-spontaneous mutation generated via human
intervention, and does not correspond to a spontaneous mutation
generated without human intervention. Non-limiting examples of
human intervention include mutagenesis (e.g., chemical mutagenesis,
ionizing radiation mutagenesis) and targeted genetic modifications
(e.g., CRISPR-based methods, TALEN-based methods, zinc finger-based
methods). Non-natural mutations and non-naturally occurring
mutations do not include spontaneous mutations that arise naturally
(e.g., via aberrant DNA replication in a germ line of a plant).
[0229] In an aspect, mutations are not natural polymorphisms that
exist in a particular tobacco variety or cultivar. It will be
appreciated that, when identifying a mutation, the reference DNA
sequence should be from the same variety of tobacco. For example,
if a modified tobacco plant comprising a mutation is from the
variety TN90, then the endogenous reference sequence must be the
endogenous TN90 sequence, not a homologous sequence from a
different tobacco variety (e.g., K326). Similarly, if a modified
tobacco cell comprising a mutation is a TN90 cell, then the
endogenous reference sequence must be the endogenous TN90 sequence,
not a homologous sequence from a tobacco cell from a different
tobacco variety (e.g., K326).
[0230] In an aspect, mutations are not natural polymorphisms that
exist in a particular Cannabis species, variety or cultivar. It
will be appreciated that, when identifying a mutation, the
reference DNA sequence should be from the same species, variety, or
cultivar of Cannabis. For example, if a modified Cannabis plant
comprising a mutation is a strain of C. indica, then the endogenous
reference sequence must be the corresponding endogenous C. indica
sequence, not a homologous sequence from a different Cannabis
species or strain (e.g. a sequence from C. sativa or a different
strain of C. indica). Similarly, if a modified Cannabis cell
comprising a mutation is a cell from C. indica, then the endogenous
reference sequence must be the corresponding endogenous C. indica
sequence, not a homologous sequence from a Cannabis cell from a
different Cannabis variety or species (e.g., a Sativa strain).
[0231] In an aspect, a tobacco plant, tobacco seed, or part
thereof, is homozygous for at least one non-natural mutation. In
another aspect, a tobacco plant, tobacco seed, or part thereof, is
heterozygous for at least one non-natural mutation. In an aspect, a
tobacco plant, tobacco seed, or part thereof is hemizygous for at
least one non-natural mutation. In another aspect, a tobacco plant,
tobacco seed, or part thereof, is homozygous for an introduced
recombinant DNA construct. In another aspect, a tobacco plant,
tobacco seed, or part thereof, is hemizygous for an introduced
recombinant DNA construct. In a further aspect, a tobacco plant,
tobacco seed, or part thereof, is heterozygous for an introduced
recombinant DNA construct.
[0232] In an aspect, a Cannabis plant, Cannabis seed, or part
thereof, is homozygous for at least one non-natural mutation. In
another aspect, a Cannabis plant, Cannabis seed, or part thereof,
is heterozygous for at least one non-natural mutation. In an
aspect, a Cannabis plant, Cannabis seed, or part thereof is
hemizygous for at least one non-natural mutation. In another
aspect, a Cannabis plant, Cannabis seed, or part thereof, is
homozygous for an introduced recombinant DNA construct. In another
aspect, a Cannabis plant, Cannabis seed, or part thereof, is
hemizygous for an introduced recombinant DNA construct. In a
further aspect, a Cannabis plant, Cannabis seed, or part thereof,
is heterozygous for an introduced recombinant DNA construct.
[0233] In an aspect, a tobacco seed, or the plant germinated
therefrom, is heterozygous for the recombinant nucleic acid
molecule. In an aspect, a tobacco seed, or the plant germinated
therefrom, is hemizygous for the recombinant nucleic acid molecule.
In an aspect, a tobacco seed, or the plant germinated therefrom, is
homozygous for the recombinant nucleic acid molecule.
[0234] In an aspect, a Cannabis seed, or the plant germinated
therefrom, is heterozygous for the recombinant nucleic acid
molecule. In an aspect, a Cannabis seed, or the plant germinated
therefrom, is hemizygous for the recombinant nucleic acid molecule.
In an aspect, a Cannabis seed, or the plant germinated therefrom,
is homozygous for the recombinant nucleic acid molecule.
[0235] In an aspect, a mutation provided herein creates a dominant
allele of the mutated locus. Dominant alleles are alleles that mask
the contribution of a second allele at the same locus. A dominant
allele can be a "dominant negative allele" or a "dominant positive
allele." Dominant negative alleles, or antimorphs, are alleles that
act in opposition to normal allelic function. A dominant negative
allele typically does not function normally and either directly
inhibits the activity of a wild-type protein (e.g., through
dimerization) or inhibits the activity of a second protein that is
required for the normal function of the wild-type protein (e.g., an
activator or a downstream component of a pathway). For example, a
dominant negative allele abrogates or reduces the normal function
of an allele in a heterozygous or homozygous state. Dominant
positive alleles can increase normal gene function (e.g., a
hypermorph) or provide new functions for a gene (e.g., a neomorph).
A semi-dominant allele occurs when penetrance of a linked phenotype
in individuals heterozygous for the allele is less than that which
is observed in individuals homozygous for the allele.
[0236] In an aspect, a mutation provided herein creates a dominant
negative allele of the mutated locus. In another aspect, a mutation
provided herein creates a dominant positive allele of a mutated
locus.
[0237] As used herein, "inducing" a mutation refers to generating a
mutation in a polynucleotide sequence via human intervention. Many
suitable methods for inducing mutations in tobacco and Cannabis are
known in the art. Non-limiting examples of such methods include use
of chemical mutagens, use of irradiation, use of nucleases, use of
transposons, and use of Agrobacterium. In an aspect, inducing a
mutation comprises the use of an agent selected from the group
consisting of a chemical mutagen, irradiation, a transposon,
Agrobacterium, and a nuclease.
[0238] In an aspect, inducing a mutation comprises the use of a
chemical mutagen. In an aspect, a chemical mutagen comprises ethyl
methanesulfonate (EMS).
[0239] In another aspect, inducing a mutation comprises the use of
irradiation. In an aspect, irradiation comprises gamma rays,
X-rays, ionizing radiation, or fast neutrons.
[0240] In an aspect, inducing a mutation comprises the use of a
transposon. In another aspect, inducing a mutation comprises the
use of Agrobacterium.
[0241] In a further aspect, inducing a mutation comprises the use
of a nuclease. In an aspect, a nuclease is selected from the group
consisting of a meganuclease, a zinc-finger nuclease, a
transcription activator-like effector nuclease, a CRISPR/Cas9
nuclease, a CRISPR/Cpf1 nuclease, a CRISPR/CasX nuclease, a
CRISPR/CasY nuclease, and a Csm1 nuclease. In an aspect, inducing a
mutation comprises the use of a CRISPR/Cas9 nuclease. In an aspect,
inducing a mutation comprises the use of a CRISPR/Cpf1 nuclease. In
an aspect, inducing a mutation comprises the use of a CRISPR/CasX
nuclease. In an aspect, inducing a mutation comprises the use of a
CRISPR/CasY nuclease. In an aspect, inducing a mutation comprises
the use of a Csm1 nuclease.
[0242] Several types of mutations are known in the art. In an
aspect, a mutation comprises an insertion. An "insertion" refers to
the addition of one or more nucleotides or amino acids to a given
polynucleotide or amino acid sequence, respectively, as compared to
an endogenous reference polynucleotide or amino acid sequence. In
another aspect, a mutation comprises a deletion. A "deletion"
refers to the removal of one or more nucleotides or amino acids to
a given polynucleotide or amino acid sequence, respectively, as
compared to an endogenous reference polynucleotide or amino acid
sequence. In another aspect, a mutation comprises a substitution. A
"substitution" refers to the replacement of one or more nucleotides
or amino acids to a given polynucleotide or amino acid sequence,
respectively, as compared to an endogenous reference polynucleotide
or amino acid sequence. In another aspect, a mutation comprises an
inversion. An "inversion" refers to when a segment of a
polynucleotide or amino acid sequence is reversed end-to-end. A
"duplication" refers to when a segment of a polynucleotide or amino
acid sequence is repeated. The repeated segment can immediately
follow the original segment, or it can be separated from the
original segment by one or more nucleotides or amino acids. In an
aspect, a mutation provided herein comprises a mutation selected
from the group consisting of an insertion, a deletion, a
substitution, a duplication, and an inversion.
[0243] In an aspect, a non-natural mutation comprises a mutation
selected from the group consisting of a substitution, a deletion,
an insertion, a duplication, and an inversion of one or more
nucleotides relative to an endogenous nucleic acid sequence
selected from the group consisting of SEQ ID NOs: 1-42, 112-139,
155-156, 158-159, and 161-190.
[0244] In an aspect, a non-natural mutation comprises a mutation
selected from the group consisting of a substitution, a deletion,
an insertion, a duplication, and an inversion of one or more
nucleotides relative to an endogenous nucleic acid sequence
encoding an amino acid sequence selected from the group consisting
of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205.
[0245] In an aspect, a non-natural mutation comprises at least one
mutation selected from the group consisting of a nonsense mutation,
a missense mutation, a frameshift mutation, a splice-site mutation,
and any combinations thereof. As used herein, a "nonsense mutation"
refers to a mutation to a nucleic acid sequence that introduces a
premature stop codon to an amino acid sequence by the nucleic acid
sequence. As used herein, a "missense mutation" refers to a
mutation to a nucleic acid sequence that causes a substitution
within the amino acid sequence encoded by the nucleic acid
sequence. As used herein, a "frameshift mutation" refers to an
insertion or deletion to a nucleic acid sequence that shifts the
frame for translating the nucleic acid sequence to an amino acid
sequence. A "splice-site mutation" refers to a mutation in a
nucleic acid sequence that causes an intron to be retained for
protein translation, or, alternatively, for an exon to be excluded
from protein translation. Splice-site mutations can cause nonsense,
missense, or frameshift mutations.
[0246] Mutations in coding regions of genes (e.g., exonic
mutations) can result in a truncated protein or polypeptide when a
mutated messenger RNA (mRNA) is translated into a protein or
polypeptide. In an aspect, this disclosure provides a mutation that
results in the truncation of a protein or polypeptide. As used
herein, a "truncated" protein or polypeptide comprises at least one
fewer amino acid as compared to an endogenous control protein or
polypeptide. For example, if endogenous Protein A comprises 100
amino acids, a truncated version of Protein A can comprise between
1 and 99 amino acids. In an aspect, a non-natural mutation results
in a truncation of a polypeptide.
[0247] Without being limited by any scientific theory, one way to
cause a protein or polypeptide truncation is by the introduction of
a premature stop codon in an mRNA transcript of an endogenous gene.
In an aspect, this disclosure provides a mutation that results in a
premature stop codon in an mRNA transcript of an endogenous gene.
As used herein, a "stop codon" refers to a nucleotide triplet
within an mRNA transcript that signals a termination of protein
translation. A "premature stop codon" refers to a stop codon
positioned earlier (e.g., on the 5'-side) than the normal stop
codon position in an endogenous mRNA transcript. Without being
limiting, several stop codons are known in the art, including
"UAG," "UAA," "UGA," "TAG," "TAA," and "TGA."
[0248] In an aspect, a mutation provided herein comprises a null
mutation. In an aspect, at least one non-natural mutation provided
herein comprises a null mutation. As used herein, a "null mutation"
refers to a mutation that confers a complete loss-of-function for a
protein encoded by a gene comprising the mutation, or,
alternatively, a mutation that confers a complete loss-of-function
for a small RNA encoded by a genomic locus. A null mutation can
cause lack of mRNA transcript production, a lack of small RNA
transcript production, a lack of protein function, or a combination
thereof.
[0249] A mutation provided herein can be positioned in any part of
an endogenous gene. In an aspect, a mutation provided herein is
positioned within an exon of an endogenous gene. In another aspect,
a mutation provided herein is positioned within an intron of an
endogenous gene. In a further aspect, a mutation provided herein is
positioned within a 5'-UTR of an endogenous gene. In still another
aspect, a mutation provided herein is positioned within a 3'-UTR of
an endogenous gene. In yet another aspect, a mutation provided
herein is positioned within a promoter of an endogenous gene. In
yet another aspect, a mutation provided herein is positioned within
a terminator of an endogenous gene. In an aspect, a non-natural
mutation provided herein comprises a mutation in a sequence region
selected from the group consisting of a promoter, a 5'-UTR, a
3'-UTR, an exon, an intron, and a terminator.
[0250] The screening and selection of mutagenized tobacco plants
can be through any methodologies known to those having ordinary
skill in the art. Examples of screening and selection methodologies
include, but are not limited to, Southern analysis, PCR
amplification for detection of a polynucleotide, Northern blots,
RNase protection, primer-extension, RT-PCR amplification for
detecting RNA transcripts, Sanger sequencing, Next Generation
sequencing technologies (e.g., Illumina, PacBio, Ion Torrent, 454,
and Oxford nanopore) enzymatic assays for detecting enzyme or
ribozyme activity of polypeptides and polynucleotides, and protein
gel electrophoresis, Western blots, immunoprecipitation, and
enzyme-linked immunoassays to detect polypeptides. Other techniques
such as in situ hybridization, enzyme staining, and immunostaining,
also can be used to detect the presence or expression of
polypeptides and/or polynucleotides. Methods for performing all of
the referenced and similar techniques are known.
Reduced Expression/Activity
[0251] In an aspect, a small RNA molecule reduces the expression of
any nucleic acid sequence to which it is capable of binding. In
another aspect, a non-natural mutation provided herein reduces the
expression of the mutated nucleic acid sequence as compared to the
non-mutated nucleic acid sequence in a control plant grown under
comparable conditions.
[0252] Reduced expression of an endogenous nucleic acid sequence
can be measured using any suitable method known in the art.
Non-limiting examples of measuring expression include quantitative
reverse transcriptase polymerase chain reaction (qRT-PCR), digital
PCR, RNA blot (e.g., a Northern blot), RNA sequencing. Differences
in expression can be described as an absolute quantification or a
relative quantification. See, for example, Livak and Schmittgen,
Methods, 25:402-408 (2001). If an endogenous nucleic acid sequence
encodes a protein, changes in expression can be inferred by
examining the accumulation of the encoded protein. Non-limiting
examples of measuring protein accumulation include Western blots
and enzyme-linked immunosorbent assays (ELISAs).
[0253] In an aspect, a reduction in expression is measured using
qRT-PCR. In another aspect, a reduction in expression is measured
using an RNA blot. In another aspect, a reduction in expression is
measured using RNA sequencing. In an aspect, a reduction in
expression is measured using digital PCR. In a further aspect, a
reduction in expression is measured using a Western blot. In yet a
further aspect, a reduction in expression is measured using an
ELISA.
[0254] In an aspect, a non-natural mutation in a nucleic acid
sequence selected from the group consisting of SEQ ID NOs: 1-42,
112-139, 155-156, 158-159, and 161-190 results in a reduced level
of expression of the nucleic acid sequence as compared to the
nucleic acid sequence lacking the non-natural mutation in a control
plant grown under comparable conditions. In an aspect, a
non-natural mutation in a nucleic acid sequence encoding an amino
acid sequence selected from the group consisting of SEQ ID NOs:
43-63, 140-153, 157, 160, and 191-205 results in a reduced level of
expression of the nucleic acid sequence as compared to the nucleic
acid sequence lacking the non-natural mutation in a control plant
grown under comparable conditions.
[0255] In an aspect, a reduction in expression comprises a
reduction of at least 1% as compared to expression in the same
tissue of a control plant grown under comparable conditions. In
another aspect, a reduction in expression comprises a reduction of
at least 5% as compared to expression in the same tissue of a
control plant grown under comparable conditions. In another aspect,
a reduction in expression comprises a reduction of at least 10% as
compared to expression in the same tissue of a control plant grown
under comparable conditions. In another aspect, a reduction in
expression comprises a reduction of at least 25% as compared to
expression in the same tissue of a control plant grown under
comparable conditions. In another aspect, a reduction in expression
comprises a reduction of at least 50% as compared to expression in
the same tissue of a control plant grown under comparable
conditions. In another aspect, a reduction in expression comprises
a reduction of at least 75% as compared to expression in the same
tissue of a control plant grown under comparable conditions. In
another aspect, a reduction in expression comprises a reduction of
at least 90% as compared to expression in the same tissue of a
control plant grown under comparable conditions. In another aspect,
a reduction in expression comprises a reduction of at least 95% as
compared to expression in the same tissue of a control plant grown
under comparable conditions.
[0256] In an aspect, a reduction in expression comprises a
reduction of between 1% and 99% as compared to expression in the
same tissue of a control plant grown under comparable conditions.
In another aspect, a reduction in expression comprises a reduction
of between 1% and 90% as compared to expression in the same tissue
of a control plant grown under comparable conditions. In another
aspect, a reduction in expression comprises a reduction of between
1% and 75% as compared to expression in the same tissue of a
control plant grown under comparable conditions. In another aspect,
a reduction in expression comprises a reduction of between 1% and
50% as compared to expression in the same tissue of a control plant
grown under comparable conditions. In another aspect, a reduction
in expression comprises a reduction of between 1% and 25% as
compared to expression in the same tissue of a control plant grown
under comparable conditions. In another aspect, a reduction in
expression comprises a reduction of between 25% and 90% as compared
to expression in the same tissue of a control plant grown under
comparable conditions. In another aspect, a reduction in expression
comprises a reduction of between 50% and 90% as compared to
expression in the same tissue of a control plant grown under
comparable conditions. In another aspect, a reduction in expression
comprises a reduction of between 25% and 75% as compared to
expression in the same tissue of a control plant grown under
comparable conditions. In an aspect, a reduction in expression
comprises a statistically significant reduction as compared to
expression in the same tissue of a control plant grown under
comparable conditions.
[0257] One of ordinary skill in the art would recognize that any
level of reduction is envisioned, so long as the level of reduction
has been determined to be statistically significant using an
accepted statistical hypothesis test. As a non-limiting example, a
Student's t-test is one statistical hypothesis test that can be
used to determine if a reduction in expression between a modified
plant and a control plant is statistically significant. As used
herein, "statistically significant" refers to a p-value of less
than or equal to 0.05.
[0258] In an aspect, a non-natural mutation results in a reduced
level of activity by a protein or polypeptide encoded by a nucleic
acid sequence provided herein as compared to the activity of a
control plant grown under comparable conditions. In another aspect,
a non-natural mutation in an endogenous nucleic acid sequence
selected from the group consisting of SEQ ID NOs: 1-42, 112-139,
155-156, 158-159, and 161-190 reduces the level of activity by a
protein or polypeptide encoded by the nucleic acid sequence as
compared to activity of a protein or polypeptide encoded by the
endogenous nucleic acid sequence in a control tobacco or Cannabis
plant when grown under comparable conditions, where the nucleic
acid sequence lacks the non-natural mutation in the control tobacco
or Cannabis plant. In another aspect, a non-natural mutation in an
endogenous nucleic acid sequence, where the endogenous nucleic acid
sequence encodes an amino acid sequence selected from the group
consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205
reduces the level of activity by a protein or polypeptide encoded
by the nucleic acid sequence as compared to activity of a protein
or polypeptide encoded by the endogenous nucleic acid sequence in a
control tobacco or Cannabis plant when grown under comparable
conditions, where the nucleic acid sequence lacks the non-natural
mutation in the control tobacco or Cannabis plant.
[0259] As used herein, when referring to a protein or polypeptide,
"activity" refers to the ability to carry out an enzymatic
function. As a non-limiting example, activity of a dehydrogenase
protein or polypeptide refers to the ability of a dehydrogenase to
remove a hydrogen group from an organic molecule substrate. Without
being limited by any scientific theory, if a mutated dehydrogenase
protein has reduced activity as compared to a non-mutated control
dehydrogenase protein, the mutated dehydrogenase (a) may be unable
to remove a hydrogen group from any organic molecule substrate; (b)
only be able to remove a hydrogen group from an organic molecule
substrate at a reduced rate as compared to a non-mutated
dehydrogenase protein; (c) only be able to remove hydrogen groups
from substrates that are not suitable substrates for a non-mutated
dehydrogenase protein; or (d) a combination of (b) and (c).
Conversely, also without being limited by any scientific theory, if
a mutated dehydrogenase protein has increased activity as compared
to a non-mutated control dehydrogenase protein, the mutated
dehydrogenase may be able to (a) remove a hydrogen group from an
organic molecule substrate at an increased rate as compared to a
non-mutated dehydrogenase protein; (b) be able to remove hydrogen
groups from substrates that are not suitable substrates for a
non-mutated dehydrogenase; or (c) both (a) and (b). The activity of
a dehydrogenase can be measured using techniques standard in the
art. For example, see Yu et al., Enzyme Microb. Technol.,
49:272-276 (2011); Seely and Pegg, J. Biol. Chem., 258:2496-2500
(1983); and Mizusaki et al., Plant and Cell Physiology, 14:103-110
(1973).
Increased Expression/Activity
[0260] In an aspect, a non-natural mutation results in increased
expression of a nucleic acid sequence. In an aspect, a non-natural
mutation results in an increased level of expression of said
nucleic acid sequence as compared to expression of said nucleic
acid sequence in the same tissue of a control tobacco or Cannabis
plant when grown under comparable conditions, where said nucleic
acid sequence lacks the at least one non-natural mutation in said
control tobacco or Cannabis plant.
[0261] In an aspect, this disclosure provides a tobacco plant
comprising increased expression of a sequence at least 70%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this
disclosure provides a tobacco plant comprising increased expression
of a sequence at least 75% identical to a sequence selected from
the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, this disclosure provides a tobacco plant
comprising increased expression of a sequence at least 80%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this
disclosure provides a tobacco plant comprising increased expression
of a sequence at least 85% identical to a sequence selected from
the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, this disclosure provides a tobacco plant
comprising increased expression of a sequence at least 90%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this
disclosure provides a tobacco plant comprising increased expression
of a sequence at least 92.5% identical to a sequence selected from
the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, this disclosure provides a tobacco plant
comprising increased expression of a sequence at least 95%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this
disclosure provides a tobacco plant comprising increased expression
of a sequence at least 96% identical to a sequence selected from
the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, this disclosure provides a tobacco plant
comprising increased expression of a sequence at least 97%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this
disclosure provides a tobacco plant comprising increased expression
of a sequence at least 98% identical to a sequence selected from
the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, this disclosure provides a tobacco plant
comprising increased expression of a sequence at least 99%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this
disclosure provides a tobacco plant comprising increased expression
of a sequence at least 99.5% identical to a sequence selected from
the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, this disclosure provides a tobacco plant
comprising increased expression of a sequence 100% identical to a
sequence selected from the group consisting of SEQ ID NOs: 1-21,
112-125, 155, 158, and 176-190.
[0262] In an aspect, this disclosure provides a Cannabis plant
comprising increased expression of a sequence at least 70%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this
disclosure provides a Cannabis plant comprising increased
expression of a sequence at least 75% identical to a sequence
selected from the group consisting of SEQ ID NOs: 22-42, 126-139,
156, 159, and 161-175. In an aspect, this disclosure provides a
Cannabis plant comprising increased expression of a sequence at
least 80% identical to a sequence selected from the group
consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In
an aspect, this disclosure provides a Cannabis plant comprising
increased expression of a sequence at least 85% identical to a
sequence selected from the group consisting of SEQ ID NOs: 22-42,
126-139, 156, 159, and 161-175. In an aspect, this disclosure
provides a Cannabis plant comprising increased expression of a
sequence at least 90% identical to a sequence selected from the
group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, this disclosure provides a Cannabis plant
comprising increased expression of a sequence at least 92.5%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this
disclosure provides a Cannabis plant comprising increased
expression of a sequence at least 95% identical to a sequence
selected from the group consisting of SEQ ID NOs: 22-42, 126-139,
156, 159, and 161-175. In an aspect, this disclosure provides a
Cannabis plant comprising increased expression of a sequence at
least 96% identical to a sequence selected from the group
consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In
an aspect, this disclosure provides a Cannabis plant comprising
increased expression of a sequence at least 97% identical to a
sequence selected from the group consisting of SEQ ID NOs: 22-42,
126-139, 156, 159, and 161-175. In an aspect, this disclosure
provides a Cannabis plant comprising increased expression of a
sequence at least 98% identical to a sequence selected from the
group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, this disclosure provides a Cannabis plant
comprising increased expression of a sequence at least 99%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this
disclosure provides a Cannabis plant comprising increased
expression of a sequence at least 99.5% identical to a sequence
selected from the group consisting of SEQ ID NOs: 22-42, 126-139,
156, 159, and 161-175. In an aspect, this disclosure provides a
Cannabis plant comprising increased expression of a sequence 100%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 22-42, 126-139, 156, 159, and 161-175.
[0263] In an aspect, this disclosure provides a tobacco plant
comprising increased expression of a sequence at least 70%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this
disclosure provides a tobacco plant comprising increased expression
of a sequence at least 75% identical to a sequence selected from
the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, this disclosure provides a tobacco plant
comprising increased expression of a sequence at least 80%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this
disclosure provides a tobacco plant comprising increased expression
of a sequence at least 85% identical to a sequence selected from
the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, this disclosure provides a tobacco plant
comprising increased expression of a sequence at least 90%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this
disclosure provides a tobacco plant comprising increased expression
of a sequence at least 92.5% identical to a sequence selected from
the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, this disclosure provides a tobacco plant
comprising increased expression of a sequence at least 95%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this
disclosure provides a tobacco plant comprising increased expression
of a sequence at least 96% identical to a sequence selected from
the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, this disclosure provides a tobacco plant
comprising increased expression of a sequence at least 97%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this
disclosure provides a tobacco plant comprising increased expression
of a sequence at least 98% identical to a sequence selected from
the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, this disclosure provides a tobacco plant
comprising increased expression of a sequence at least 99%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this
disclosure provides a tobacco plant comprising increased expression
of a sequence at least 99.5% identical to a sequence selected from
the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, this disclosure provides a tobacco plant
comprising increased expression of a sequence 100% identical to a
sequence selected from the group consisting of SEQ ID NOs: 22-42,
126-139, 156, 159, and 161-175.
[0264] In an aspect, this disclosure provides a Cannabis plant
comprising increased expression of a sequence at least 70%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this
disclosure provides a Cannabis plant comprising increased
expression of a sequence at least 75% identical to a sequence
selected from the group consisting of SEQ ID NOs: 1-21, 112-125,
155, 158, and 176-190. In an aspect, this disclosure provides a
Cannabis plant comprising increased expression of a sequence at
least 80% identical to a sequence selected from the group
consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In
an aspect, this disclosure provides a Cannabis plant comprising
increased expression of a sequence at least 85% identical to a
sequence selected from the group consisting of SEQ ID NOs: 1-21,
112-125, 155, 158, and 176-190. In an aspect, this disclosure
provides a Cannabis plant comprising increased expression of a
sequence at least 90% identical to a sequence selected from the
group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, this disclosure provides a Cannabis plant
comprising increased expression of a sequence at least 92.5%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this
disclosure provides a Cannabis plant comprising increased
expression of a sequence at least 95% identical to a sequence
selected from the group consisting of SEQ ID NOs: 1-21, 112-125,
155, 158, and 176-190. In an aspect, this disclosure provides a
Cannabis plant comprising increased expression of a sequence at
least 96% identical to a sequence selected from the group
consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In
an aspect, this disclosure provides a Cannabis plant comprising
increased expression of a sequence at least 97% identical to a
sequence selected from the group consisting of SEQ ID NOs: 1-21,
112-125, 155, 158, and 176-190. In an aspect, this disclosure
provides a Cannabis plant comprising increased expression of a
sequence at least 98% identical to a sequence selected from the
group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, this disclosure provides a Cannabis plant
comprising increased expression of a sequence at least 99%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this
disclosure provides a Cannabis plant comprising increased
expression of a sequence at least 99.5% identical to a sequence
selected from the group consisting of SEQ ID NOs: 1-21, 112-125,
155, 158, and 176-190. In an aspect, this disclosure provides a
Cannabis plant comprising increased expression of a sequence 100%
identical to a sequence selected from the group consisting of SEQ
ID NOs: 1-21, 112-125, 155, 158, and 176-190.
[0265] In another aspect, this disclosure provides increased
expression of a sequence encoding a protein at least 70% identical
or similar to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In
another aspect, this disclosure provides increased expression of a
sequence encoding a protein at least 75% identical or similar to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, this
disclosure provides increased expression of a sequence encoding a
protein at least 80% identical or similar to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 43-63, 140-153,
157, 160, and 191-205. In another aspect, this disclosure provides
increased expression of a sequence encoding a protein at least 85%
identical or similar to an amino acid sequence selected from the
group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and
191-205. In another aspect, this disclosure provides increased
expression of a sequence encoding a protein at least 90% identical
or similar to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In
another aspect, this disclosure provides increased expression of a
sequence encoding a protein at least 92.5% identical or similar to
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, this
disclosure provides increased expression of a sequence encoding a
protein at least 95% identical or similar to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 43-63, 140-153,
157, 160, and 191-205. In another aspect, this disclosure provides
increased expression of a sequence encoding a protein at least 96%
identical or similar to an amino acid sequence selected from the
group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and
191-205. In another aspect, this disclosure provides increased
expression of a sequence encoding a protein at least 97% identical
or similar to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In
another aspect, this disclosure provides increased expression of a
sequence encoding a protein at least 98% identical or similar to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, this
disclosure provides increased expression of a sequence encoding a
protein at least 99% identical or similar to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 43-63, 140-153,
157, 160, and 191-205. In another aspect, this disclosure provides
increased expression of a sequence encoding a protein at least
99.5% identical or similar to an amino acid sequence selected from
the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and
191-205. In another aspect, this disclosure provides increased
expression of a sequence encoding a protein 100% identical or
similar to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and
191-205.
[0266] In an aspect, an increased level of expression comprises an
increase of at least 1% as compared to expression in the same
tissue of a control plant grown under comparable conditions. In an
aspect, an increased level of expression comprises an increase of
at least 5% as compared to expression in the same tissue of a
control plant grown under comparable conditions. In another aspect,
an increased level of expression comprises an increase of at least
10% as compared to expression in the same tissue of a control plant
grown under comparable conditions. In another aspect, an increased
level of expression comprises an increase of at least 25% as
compared to expression in the same tissue of a control plant grown
under comparable conditions. In another aspect, an increased level
of expression comprises an increase of at least 50% as compared to
expression in the same tissue of a control plant grown under
comparable conditions. In another aspect, an increased level of
expression comprises an increase of at least 75% as compared to
expression in the same tissue of a control plant grown under
comparable conditions. In another aspect, an increased level of
expression comprises an increase of at least 100% as compared to
expression in the same tissue of a control plant grown under
comparable conditions. In another aspect, an increased level of
expression comprises an increase of at least 200% as compared to
expression in the same tissue of a control plant grown under
comparable conditions. In another aspect, an increased level of
expression comprises an increase of at least 500% as compared to
expression in the same tissue of a control plant grown under
comparable conditions.
[0267] In an aspect, a non-natural mutation results in an increased
level of activity by a protein or polypeptide encoded by said
nucleic acid sequence as compared to activity of a protein or
polypeptide encoded by said nucleic acid sequence in a control
tobacco plant or a control Cannabis plant when grown under
comparable conditions, where said nucleic acid sequence lacks the
at least one non-natural mutation in said control tobacco or
Cannabis plant.
Nucleic Acids and Amino Acids
[0268] As used herein, "heterologous" refers to a sequence (nucleic
acid or amino acid) that originates from a foreign species, or, if
from the same species, is substantially modified from its native
form in composition and/or genomic locus by deliberate human
intervention. The term also is applicable to nucleic acid
constructs, also referred to herein as "polynucleotide constructs."
In this manner, a "heterologous" nucleic acid construct is intended
to mean a construct that originates from a foreign species, or, if
from the same species, is substantially modified from its native
form in composition and/or genomic locus by deliberate human
intervention. Heterologous nucleic acid constructs include, but are
not limited to, recombinant nucleotide constructs that have been
introduced into a plant or plant part thereof, for example, via
transformation methods or subsequent breeding of a transgenic plant
with another plant of interest. It will be appreciated that an
endogenous promoter can be considered heterologous to an operably
linked endogenous gene if the endogenous promoter and endogenous
gene are not naturally operably linked (e.g., human intervention is
required to put them in operable linkage). As used herein, an
"endogenous" nucleic acid sequence refers to a nucleic acid
sequence that occurs naturally in the genome of an organism.
[0269] In an aspect, a heterologous polynucleotide comprises a
gene. In an aspect, a heterologous polynucleotide encodes a small
RNA molecule or a precursor thereof. In an aspect, a heterologous
polynucleotide encodes a polypeptide.
[0270] As used herein, a "gene" refers to a polynucleotide that can
produce a functional unit (e.g., without being limiting, for
example, a polypeptide, or a small RNA molecule). A gene can
comprise a promoter, an enhancer sequence, a leader sequence, a
transcriptional start site, a transcriptional stop site, a
polyadenylation site, one or more exons, one or more introns, a
5'-UTR, a 3'-UTR, or any combination thereof. A "gene sequence" can
comprise a polynucleotide sequence encoding a promoter, an enhancer
sequence, a leader sequence, a transcriptional start site, a
transcriptional stop site, a polyadenylation site, one or more
exons, one or more introns, a 5'-UTR, a 3'-UTR, or any combination
thereof. In one aspect, a gene encodes a small RNA molecule or a
precursor thereof. In another aspect, a gene encodes a
polypeptide.
[0271] In an aspect, a gene comprises a nucleic acid sequence at
least 70% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, a gene comprises a nucleic acid sequence at
least 75% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, a gene comprises a nucleic acid sequence at
least 80% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, a gene comprises a nucleic acid sequence at
least 85% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, a gene comprises a nucleic acid sequence at
least 90% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, a gene comprises a nucleic acid sequence at
least 92.5% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, a gene comprises a nucleic acid sequence at
least 95% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, a gene comprises a nucleic acid sequence at
least 96% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, a gene comprises a nucleic acid sequence at
least 97% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, a gene comprises a nucleic acid sequence at
least 98% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, a gene comprises a nucleic acid sequence at
least 99% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, a gene comprises a nucleic acid sequence at
least 99.5% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and
176-190. In an aspect, a gene comprises a nucleic acid sequence
100% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190.
[0272] In an aspect, a gene comprises a nucleic acid sequence at
least 70% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, a gene comprises a nucleic acid sequence at
least 75% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, a gene comprises a nucleic acid sequence at
least 80% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, a gene comprises a nucleic acid sequence at
least 85% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, a gene comprises a nucleic acid sequence at
least 90% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, a gene comprises a nucleic acid sequence at
least 92.5% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, a gene comprises a nucleic acid sequence at
least 95% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, a gene comprises a nucleic acid sequence at
least 96% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, a gene comprises a nucleic acid sequence at
least 97% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, a gene comprises a nucleic acid sequence at
least 98% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, a gene comprises a nucleic acid sequence at
least 99% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, a gene comprises a nucleic acid sequence at
least 99.5% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175. In an aspect, a gene comprises a nucleic acid sequence
100% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and
161-175.
[0273] In an aspect, a gene comprises a nucleic acid sequence
encoding an amino acid sequence at least 70% identical or similar
to an amino acid sequence selected from the group consisting of SEQ
ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a gene
comprises a nucleic acid sequence encoding an amino acid sequence
at least 75% identical or similar to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 43-63, 140-153,
157, 160, and 191-205. In an aspect, a gene comprises a nucleic
acid sequence encoding an amino acid sequence at least 80%
identical or similar to an amino acid sequence selected from the
group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and
191-205. In an aspect, a gene comprises a nucleic acid sequence
encoding an amino acid sequence at least 85% identical or similar
to an amino acid sequence selected from the group consisting of SEQ
ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a gene
comprises a nucleic acid sequence encoding an amino acid sequence
at least 90% identical or similar to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 43-63, 140-153,
157, 160, and 191-205. In an aspect, a gene comprises a nucleic
acid sequence encoding an amino acid sequence at least 92.5%
identical or similar to an amino acid sequence selected from the
group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and
191-205. In an aspect, a gene comprises a nucleic acid sequence
encoding an amino acid sequence at least 95% identical or similar
to an amino acid sequence selected from the group consisting of SEQ
ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a gene
comprises a nucleic acid sequence encoding an amino acid sequence
at least 96% identical or similar to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 43-63, 140-153,
157, 160, and 191-205. In an aspect, a gene comprises a nucleic
acid sequence encoding an amino acid sequence at least 97%
identical or similar to an amino acid sequence selected from the
group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and
191-205. In an aspect, a gene comprises a nucleic acid sequence
encoding an amino acid sequence at least 98% identical or similar
to an amino acid sequence selected from the group consisting of SEQ
ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a gene
comprises a nucleic acid sequence encoding an amino acid sequence
at least 99% identical or similar to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 43-63, 140-153,
157, 160, and 191-205. In an aspect, a gene comprises a nucleic
acid sequence encoding an amino acid sequence at least 99.9%
identical or similar to an amino acid sequence selected from the
group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and
191-205. In an aspect, a gene comprises a nucleic acid sequence
encoding an amino acid sequence 100% identical or similar to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 43-63, 140-153, 157, 160, and 191-205.
[0274] In an aspect, a polypeptide comprises an amino acid sequence
at least 70% identical or similar to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 43-63, 140-153,
157, 160, and 191-205. In an aspect, a polypeptide comprises an
amino acid sequence at least 75% identical or similar to an amino
acid sequence selected from the group consisting of SEQ ID NOs:
43-63, 140-153, 157, 160, and 191-205. In an aspect, a polypeptide
comprises an amino acid sequence at least 80% identical or similar
to an amino acid sequence selected from the group consisting of SEQ
ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a
polypeptide comprises an amino acid sequence at least 85% identical
or similar to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In
an aspect, a polypeptide comprises an amino acid sequence at least
90% identical or similar to an amino acid sequence selected from
the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and
191-205. In an aspect, a polypeptide comprises an amino acid
sequence at least 92.5% identical or similar to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 43-63,
140-153, 157, 160, and 191-205. In an aspect, a polypeptide
comprises an amino acid sequence at least 95% identical or similar
to an amino acid sequence selected from the group consisting of SEQ
ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a
polypeptide comprises an amino acid sequence at least 96% identical
or similar to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In
an aspect, a polypeptide comprises an amino acid sequence at least
97% identical or similar to an amino acid sequence selected from
the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and
191-205. In an aspect, a polypeptide comprises an amino acid
sequence at least 98% identical or similar to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 43-63,
140-153, 157, 160, and 191-205. In an aspect, a polypeptide
comprises an amino acid sequence at least 99% identical or similar
to an amino acid sequence selected from the group consisting of SEQ
ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a
polypeptide comprises an amino acid sequence at least 99.9%
identical or similar to an amino acid sequence selected from the
group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and
191-205. In an aspect, a polypeptide comprises an amino acid
sequence 100% identical or similar to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 43-63, 140-153,
157, 160, and 191-205.
[0275] The terms "percent identity" or "percent identical" as used
herein in reference to two or more nucleotide or amino acid
sequences is calculated by (i) comparing two optimally aligned
sequences (nucleotide or amino acid) over a window of comparison
(the "alignable" region or regions), (ii) determining the number of
positions at which the identical nucleic acid base (for nucleotide
sequences) or amino acid residue (for proteins and polypeptides)
occurs in both sequences to yield the number of matched positions,
(iii) dividing the number of matched positions by the total number
of positions in the window of comparison, and then (iv) multiplying
this quotient by 100% to yield the percent identity. If the
"percent identity" is being calculated in relation to a reference
sequence without a particular comparison window being specified,
then the percent identity is determined by dividing the number of
matched positions over the region of alignment by the total length
of the reference sequence. Accordingly, for purposes of the present
application, when two sequences (query and subject) are optimally
aligned (with allowance for gaps in their alignment), the "percent
identity" for the query sequence is equal to the number of
identical positions between the two sequences divided by the total
number of positions in the query sequence over its length (or a
comparison window), which is then multiplied by 100%.
[0276] When percentage of sequence identity is used in reference to
amino acids it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. When sequences differ in conservative
substitutions, the percent sequence identity can be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity."
[0277] For optimal alignment of sequences to calculate their
percent identity, various pair-wise or multiple sequence alignment
algorithms and programs are known in the art, such as ClustalW or
Basic Local Alignment Search Tool.RTM. (BLAST.TM.), etc., that can
be used to compare the sequence identity or similarity between two
or more nucleotide or amino acid sequences. Although other
alignment and comparison methods are known in the art, the
alignment and percent identity between two sequences (including the
percent identity ranges described above) can be as determined by
the Clustal W algorithm, see, e.g., Chenna et al., "Multiple
sequence alignment with the Clustal series of programs," Nucleic
Acids Research 31: 3497-3500 (2003); Thompson et al., "Clustal W:
Improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, position-specific gap
penalties and weight matrix choice," Nucleic Acids Research 22:
4673-4680 (1994); Larkin M A et al., "Clustal W and Clustal X
version 2.0," Bioinformatics 23: 2947-48 (2007); and Altschul et
al. "Basic local alignment search tool." J. Mol. Biol. 215:403-410
(1990), the entire contents and disclosures of which are
incorporated herein by reference.
[0278] The terms "percent complementarity" or "percent
complementary" as used herein in reference to two nucleotide
sequences is similar to the concept of percent identity but refers
to the percentage of nucleotides of a query sequence that optimally
base-pair or hybridize to nucleotides a subject sequence when the
query and subject sequences are linearly arranged and optimally
base paired without secondary folding structures, such as loops,
stems or hairpins. Such a percent complementarity can be between
two DNA strands, two RNA strands, or a DNA strand and a RNA strand.
The "percent complementarity" can be calculated by (i) optimally
base-pairing or hybridizing the two nucleotide sequences in a
linear and fully extended arrangement (i.e., without folding or
secondary structures) over a window of comparison, (ii) determining
the number of positions that base-pair between the two sequences
over the window of comparison to yield the number of complementary
positions, (iii) dividing the number of complementary positions by
the total number of positions in the window of comparison, and (iv)
multiplying this quotient by 100% to yield the percent
complementarity of the two sequences. Optimal base pairing of two
sequences can be determined based on the known pairings of
nucleotide bases, such as G-C, A-T, and A-U, through hydrogen
binding. If the "percent complementarity" is being calculated in
relation to a reference sequence without specifying a particular
comparison window, then the percent identity is determined by
dividing the number of complementary positions between the two
linear sequences by the total length of the reference sequence.
Thus, for purposes of the present application, when two sequences
(query and subject) are optimally base-paired (with allowance for
mismatches or non-base-paired nucleotides), the "percent
complementarity" for the query sequence is equal to the number of
base-paired positions between the two sequences divided by the
total number of positions in the query sequence over its length,
which is then multiplied by 100%.
[0279] The use of the term "polynucleotide" or "nucleic acid
molecule" is not intended to limit the present disclosure to
polynucleotides comprising deoxyribonucleic acid (DNA). For
example, ribonucleic acid (RNA) molecules are also envisioned.
Those of ordinary skill in the art will recognize that
polynucleotides and nucleic acid molecules can comprise
ribonucleotides and combinations of ribonucleotides and
deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides
include both naturally occurring molecules and synthetic analogues.
The polynucleotides of the present disclosure also encompass all
forms of sequences including, but not limited to, single-stranded
forms, double-stranded forms, hairpins, stem-and-loop structures,
and the like. In an aspect, a nucleic acid molecule provided herein
is a DNA molecule. In another aspect, a nucleic acid molecule
provided herein is an RNA molecule. In an aspect, a nucleic acid
molecule provided herein is single-stranded. In another aspect, a
nucleic acid molecule provided herein is double-stranded. A nucleic
acid molecule can encode a polypeptide or a small RNA.
[0280] As used herein, a "recombinant nucleic acid molecule" refers
to a nucleic acid molecule formed by laboratory methods of genetic
recombination, such as, without being limiting, molecular cloning.
Similarly, a "recombinant DNA construct" refers to a DNA molecule
formed by laboratory methods of genetic recombination.
[0281] In one aspect, this disclosure provides a recombinant
nucleic acid molecule comprising a nucleic acid sequence encoding a
polypeptide involved in the biosynthesis of at least one terpene
operably linked to a heterologous promoter. In one aspect, this
disclosure provides a recombinant nucleic acid molecule comprising
a nucleic acid sequence encoding a polypeptide involved in the
biosynthesis of at least one terpene operably linked to a
heterologous promoter. In one aspect, this disclosure provides a
recombinant nucleic acid molecule comprising a nucleic acid
sequence encoding a polypeptide involved in the biosynthesis of at
least one terpene operably linked to a heterologous promoter.
[0282] Nucleic acids can be isolated using techniques routine in
the art. For example, nucleic acids can be isolated using any
method including, without limitation, recombinant nucleic acid
technology, and/or the polymerase chain reaction (PCR). General PCR
techniques are described, for example in PCR Primer: A Laboratory
Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor
Laboratory Press, 1995. Recombinant nucleic acid techniques
include, for example, restriction enzyme digestion and ligation,
which can be used to isolate a nucleic acid. Isolated nucleic acids
also can be chemically synthesized, either as a single nucleic acid
molecule or as a series of oligonucleotides. Non-limiting examples
of primers that can be used to amplify specific candidate genes
provided in this disclosure are provided in Table 1 (SEQ ID NOs:
64-103).
[0283] In an aspect, this disclosure provides methods of detecting
mRNA expression of an endogenous gene encoding a polypeptide
involved in the biosynthesis of at least one terpene. Messenger RNA
(mRNA) levels can be measured by techniques standard in the art,
such as reverse transcription polymerase chain reaction (RT-PCR)
and microarray analysis. RNA can be reverse transcribed to cDNA
(complementary DNA) by methods known in the art. Synthesized cDNA
may be amplified by methods known in the art, such as real-time PCR
and digital PCR for relative or absolute quantification of mRNA
transcript targets.
[0284] As used herein, the term "polypeptide" refers to a chain of
at least two covalently linked amino acids. Polypeptides can be
encoded by polynucleotides provided herein. Proteins provided
herein can be encoded by nucleic acid molecules provided herein.
Proteins can comprise polypeptides provided herein. As used herein,
a "protein" refers to a chain of amino acid residues that is
capable of providing structure or enzymatic activity to a cell.
[0285] Polypeptides can be purified from natural sources (e.g., a
biological sample) by known methods such as DEAE ion exchange, gel
filtration, and hydroxyapatite chromatography. A polypeptide also
can be purified, for example, by expressing a nucleic acid in an
expression vector. In addition, a purified polypeptide can be
obtained by chemical synthesis. The extent of purity of a
polypeptide can be measured using any appropriate method, e.g.,
column chromatography, polyacrylamide gel electrophoresis, or HPLC
analysis.
[0286] In one aspect, this disclosure provides methods of detecting
recombinant nucleic acids and polypeptides in plant cells. Without
being limiting, nucleic acids also can be detected using
hybridization. Hybridization between nucleic acids is discussed in
detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory
Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.). mRNA expression can be localized in tissue samples
using in situ hybridization.
[0287] In an aspect, this disclosure provides methods for detecting
accumulation of a polypeptide in a modified tobacco plant as
compared to a control tobacco plant lacking at least one
non-natural mutation when grown under comparable conditions.
Increased accumulation of a polypeptide in a modified plant is
determined relative to detection of the same polypeptide in a
control plant lacking the non-natural mutation, and grown under
comparable conditions.
[0288] Polypeptides can be detected using antibodies. Techniques
for detecting polypeptides using antibodies include enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations
and immunofluorescence. An antibody provided herein can be a
polyclonal antibody or a monoclonal antibody. An antibody having
specific binding affinity for a polypeptide provided herein can be
generated using methods well known in the art. An antibody provided
herein can be attached to a solid support such as a microtiter
plate using methods known in the art.
[0289] Detection (e.g., of an amplification product, of a
hybridization complex, of a polypeptide) can be accomplished using
detectable labels. The term "label" is intended to encompass the
use of direct labels as well as indirect labels. Detectable labels
include enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials.
[0290] In an aspect, this disclosure provides a modified tobacco or
Cannabis plant, tobacco or Cannabis seed, or part thereof,
comprising a recombinant DNA construct comprising a heterologous
promoter operably linked to a nucleic acid encoding at least one
small RNA molecule capable of binding to and reducing the
expression of a nucleic acid sequence encoding a polypeptide
involved in the biosynthesis of at least one terpene.
[0291] In an aspect, this disclosure provides a small RNA molecule,
or a precursor thereof. As used herein, a "small RNA molecule"
refers to a non-coding RNA molecule of between 16 nucleotides and
50 nucleotides in length. In an aspect, a small RNA molecule
comprises between 16 nucleotides and 40 nucleotides. In another
aspect, a small RNA molecule comprises between 16 nucleotides and
30 nucleotides. In another aspect, a small RNA molecule comprises
between 18 nucleotides and 50 nucleotides. In another aspect, a
small RNA molecule comprises between 18 nucleotides and 40
nucleotides. In another aspect, a small RNA molecule comprises
between 18 nucleotides and 30 nucleotides. In another aspect, a
small RNA molecule comprises between 18 nucleotides and 25
nucleotides. In another aspect, a small RNA molecule comprises
between 20 nucleotides and 28 nucleotides. In another aspect, a
small RNA molecule comprises between 20 nucleotides and 24
nucleotides. In another aspect, a small RNA molecule comprises
between 21 nucleotides and 23 nucleotides. In another aspect, a
small RNA molecule comprises 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.
[0292] In an aspect, a small RNA molecule is at least 75% identical
or complementary to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and
161-190. In an aspect, a small RNA molecule is at least 80%
identical or complementary to a nucleic acid sequence selected from
the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156,
158-159, and 161-190. In an aspect, a small RNA molecule is at
least 85% identical or complementary to a nucleic acid sequence
selected from the group consisting of SEQ ID NOs: 1-42, 112-139,
155-156, 158-159, and 161-190. In an aspect, a small RNA molecule
is at least 90% identical or complementary to a nucleic acid
sequence selected from the group consisting of SEQ ID NOs: 1-42,
112-139, 155-156, 158-159, and 161-190. In an aspect, a small RNA
molecule is at least 92.5% identical or complementary to a nucleic
acid sequence selected from the group consisting of SEQ ID NOs:
1-42, 112-139, 155-156, 158-159, and 161-190. In an aspect, a small
RNA molecule is at least 95% identical or complementary to a
nucleic acid sequence selected from the group consisting of SEQ ID
NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In an aspect, a
small RNA molecule is at least 97.5% identical or complementary to
a nucleic acid sequence selected from the group consisting of SEQ
ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In an aspect,
a small RNA molecule is at least 99% identical or complementary to
a nucleic acid sequence selected from the group consisting of SEQ
ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In an aspect,
a small RNA molecule is 100% identical or complementary to a
nucleic acid sequence selected from the group consisting of SEQ ID
NOs: 1-42, 112-139, 155-156, 158-159, and 161-190.
[0293] In an aspect, a small RNA molecule is at least 75% identical
or complementary to a polynucleotide encoding a polypeptide
selected from the group consisting of SEQ ID NOs: 43-63, 140-153,
157, 160, and 191-205. In an aspect, a small RNA molecule is at
least 80% identical or complementary to a polynucleotide encoding a
polypeptide selected from the group consisting of SEQ ID NOs:
43-63, 140-153, 157, 160, and 191-205. In an aspect, a small RNA
molecule is at least 85% identical or complementary to a
polynucleotide encoding a polypeptide selected from the group
consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In
an aspect, a small RNA molecule is at least 90% identical or
complementary to a polynucleotide encoding a polypeptide selected
from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160,
and 191-205. In an aspect, a small RNA molecule is at least 92.5%
identical or complementary to a polynucleotide encoding a
polypeptide selected from the group consisting of SEQ ID NOs:
43-63, 140-153, 157, 160, and 191-205. In an aspect, a small RNA
molecule is at least 95% identical or complementary to a
polynucleotide encoding a polypeptide selected from the group
consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In
an aspect, a small RNA molecule is at least 97.5% identical or
complementary to a polynucleotide encoding a polypeptide selected
from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160,
and 191-205. In an aspect, a small RNA molecule is at least 99%
identical or complementary to a polynucleotide encoding a
polypeptide selected from the group consisting of SEQ ID NOs:
43-63, 140-153, 157, 160, and 191-205. In an aspect, a small RNA
molecule is 100% identical or complementary to a polynucleotide
encoding a polypeptide selected from the group consisting of SEQ ID
NOs: 43-63, 140-153, 157, 160, and 191-205.
[0294] In an aspect, a small RNA provided herein comprises a
nucleic acid sequence at least 88.7% identical or complementary to
at least 18 contiguous nucleotides of a nucleic acid sequence
selected from the group consisting of SEQ ID NOs: 1-42, 112-139,
155-156, 158-159, and 161-190. In another aspect, a small RNA
provided herein comprises a nucleic acid sequence at least 94.3%
identical or complementary to at least 18 contiguous nucleotides of
a nucleic acid sequence selected from the group consisting of SEQ
ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In another
aspect, a small RNA provided herein comprises a nucleic acid
sequence 100% identical or complementary to at least 18 contiguous
nucleotides of a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and
161-190. In another aspect, a small RNA provided herein comprises a
nucleic acid sequence at least 85% identical or complementary to at
least 20 contiguous nucleotides of a nucleic acid sequence selected
from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156,
158-159, and 161-190. In another aspect, a small RNA provided
herein comprises a nucleic acid sequence at least 90% identical or
complementary to at least 20 contiguous nucleotides of a nucleic
acid sequence selected from the group consisting of SEQ ID NOs:
1-42, 112-139, 155-156, 158-159, and 161-190. In another aspect, a
small RNA provided herein comprises a nucleic acid sequence at
least 95% identical or complementary to at least 20 contiguous
nucleotides of a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and
161-190. In another aspect, a small RNA provided herein comprises a
nucleic acid sequence 100% identical or complementary to at least
20 contiguous nucleotides of a nucleic acid sequence selected from
the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156,
158-159, and 161-190.
[0295] In an aspect, a small RNA molecule is selected from the
group consisting of a double-stranded RNA, a small interfering RNA
(siRNA), a trans-acting siRNA, and a microRNA (miRNA).
[0296] miRNAs are generally of between about 19 to about 25
nucleotides (commonly about 20-24 nucleotides in plants), that
guide cleavage in trans of target transcripts, negatively
regulating the expression of genes involved in various regulation
and development pathways. In some cases, miRNAs serve to guide
in-phase processing of siRNA primary transcripts.
[0297] Many microRNA genes (MIR genes) have been identified and
made publicly available in a database ("miRBase," available online
at microrna[dot]sanger[dot]ac[dot]uk/sequences; also see
Griffiths-Jones et al. (2003) Nucleic Acids Res., 31:439-441). MIR
genes have been reported to occur in intergenic regions, both
isolated and in clusters in the genome, but can also be located
entirely or partially within introns of other genes (both
protein-coding and non-protein-coding). For a review of miRNA
biogenesis, see Kim (2005) Nature Rev. Mol. Cell. Biol., 6:376-385.
Transcription of MIR genes can be, at least in some cases, under
promotional control of a MIR gene's own promoter. The primary
transcript, termed a "pri-miRNA," can be quite large (several
kilobases) and can be polycistronic, containing one or more
pre-miRNAs (fold-back structures containing a stem-loop arrangement
that is processed to the mature miRNA) as well as the usual 5'
"cap" and polyadenylated tail of an mRNA.
[0298] Maturation of a mature miRNA from its corresponding
precursors (pri-miRNAs and pre-miRNAs) differs significantly
between animals and plants. For example, in plant cells, microRNA
precursor molecules are believed to be largely processed to the
mature miRNA entirely in the nucleus, whereas in animal cells, the
pri-miRNA transcript is processed in the nucleus by the
animal-specific enzyme Drosha, followed by export of the pre-miRNA
to the cytoplasm where it is further processed to the mature miRNA.
Mature miRNAs in plants are typically 21 nucleotides in length.
[0299] Transgenic expression of miRNAs (whether a naturally
occurring sequence or an artificial sequence) can be employed to
regulate expression of the miRNA's target gene or genes. Inclusion
of a miRNA recognition site in a transgenically expressed
transcript is also useful in regulating expression of the
transcript. Recognition sites of miRNAs have been validated in all
regions of an mRNA, including the 5' untranslated region, coding
region, and 3' untranslated region, indicating that the position of
the miRNA target site relative to the coding sequence may not
necessarily affect suppression. Because miRNAs are important
regulatory elements in eukaryotes, transgenic suppression of miRNAs
is useful for manipulating biological pathways and responses.
Finally, promoters of MIR genes can have very specific expression
patterns (e.g., cell-specific, tissue-specific, temporally
specific, or inducible), and thus are useful in recombinant
constructs to induce such specific transcription of a DNA sequence
to which they are operably linked. Various utilities of miRNAs,
their precursors, their recognition sites, and their promoters are
described in detail in U.S. Patent Application Publication
2006/0200878 A1, incorporated by reference herein. Non-limiting
examples of these utilities include: (1) the expression of a native
miRNA or miRNA precursor sequence to suppress a target gene; (2)
the expression of an artificial miRNA or miRNA precursor sequence
to suppress a target gene; (3) expression of a transgene with a
miRNA recognition site, where the transgene is suppressed when the
mature miRNA is expressed; (4) expression of a transgene driven by
a miRNA promoter.
[0300] Designing an artificial miRNA sequence can be as simple as
substituting sequence that is complementary to the intended target
for nucleotides in the miRNA stem region of the miRNA precursor, as
demonstrated by Zeng et al. (2002) Mol. Cell, 9:1327-1333. One
non-limiting example of a general method for determining nucleotide
changes in the native miRNA sequence to produce the engineered
miRNA precursor includes the following steps: (a) Selecting a
unique target sequence of at least 18 nucleotides specific to the
target gene, e.g., by using sequence alignment tools such as BLAST
(see, for example, Altschul et al. (1990) J. Mol. Biol.,
215:403-410; Altschul et al. (1997) Nucleic Acids Res.,
25:3389-3402), for example, of both tobacco cDNA and genomic DNA
databases, to identify target transcript orthologues and any
potential matches to unrelated genes, thereby avoiding
unintentional silencing of non-target sequences; (b) Analyzing the
target gene for undesirable sequences (e.g., matches to sequences
from non-target species), and score each potential 19-mer segment
for GC content, Reynolds score (see Reynolds et al. (2004) Nature
Biotechnol., 22:326-330), and functional asymmetry characterized by
a negative difference in free energy (".DELTA..DELTA.G" or
".DELTA..DELTA.G") (see Khvorova et al. (2003) Cell, 115:209-216).
Preferably 19-mers are selected that have all or most of the
following characteristics: (1) a Reynolds score>4, (2) a GC
content between about 40% to about 60%, (3) a negative
.DELTA..DELTA.G, (4) a terminal adenosine, (5) lack of a
consecutive run of 4 or more of the same nucleotide; (6) a location
near the 3' terminus of the target gene; (7) minimal differences
from the miRNA precursor transcript. Positions at every third
nucleotide in an siRNA have been reported to be especially
important in influencing RNAi efficacy and an algorithm,
"siExplorer" is publicly available at
rna[dot]chem[dot]t[dot]u-tokyo[dot]ac[dot]jp/siexplorer.htm (see
Katoh and Suzuki (2007) Nucleic Acids Res., 10.1093/nar/gkl1120);
(c) Determining the reverse complement of the selected 19-mers to
use in making a modified mature miRNA. The additional nucleotide at
position 20 is preferably matched to the selected target sequence,
and the nucleotide at position 21 is preferably chosen to either be
unpaired to prevent spreading of silencing on the target transcript
or paired to the target sequence to promote spreading of silencing
on the target transcript; and (d) transforming the artificial miRNA
into a plant.
[0301] In an aspect, a vector can be used for overexpression and
knockdown, e.g., of candidate terpene biosynthesis genes. A
non-limiting example of a vector is pGWB551. In an aspect, a vector
comprises a nucleic acid sequence at least 90% identical to SEQ ID
NO: 154. In an aspect, a vector comprises a nucleic acid sequence
at least 95% identical to SEQ ID NO: 154. In an aspect, a vector
comprises a nucleic acid sequence 100% identical to SEQ ID NO:
154.
Products
[0302] In an aspect, this disclosure provides plant material from
any plant or plant part provided herein. In an aspect, this
disclosure provides Cannabis material from any Cannabis plant or
Cannabis plant part provided herein. In an aspect, this disclosure
provides cured plant material from any plant or plant part provided
herein. In an aspect, this disclosure provides cured tobacco
material from any tobacco plant or tobacco plant part provided
herein. In an aspect, this disclosure provides cured Cannabis
material from any Cannabis plant or Cannabis plant part provided
herein.
[0303] In an aspect, this disclosure provides a method comprising
preparing a tobacco product or a Cannabis product using cured
tobacco material or Cannabis material from a modified tobacco or
Cannabis plant, where the modified tobacco or Cannabis plant
comprises a recombinant nucleic acid molecule comprising a nucleic
acid sequence encoding a polypeptide involved in the biosynthesis
of at least one terpene operably linked to a heterologous promoter.
In an aspect, this disclosure provides a method comprising
preparing a tobacco product or a Cannabis product using cured
tobacco material or Cannabis material from a modified tobacco or
Cannabis plant, where the modified tobacco or Cannabis plant
comprises at least one non-natural mutation in an endogenous gene
encoding a polypeptide involved in the biosynthesis of at least one
terpene, where the modified tobacco or Cannabis plant exhibits
increased mRNA expression of the endogenous gene or increased
accumulation of the polypeptide as compared to a control tobacco or
Cannabis plant lacking the at least one non-natural mutation when
grown under comparable conditions.
[0304] In an aspect, cured tobacco plant material is made by a
curing process selected from the group consisting of flue curing,
air curing, fire curing, and sun curing. In an aspect, cured
tobacco material is made by a curing process selected from the
group consisting of flue curing, air curing, fire curing, and sun
curing.
[0305] "Curing" is the aging process that reduces moisture and
brings about the destruction of chlorophyll giving tobacco leaves a
golden color and by which starch is converted to sugar. Cured
tobacco therefore has a higher reducing sugar content and a lower
starch content compared to harvested green leaf. In one aspect,
tobacco plants or plant components provided herein can be cured
using conventional means, e.g., flue-cured, barn-cured, fire-cured,
air-cured or sun-cured. See, for example, Tso (1999, Chapter 1 in
Tobacco, Production, Chemistry and Technology, Davis & Nielsen,
eds., Blackwell Publishing, Oxford) for a description of different
types of curing methods. Cured tobacco is usually aged in a wooden
drum (e.g., a hogshead) or cardboard cartons in compressed
conditions for several years (e.g., two to five years), at a
moisture content ranging from 10% to about 25%. See, U.S. Pat. Nos.
4,516,590 and 5,372,149. Cured and aged tobacco then can be further
processed. Further processing includes conditioning the tobacco
under vacuum with or without the introduction of steam at various
temperatures, pasteurization, and fermentation.
[0306] Following Cannabis plant harvest, Cannabis plant material is
typically dried, prior to curing. Cannabis can be dried in a dark
room with temperatures kept between about 15.degree. C. and about
22.degree. C. and humidity between 45% and 55%, with a fan for
gentle air circulation. An initial drying cycle ranges between 5
days and 15 days. Dried Cannabis can then be cured to increase
flavor, aroma, and potency. Length of Cannabis curing is
strain-dependent. For example, some Cannabis strains benefit from 2
to 3 weeks of curing, other Cannabis strains benefit from 4 to 8
weeks of curing, while other Cannabis strains benefit from 6 months
or more of curing.
[0307] Information regarding the harvesting of burley and dark
tobacco varieties can be found in the 2019-2020 Burley and Dark
Tobacco Production Guide (December 2018) published by the
University of Kentucky, The University of Tennessee, Virginia Tech,
and North Carolina State University, which is incorporated herein
by reference in its entirety.
[0308] In an aspect, cured tobacco material comprises tobacco
material selected from the group selected from cured leaf material,
cured stem material, cured bud material, cured flower material, and
cured root material. In an aspect, cured tobacco material comprises
cured leaf material, cured stem material, or both. In an aspect,
cured tobacco material comprises cured leaf material. In an aspect,
cured tobacco material comprises cured stem material.
[0309] In an aspect, Cannabis material comprises Cannabis material
selected from the group selected from leaf material, stem material,
bud material, flower material, root material, hairy roots, cell
culture, and callus. In an aspect, Cannabis material comprises leaf
material, stem material, or both. In an aspect, Cannabis material
comprises leaf material. In an aspect, Cannabis material comprises
stem material.
[0310] In an aspect, cured tobacco material comprises flue-cured
tobacco material. In an aspect, cured tobacco material comprises
air-cured tobacco material. In an aspect, cured tobacco material
comprises fire-cured tobacco material. In an aspect, cured tobacco
material comprises sun-cured tobacco material. In an aspect, cured
tobacco material provided herein is selected from the group
consisting of air-cured tobacco material, fire-cured tobacco
material, sun-cured tobacco material, and flue-cured tobacco
material. In an aspect, cured tobacco material is from a tobacco
variety selected from the group consisting of a flue-cured variety,
a bright variety, a Burley variety, a Virginia variety, a Maryland
variety, a dark variety, an Oriental variety, and a Turkish
variety.
[0311] In an aspect, cured tobacco leaf provided herein is selected
from the group consisting of air-cured tobacco leaf, fire-cured
tobacco leaf, sun-cured tobacco leaf, and flue-cured tobacco leaf.
In an aspect, cured tobacco leaf is from a tobacco variety selected
from the group consisting of a flue-cured variety, a bright
variety, a Burley variety, a Virginia variety, a Maryland variety,
a dark variety, an Oriental variety, and a Turkish variety.
[0312] Fermentation typically is characterized by high initial
moisture content, heat generation, and a 10 to 20% loss of dry
weight. See, for example, U.S. Pat. Nos. 4,528,993, 4,660,577,
4,848,373, 5,372,149; U.S. Publication No. 2005/0178398; and Tso
(1999, Chapter 1 in Tobacco, Production, Chemistry and Technology,
Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured,
aged, and fermented tobacco can be further processed (e.g., cut,
shredded, expanded, or blended). See, for example, U.S. Pat. Nos.
4,528,993; 4,660,577; and 4,987,907. Cured, aged, and fermented
Cannabis can be further processed (e.g., cut, shredded, expanded,
or blended). In an aspect, this disclosure provides fermented
tobacco material from any tobacco plant, or part thereof, provided
herein. In another aspect, this disclosure provides fermented
tobacco material from any modified tobacco plant, or part thereof,
provided herein. In an aspect, this disclosure provides fermented
Cannabis material from any Cannabis plant, or part thereof,
provided herein. In another aspect, this disclosure provides
fermented Cannabis material from any modified Cannabis plant, or
part thereof, provided herein.
[0313] Tobacco material obtained from the tobacco lines, varieties
or hybrids of the present disclosure can be used to make tobacco
products. As used herein, "tobacco product" is defined as any
product made or derived from tobacco that is intended for human use
or consumption. In an aspect, this disclosure provides a tobacco
product comprising plant material from a tobacco plant provided
herein. In another aspect, this disclosure provides a tobacco
product comprising plant material from a modified tobacco plant
provided herein. In another aspect, this disclosure provides a
tobacco product comprising cured tobacco material. In another
aspect, this disclosure provides a tobacco product comprising
fermented tobacco material. In another aspect, this disclosure
provides a tobacco product comprising a tobacco blend.
[0314] Cannabis material obtained from the Cannabis lines, strains,
varieties or hybrids of the present disclosure can be used to make
Cannabis products. As used herein, "Cannabis product" is defined as
any product made or derived from Cannabis that is intended for
human use or consumption. In an aspect, this disclosure provides a
Cannabis product comprising plant material from a Cannabis plant
provided herein. In another aspect, this disclosure provides a
Cannabis product comprising plant material from a modified Cannabis
plant provided herein. In another aspect, this disclosure provides
a Cannabis product comprising Cannabis material. In another aspect,
this disclosure provides a Cannabis product comprising cured
Cannabis material. In another aspect, this disclosure provides a
Cannabis product comprising fermented Cannabis material. In another
aspect, this disclosure provides a Cannabis product comprising a
Cannabis blend.
[0315] Tobacco products include, without limitation, cigarette
products (e.g., cigarettes and bidi cigarettes), cigar products
(e.g., cigar wrapping tobacco and cigarillos), pipe tobacco
products, products derived from tobacco, tobacco-derived nicotine
products, smokeless tobacco products (e.g., moist snuff, dry snuff,
and chewing tobacco), films, chewables, tabs, shaped parts, gels,
consumable units, insoluble matrices, hollow shapes, reconstituted
tobacco, expanded tobacco, and the like. See, e.g., U.S. Patent
Publication No. US 2006/0191548.
[0316] As used herein, "cigarette" refers a tobacco product having
a "rod" and "filler". The cigarette "rod" includes the cigarette
paper, filter, plug wrap (used to contain filtration materials),
tipping paper that holds the cigarette paper (including the filler)
to the filter, and all glues that hold these components together.
The "filler" includes (1) all tobaccos, including but not limited
to reconstituted and expanded tobacco, (2) non-tobacco substitutes
(including but not limited to herbs, non-tobacco plant materials
and other spices that may accompany tobaccos rolled within the
cigarette paper), (3) casings, (4) flavorings, and (5) all other
additives (that are mixed into tobaccos and substitutes and rolled
into the cigarette).
[0317] In an aspect, a tobacco product comprises reconstituted
tobacco. In another aspect, this disclosure provides reconstituted
tobacco comprising cured tobacco material. As used herein,
"reconstituted tobacco" refers to a part of tobacco filler made
from tobacco dust and other tobacco scrap material, processed into
sheet form and cut into strips to resemble tobacco. In addition to
the cost savings, reconstituted tobacco is very important for its
contribution to cigarette taste from processing flavor development
using reactions between ammonia and sugars.
[0318] In an aspect, a Cannabis product comprises reconstituted
Cannabis. In another aspect, this disclosure provides reconstituted
Cannabis comprising Cannabis material. As used herein,
"reconstituted Cannabis" refers to a part of Cannabis filler made
from Cannabis dust and other Cannabis scrap material, processed
into sheet form and cut into strips to resemble Cannabis.
[0319] In an aspect, a tobacco product comprises expanded tobacco.
As used herein, "expanded tobacco" refers to a part of tobacco
filler which is processed through expansion of suitable gases so
that the tobacco is "puffed" resulting in reduced density and
greater filling capacity. It reduces the weight of tobacco used in
cigarettes.
[0320] In an aspect, a Cannabis product comprises expanded
Cannabis. As used herein, "expanded Cannabis" refers to a part of
Cannabis filler which is processed through expansion of suitable
gases so that the Cannabis is "puffed" resulting in reduced density
and greater filling capacity.
[0321] Tobacco products derived from plants of the present
disclosure also include cigarettes and other smoking articles,
particularly those smoking articles including filter elements,
where the rod of smokable material includes cured tobacco within a
tobacco blend. In an aspect, a tobacco product of the present
disclosure is selected from the group consisting of a kretek, a
bidi cigarette, a cigarillo, a non-ventilated recess filter
cigarette, a vented recess filter cigarette, a cigar, snuff, pipe
tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf
tobacco, hookah tobacco, shredded tobacco, and cut tobacco.
[0322] Cannabis products derived from plants of the present
disclosure also include cigarettes and other smoking articles,
particularly those smoking articles including filter elements,
where the rod of smokable material includes cured Cannabis within a
Cannabis blend. In an aspect, a Cannabis product of the present
disclosure is selected from the group consisting of a kretek, a
cigarillo, a cigar, snuff, pipe Cannabis, cigar Cannabis, cigarette
Cannabis, chewing Cannabis, hookah Cannabis, shredded Cannabis, and
cut Cannabis.
[0323] In an aspect, a tobacco product of the present disclosure is
selected from the group consisting of a cigarette, a heated tobacco
product, a kretek, a bidi cigarette, a cigar, a cigarillo, a
non-ventilated cigarette, a vented recess filter cigarette, pipe
tobacco, snuff, snus, chewing tobacco, moist smokeless tobacco,
fine cut chewing tobacco, long cut chewing tobacco, pouched chewing
tobacco product, gum, a tablet, a lozenge, and a dissolving
strip.
[0324] In an aspect, a Cannabis product of the present disclosure
is selected from the group consisting of a cigarette, a heated
Cannabis product, a kretek, a cigar, a cigarillo, pipe Cannabis,
snuff, snus, chewing Cannabis, moist smokeless Cannabis, fine cut
chewing Cannabis, long cut chewing Cannabis, pouched chewing
Cannabis product, gum, a tablet, a lozenge, and a dissolving
strip.
[0325] In an aspect, a Cannabis product of the present disclosure
is a consumable product. In a further aspect, a consumable product
is an edible product. In an aspect, a consumable product is an
oil.
[0326] In an aspect, a tobacco product of the present disclosure is
a smokeless tobacco product. In an aspect, a smokeless tobacco
product is selected from the group consisting of loose leaf chewing
tobacco, plug chewing tobacco, moist snuff, nasal snuff, dry snuff,
and snus.
[0327] In an aspect, a Cannabis product of the present disclosure
is a smokeless Cannabis product. In an aspect, a smokeless Cannabis
product is selected from the group consisting of chewing Cannabis,
moist snuff, dry snuff, and snus.
[0328] Smokeless tobacco or Cannabis products are not combusted and
include, but not limited to, chewing tobacco, moist smokeless
tobacco, moist smokeless Cannabis, snus, and dry snuff. Chewing
tobacco or Cannabis is coarsely divided tobacco or Cannabis leaf
that is typically packaged in a large pouch-like package and used
in a plug or twist. Moist smokeless tobacco or Cannabis is a moist,
more finely divided tobacco or Cannabis that is provided in loose
form or in pouch form and is typically packaged in round cans and
used as a pinch or in a pouch placed between an adult tobacco or
Cannabis consumer's cheek and gum. Snus is a heat-treated smokeless
tobacco or Cannabis. Dry snuff is finely ground tobacco or Cannabis
that is placed in the mouth or used nasally.
[0329] In yet another aspect, a tobacco or Cannabis product of the
present disclosure is selected from the group consisting of an
electronically heated cigarette, an e-cigarette, an electronic
vaporing device.
[0330] In an aspect, a tobacco or Cannabis product of the present
disclosure can be a blended tobacco or Cannabis product.
[0331] In another aspect, this disclosure provides a tobacco blend
comprising cured tobacco material. A tobacco blend can comprise any
combination of cured tobacco, uncured tobacco, fermented tobacco,
unfermented tobacco, expanded tobacco, and reconstituted
tobacco.
[0332] In another aspect, this disclosure provides a Cannabis blend
comprising Cannabis material. A Cannabis blend can comprise any
combination of fermented Cannabis, unfermented Cannabis, expanded
Cannabis, and reconstituted Cannabis.
[0333] In an aspect, a tobacco blend comprises at least 5% cured
tobacco by weight. In an aspect, a tobacco blend comprises at least
10% cured tobacco by weight. In an aspect, a tobacco blend
comprises at least 15% cured tobacco by weight. In an aspect, a
tobacco blend comprises at least 20% cured tobacco by weight. In an
aspect, a tobacco blend comprises at least 25% cured tobacco by
weight. In an aspect, a tobacco blend comprises at least 30% cured
tobacco by weight. In an aspect, a tobacco blend comprises at least
35% cured tobacco by weight. In an aspect, a tobacco blend
comprises at least 40% cured tobacco by weight. In an aspect, a
tobacco blend comprises at least 45% cured tobacco by weight. In an
aspect, a tobacco blend comprises at least 50% cured tobacco by
weight. In an aspect, a tobacco blend comprises at least 55% cured
tobacco by weight. In an aspect, a tobacco blend comprises at least
60% cured tobacco by weight. In an aspect, a tobacco blend
comprises at least 65% cured tobacco by weight. In an aspect, a
tobacco blend comprises at least 70% cured tobacco by weight. In an
aspect, a tobacco blend comprises at least 75% cured tobacco by
weight. In an aspect, a tobacco blend comprises at least 80% cured
tobacco by weight. In an aspect, a tobacco blend comprises at least
85% cured tobacco by weight. In an aspect, a tobacco blend
comprises at least 90% cured tobacco by weight. In an aspect, a
tobacco blend comprises at least 95% cured tobacco by weight.
[0334] In an aspect, a Cannabis blend comprises at least 5%
Cannabis by weight. In an aspect, a Cannabis blend comprises at
least 10% Cannabis by weight. In an aspect, a Cannabis blend
comprises at least 15% Cannabis by weight. In an aspect, a Cannabis
blend comprises at least 20% Cannabis by weight. In an aspect, a
Cannabis blend comprises at least 25% Cannabis by weight. In an
aspect, a Cannabis blend comprises at least 30% Cannabis by weight.
In an aspect, a Cannabis blend comprises at least 35% Cannabis by
weight. In an aspect, a Cannabis blend comprises at least 40%
Cannabis by weight. In an aspect, a Cannabis blend comprises at
least 45% Cannabis by weight. In an aspect, a Cannabis blend
comprises at least 50% Cannabis by weight. In an aspect, a Cannabis
blend comprises at least 55% Cannabis by weight. In an aspect, a
Cannabis blend comprises at least 60% Cannabis by weight. In an
aspect, a Cannabis blend comprises at least 65% Cannabis by weight.
In an aspect, a Cannabis blend comprises at least 70% Cannabis by
weight. In an aspect, a Cannabis blend comprises at least 75%
Cannabis by weight. In an aspect, a Cannabis blend comprises at
least 80% Cannabis by weight. In an aspect, a Cannabis blend
comprises at least 85% Cannabis by weight. In an aspect, a Cannabis
blend comprises at least 90% Cannabis by weight. In an aspect, a
Cannabis blend comprises at least 95% Cannabis by weight.
[0335] In an aspect, a tobacco blend comprises at least 5% cured
tobacco by volume. In an aspect, a tobacco blend comprises at least
10% cured tobacco by volume. In an aspect, a tobacco blend
comprises at least 15% cured tobacco by volume. In an aspect, a
tobacco blend comprises at least 20% cured tobacco by volume. In an
aspect, a tobacco blend comprises at least 25% cured tobacco by
volume. In an aspect, a tobacco blend comprises at least 30% cured
tobacco by volume. In an aspect, a tobacco blend comprises at least
35% cured tobacco by volume. In an aspect, a tobacco blend
comprises at least 40% cured tobacco by volume. In an aspect, a
tobacco blend comprises at least 45% cured tobacco by volume. In an
aspect, a tobacco blend comprises at least 50% cured tobacco by
volume. In an aspect, a tobacco blend comprises at least 55% cured
tobacco by volume. In an aspect, a tobacco blend comprises at least
60% cured tobacco by volume. In an aspect, a tobacco blend
comprises at least 65% cured tobacco by volume. In an aspect, a
tobacco blend comprises at least 70% cured tobacco by volume. In an
aspect, a tobacco blend comprises at least 75% cured tobacco by
volume. In an aspect, a tobacco blend comprises at least 80% cured
tobacco by volume. In an aspect, a tobacco blend comprises at least
85% cured tobacco by volume. In an aspect, a tobacco blend
comprises at least 90% cured tobacco by volume. In an aspect, a
tobacco blend comprises at least 95% cured tobacco by volume.
[0336] In an aspect, a Cannabis blend comprises at least 5%
Cannabis by volume. In an aspect, a Cannabis blend comprises at
least 10% Cannabis by volume. In an aspect, a Cannabis blend
comprises at least 15% Cannabis by volume. In an aspect, a Cannabis
blend comprises at least 20% Cannabis by volume. In an aspect, a
Cannabis blend comprises at least 25% Cannabis by volume. In an
aspect, a Cannabis blend comprises at least 30% Cannabis by volume.
In an aspect, a Cannabis blend comprises at least 35% Cannabis by
volume. In an aspect, a Cannabis blend comprises at least 40%
Cannabis by volume. In an aspect, a Cannabis blend comprises at
least 45% Cannabis by volume. In an aspect, a Cannabis blend
comprises at least 50% Cannabis by volume. In an aspect, a Cannabis
blend comprises at least 55% Cannabis by volume. In an aspect, a
Cannabis blend comprises at least 60% Cannabis by volume. In an
aspect, a Cannabis blend comprises at least 65% Cannabis by volume.
In an aspect, a Cannabis blend comprises at least 70% Cannabis by
volume. In an aspect, a Cannabis blend comprises at least 75%
Cannabis by volume. In an aspect, a Cannabis blend comprises at
least 80% Cannabis by volume. In an aspect, a Cannabis blend
comprises at least 85% Cannabis by volume. In an aspect, a Cannabis
blend comprises at least 90% Cannabis by volume. In an aspect, a
Cannabis blend comprises at least 95% Cannabis by volume.
[0337] In an aspect, this disclosure provides a method comprising
preparing a tobacco or Cannabis product using cured tobacco or
Cannabis material from a modified tobacco or Cannabis plant, where
the modified tobacco or Cannabis plant comprises a recombinant DNA
construct comprising a heterologous promoter operably linked to a
nucleic acid encoding at least one small RNA molecule capable of
binding to and reducing the expression of a nucleic acid sequence
encoding a polypeptide involved in the biosynthesis of at least one
terpene.
EMBODIMENTS
[0338] The following examples of non-limiting embodiments are
envisioned: [0339] 1. A modified tobacco or Cannabis plant, tobacco
or Cannabis seed, or part thereof comprising a recombinant nucleic
acid molecule comprising a nucleic acid sequence encoding a
polypeptide involved in the biosynthesis of at least one terpene
operably linked to a heterologous promoter. [0340] 2. A modified
tobacco or Cannabis plant, tobacco or Cannabis seed, or part
thereof comprising at least one non-natural mutation in an
endogenous gene encoding a polypeptide involved in the biosynthesis
of at least one terpene, where the modified tobacco or Cannabis
plant exhibits increased mRNA expression of the endogenous gene or
increased accumulation of the polypeptide as compared to a control
tobacco or Cannabis plant lacking the at least one non-natural
mutation when grown under comparable conditions. [0341] 3. A
modified tobacco or Cannabis plant, tobacco or Cannabis seed, or
part thereof, comprising a recombinant DNA construct comprising a
heterologous promoter operably linked to a nucleic acid encoding at
least one small RNA molecule capable of binding to and reducing the
expression of a nucleic acid sequence encoding a polypeptide
involved in the biosynthesis of at least one terpene. [0342] 4. The
modified tobacco or Cannabis plant, tobacco or Cannabis seed, or
part thereof of any one of embodiments [0059]-3, where the
polypeptide is selected from the group consisting of geranyl
diphosphate synthase (GDP), limonene synthase (LS), limonene
3-hydroxylase (L30H), isopiperitenol dehydrogenase (IPD), pulegone
reductase, menthofuran synthase (MFS), geranylgeranyl diphosphate
synthase (GGPPS), neomenthol dehydrogenase (NtNMD), phylloplanin,
premnaspirodiene oxygenase (PSO), kolavenyl diphosphate synthase
(KPS), solanesyl phosphate synthase 3, terpene synthase 10-like,
and germacrene D synthase. [0343] 5. The modified tobacco or
Cannabis plant, tobacco or Cannabis seed, or part thereof of any
one of embodiments [0059]-3, where the at least one terpene is
menthol or a related compound. [0344] 6. The modified tobacco or
Cannabis plant, tobacco or Cannabis seed, or part thereof of any
one of embodiments [0059]-3, where the polypeptide is selected from
the group consisting of geranylgeranyl diphosphate synthase
(GGPPS2), 8-hydroxy-copalyl diphosphate synthase, and cis-abienol
synthase. [0345] 7. The modified tobacco or Cannabis plant, tobacco
or Cannabis seed, or part thereof of embodiment 6, where the
cis-abienol synthase is selected from the group consisting of
cis-abienol synthase ISOFORM1 and cis-abienol synthase ISOFORM2.
[0346] 8. The modified tobacco or Cannabis plant, tobacco or
Cannabis seed, or part thereof of any one of embodiments [0059]-3,
where the at least one terpene is a labdanoid. [0347] 9. The
modified tobacco or Cannabis plant, tobacco or Cannabis seed, or
part thereof of any one of embodiments [0059]-3, where the at least
one terpene is neophytadiene. [0348] 10. The modified tobacco or
Cannabis plant, tobacco or Cannabis seed, or part thereof of
embodiment 8, where the labdanoid is cis-abienol. [0349] 11. The
modified tobacco or Cannabis plant, tobacco or Cannabis seed, or
part thereof of any one of embodiments [0059]-3, where the
polypeptide is cembratrienol synthase 2a. [0350] 12. The modified
tobacco or Cannabis plant, tobacco or Cannabis seed, or part
thereof of any one of embodiments [0059]-3, where the at least one
terpene is cembratrienediol. [0351] 13. The modified tobacco or
Cannabis plant, tobacco or Cannabis seed, or part thereof of any
one of embodiments [0059]-3, where the polypeptide is
levopimaradiene synthetase. [0352] 14. The modified tobacco or
Cannabis plant, tobacco or Cannabis seed, or part thereof of any
one of embodiments [0059]-3, where the at least one terpene is
levopimaric acid. [0353] 15. The modified tobacco or Cannabis
plant, tobacco or Cannabis seed, or part thereof of any one of
embodiments [0059]-3, where the polypeptide is selected from the
group consisting of 2-isopropylmalate synthetase (IPS),
2-oxoisovalerate dehydrogenase, and 2-alkenal reductase. [0354] 16.
The modified tobacco or Cannabis plant, tobacco or Cannabis seed,
or part thereof of any one of embodiments [0059]-3, where the at
least one terpene is L-leucine. [0355] 17. The modified tobacco or
Cannabis plant, tobacco or Cannabis seed, or part thereof of
embodiment [0059], where the nucleic acid sequence comprises a
sequence at least 70% identical to a nucleic acid sequence selected
from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158,
and 176-190. [0356] 18. The modified tobacco or Cannabis plant,
tobacco or Cannabis seed, or part thereof of embodiment 2, where
the endogenous gene comprises a sequence at least 70% identical to
a nucleic acid sequence selected from the group consisting of SEQ
ID NOs: 22-42, 126-139, 156, 159, and 161-175. [0357] 19. The
modified tobacco or Cannabis plant, tobacco or Cannabis seed, or
part thereof of any one of embodiments [0059]-3, where the
polypeptide comprises an amino acid sequence at least 70% identical
or similar to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205.
[0358] 20. The modified tobacco or Cannabis plant, tobacco or
Cannabis seed, or part thereof of any one of embodiments [0059]-3,
where the heterologous promoter comprises a constitutive promoter.
[0359] 21. The modified tobacco or Cannabis plant, tobacco or
Cannabis seed, or part thereof of any one of embodiments [0059]-3,
where the heterologous promoter comprises a tissue-preferred
promoter or tissue-specific promoter. [0360] 22. The modified
tobacco or Cannabis plant, tobacco or Cannabis seed, or part
thereof of embodiment 21, where the tissue-preferred promoter or
tissue-specific promoter comprises a trichome-preferred promoter or
a trichome-specific promoter. [0361] 23. The modified tobacco or
Cannabis plant, tobacco or Cannabis seed, or part thereof of any
one of embodiments [0059]-3, where the heterologous promoter
comprises a nucleic acid sequence at least 90% identical to SEQ ID
NOs: 104-111, 206-208, or a functional fragment thereof. [0362] 24.
The modified tobacco or Cannabis plant, tobacco or Cannabis seed,
or part thereof of embodiment [0059], where the modified tobacco or
Cannabis plant comprises an increased amount of the at least one
terpene as compared to a control tobacco or Cannabis plant lacking
the recombinant nucleic acid molecule when grown under comparable
conditions. [0363] 25. The modified tobacco or Cannabis plant,
tobacco or Cannabis seed, or part thereof of embodiment 2, where
the modified tobacco or Cannabis plant comprises an increased
amount of the at least one terpene as compared to the control
tobacco or Cannabis plant. [0364] 26. The modified tobacco or
Cannabis plant, tobacco or Cannabis seed, or part thereof of
embodiment 24 or 25, where the increased amount of the at least one
terpene comprises an increase of at least 5% as compared to the
control tobacco or Cannabis plant. [0365] 27. The modified tobacco
plant, tobacco seed, or part thereof of any one of embodiments
[0059]-3, where the tobacco plant, tobacco seed, or part thereof is
of a tobacco variety selected from the group consisting of a
flue-cured variety, a bright variety, a Burley variety, a Virginia
variety, a Maryland variety, a dark variety, a Galpao variety, an
Oriental variety, and a Turkish variety. [0366] 28. The modified
tobacco or Cannabis plant, tobacco or Cannabis seed, or part
thereof of any one of embodiments [0059]-3, where the tobacco or
Cannabis plant is male sterile or cytoplasmically male sterile.
[0367] 29. The modified tobacco or Cannabis plant, tobacco or
Cannabis seed, or part thereof, of embodiment 2, where the modified
tobacco or Cannabis plant, tobacco or Cannabis seed, or part
thereof is homozygous for the at least one non-natural mutation.
[0368] 30. The modified tobacco or Cannabis plant, tobacco or
Cannabis seed, or part thereof, of embodiment 2, where the modified
tobacco or Cannabis plant, tobacco or Cannabis seed, or part
thereof is heterozygous for the at least one non-natural mutation.
[0369] 31. The modified tobacco or Cannabis plant, tobacco or
Cannabis seed, or part thereof, of embodiment 2, where the at least
one non-natural mutation comprises a mutation selected from the
group consisting of an insertion, a deletion, a substitution, a
duplication, and an inversion. [0370] 32. The modified tobacco or
Cannabis plant, tobacco or Cannabis seed, or part thereof, of
embodiment 2, where the at least one non-natural mutation comprises
at least one mutation selected from the group consisting of a
nonsense mutation, a missense mutation, a frameshift mutation, a
splice-site mutation, and any combination thereof. [0371] 33. The
modified tobacco or Cannabis plant, tobacco or Cannabis seed, or
part thereof, of embodiment 2, where the at least one non-natural
mutation comprises a null mutation. [0372] 34. The modified tobacco
or Cannabis plant, tobacco or Cannabis seed, or part thereof, of
embodiment 2, where the at least one non-natural mutation results
in a truncation of the polypeptide. [0373] 35. The modified tobacco
or Cannabis plant, tobacco or Cannabis seed, or part thereof, of
embodiment 2, where the at least one non-natural mutation comprises
a mutation in a sequence region selected from the group consisting
of a promoter, a 5'-untranslated region (UTR), an exon, an intron,
a 3' -UTR, and a terminator. [0374] 36. Cured tobacco material or
Cannabis material from the modified tobacco or Cannabis plant,
tobacco or Cannabis seed, or part thereof of any one of embodiments
[0059]-35. [0375] 37. The cured tobacco material of embodiment 36,
where the cured tobacco material is made by a curing process
selected from the group consisting of flue curing, air curing, fire
curing, and sun curing. [0376] 38. A tobacco product comprising the
cured tobacco material or the Cannabis material of embodiment 36 or
37, where the tobacco or Cannabis product is selected from the
group consisting of a kretek, a bidi cigarette, a cigarillo, a
non-ventilated recess filter cigarette, a vented recess filter
cigarette, a cigar, snuff, pipe tobacco, pipe Cannabis, cigar
tobacco, cigar Cannabis, cigarette tobacco, cigarette Cannabis,
chewing tobacco, leaf tobacco, hookah tobacco, hookah Cannabis,
shredded tobacco, shredded Cannabis, cut tobacco, and cut Cannabis.
[0377] 39. The tobacco or Cannabis product of embodiment 38, where
the tobacco or Cannabis product is a smokeless tobacco or Cannabis
product. [0378] 40. The tobacco or Cannabis product of embodiment
39, where the smokeless tobacco or Cannabis product is selected
from the group consisting of loose leaf chewing tobacco, loose leaf
chewing Cannabis, plug chewing tobacco, plug chewing Cannabis,
moist snuff, nasal snuff, dry snuff, and snus. [0379] 41. A
reconstituted tobacco or Cannabis comprising the cured tobacco
material or the Cannabis material of embodiment 36 or 37. [0380]
42. A fermented tobacco or a fermented Cannabis comprising the
cured tobacco material or the Cannabis material of embodiment 36 or
37. [0381] 43. The tobacco or Cannabis product of embodiment 38,
where the tobacco or Cannabis product comprises reconstituted
tobacco or reconstituted Cannabis. [0382] 44. The tobacco or
Cannabis product of embodiment 38, where the tobacco or Cannabis
product comprises fermented tobacco or fermented Cannabis. [0383]
45. A recombinant nucleic acid molecule comprising a nucleic acid
sequence encoding a polypeptide involved in the biosynthesis of at
least one terpene operably linked to a heterologous promoter.
[0384] 46. A method of producing a modified tobacco or Cannabis
plant comprising: (a) introducing a recombinant nucleic acid
molecule to at least one tobacco or Cannabis cell, where the
recombinant nucleic acid molecule comprises a nucleic acid sequence
encoding a polypeptide involved in terpene biosynthesis operably
linked to a heterologous promoter; (b) selecting at least one
tobacco or Cannabis cell comprising the recombinant nucleic acid
molecule; and (c) regenerating a modified tobacco or Cannabis plant
from the at least one tobacco or Cannabis cell selected in step (b)
where the modified tobacco or Cannabis plant comprises an increased
amount of at least one terpene in at least one tissue as compared
to a control tobacco or Cannabis plant lacking the recombinant
nucleic acid molecule when grown under comparable conditions.
[0385] 47. A method of producing a modified tobacco or Cannabis
plant comprising: (a) inducing at least one non-natural mutation in
at least one tobacco or Cannabis cell in an endogenous gene
encoding a polypeptide involved in terpene biosynthesis; (b)
selecting at least one tobacco or Cannabis plant comprising the at
least one non-natural mutation from step (a); and (c) regenerating
a modified tobacco or Cannabis plant from the at least one tobacco
or Cannabis cell selected in step (b), where the modified tobacco
or Cannabis plant comprises an increased amount of at least one
terpene in at least one tissue as compared to a control tobacco or
Cannabis plant lacking the at least one non-natural mutation when
grown under comparable conditions. [0386] 48. A method of producing
a modified tobacco or Cannabis plant comprising: (a) introducing a
recombinant DNA construct to at least one tobacco or Cannabis cell,
where said recombinant DNA construct comprises a heterologous
promoter operably linked to a nucleic acid encoding at least one
small RNA molecule capable of binding to and reducing the
expression of a nucleic acid sequence encoding a polypeptide
involved in the biosynthesis of at least one terpene; (b) selecting
at least one tobacco or Cannabis cell comprising said recombinant
DNA construct; and (c) regenerating at least one modified tobacco
or Cannabis plant from said at least one tobacco or Cannabis cell
selected in step (b), where the modified tobacco or Cannabis plant
comprises a reduced amount of at least one terpene in at least one
tissue as compared to a control tobacco or Cannabis plant lacking
the recombinant DNA construct when grown under comparable
conditions. [0387] 49. A method comprising preparing a tobacco or
Cannabis product using cured tobacco material from a modified
tobacco or Cannabis plant, where the modified tobacco or Cannabis
plant comprises a recombinant nucleic acid molecule comprising a
nucleic acid sequence encoding a polypeptide involved in the
biosynthesis of at least one terpene operably linked to a
heterologous promoter. [0388] 50. A method comprising preparing a
tobacco or Cannabis product using cured tobacco or Cannabis
material from a modified tobacco or Cannabis plant, where the
modified tobacco or Cannabis plant comprises at least one
non-natural mutation in an endogenous gene encoding a polypeptide
involved in the biosynthesis of at least one terpene, where the
modified tobacco or
Cannabis plant exhibits increased mRNA expression of the endogenous
gene or increased accumulation of the polypeptide as compared to a
control tobacco or Cannabis plant lacking the at least one
non-natural mutation when grown under comparable conditions. [0389]
51. A method comprising preparing a tobacco or Cannabis product
using cured tobacco or Cannabis material from a modified tobacco or
Cannabis plant, where the modified tobacco or Cannabis plant
comprises a recombinant DNA construct comprising a heterologous
promoter operably linked to a nucleic acid encoding at least one
small RNA molecule capable of binding to and reducing the
expression of a nucleic acid sequence encoding a polypeptide
involved in the biosynthesis of at least one terpene. [0390] 52. A
method comprising transforming a tobacco or Cannabis cell with a
recombinant nucleic acid molecule, where the recombinant nucleic
acid molecule comprising a nucleic acid sequence encoding a
polypeptide involved in the biosynthesis of at least one terpene
operably linked to a heterologous promoter. [0391] 53. A method
comprising transforming a tobacco or Cannabis cell with a
recombinant nucleic acid molecule, where the recombinant nucleic
acid molecule comprises a heterologous promoter operably linked to
a nucleic acid encoding at least one small RNA molecule capable of
binding to and reducing the expression of a nucleic acid sequence
encoding a polypeptide involved in the biosynthesis of at least one
terpene. [0392] 54. A method for producing a tobacco or Cannabis
plant, the method comprising: (a) crossing at least one tobacco or
Cannabis plant of a first tobacco variety with at least one tobacco
or Cannabis plant of a second tobacco or Cannabis variety to
produce at least one progeny tobacco or Cannabis seed, where the at
least one tobacco or Cannabis plant of the first tobacco or
Cannabis variety comprises a recombinant nucleic acid molecule
comprising a nucleic acid sequence encoding a polypeptide involved
in the biosynthesis of at least one terpene operably linked to a
heterologous promoter; and (b) selecting for at least one progeny
tobacco or Cannabis seed, or a plant germinated therefrom, where
the at least one progeny tobacco or Cannabis seed or plant
germinated therefrom comprises the recombinant nucleic acid
molecule. [0393] 55. A method for producing a tobacco or Cannabis
plant, the method comprising: (a) crossing at least one tobacco or
Cannabis plant of a first tobacco or Cannabis variety with at least
one tobacco plant of a second tobacco or Cannabis variety to
produce at least one progeny tobacco or Cannabis seed, wherein the
at least one tobacco or Cannabis plant of the first tobacco or
Cannabis variety comprises a recombinant nucleic acid molecule
comprising a heterologous promoter operably linked to a nucleic
acid encoding at least one small RNA molecule capable of binding to
and reducing the expression of a nucleic acid sequence encoding a
polypeptide involved in the biosynthesis of at least one terpene;
and (b) selecting for at least one progeny tobacco seed, or a plant
germinated therefrom, wherein the at least one progeny tobacco or
Cannabis seed or plant germinated therefrom comprises the
recombinant nucleic acid molecule. [0394] 56. A method for
producing a tobacco or Cannabis plant, the method comprising: (a)
crossing at least one tobacco or Cannabis plant of a first tobacco
variety with at least one tobacco or Cannabis plant of a second
tobacco or Cannabis variety to produce at least one progeny tobacco
or Cannabis seed, where the at least one tobacco or Cannabis plant
of the first tobacco variety comprises at least one non-natural
mutation in an endogenous gene encoding a polypeptide involved in
the biosynthesis of at least one terpene, where the at least one
tobacco or Cannabis plant of the first tobacco or Cannabis variety
exhibits increased mRNA expression of the endogenous gene or
increased accumulation of the polypeptide as compared to a control
tobacco or Cannabis plant of the first tobacco or Cannabis variety
lacking the at least one non-natural mutation when grown under
comparable conditions; and (b) selecting for at least one progeny
tobacco or Cannabis seed, or a plant germinated therefrom, where
the at least one progeny tobacco or Cannabis seed or plant
germinated therefrom comprises the at least one non-natural
mutation. [0395] 57. The method of any one of embodiments 54-56,
where the first tobacco variety and the second tobacco variety are
the same tobacco variety, or the first Cannabis variety and the
second Cannabis variety are the same Cannabis variety. [0396] 58.
The method of any one of embodiments 54-55, where the at least one
tobacco or Cannabis plant of a second tobacco or Cannabis variety
comprises the recombinant nucleic acid molecule. [0397] 59. The
method of any one of embodiments 54-55, where the at least one
progeny tobacco or Cannabis seed, or the plant germinated
therefrom, is heterozygous for the recombinant nucleic acid
molecule. [0398] 60. The method of any one of embodiments 54-55,
where the at least one progeny tobacco or Cannabis seed, or the
plant germinated therefrom, is homozygous for the recombinant
nucleic acid molecule. [0399] 61. The method of embodiment 56,
where the at least one tobacco or Cannabis plant of a second
tobacco or Cannabis variety comprises the at least one non-natural
mutation. [0400] 62. The method of embodiment 56, where the at
least one progeny tobacco or Cannabis seed, or the plant germinated
therefrom, is heterozygous for the at least one non-natural
mutation. [0401] 63. The method of embodiment 56, where the at
least one progeny tobacco or Cannabis seed, or the plant germinated
therefrom, is homozygous for the at least one non-natural
mutation.
[0402] Having now generally described the disclosure, the same will
be more readily understood through reference to the following
examples that are provided by way of illustration, and are not
intended to be limiting of the present disclosure, unless
specified.
EXAMPLES
Example 1. Identification of Candidate Trichome/Terpenoid Genes
[0403] Proteins that carry out the biosynthetic steps for producing
for several terpenes (see FIGS. 1-6) are largely unknown. Published
genes involved in terpene biosynthesis were used as a query in a
National Center for Biotechnology Information (NCBI) database.
Resulting sequences were then extracted from a second, internal
database. This methodology produced gene candidates and homologs
putatively involved in terpene biosynthesis (SEQ ID NOs: 1-63,
112-153, and 155-205. See Table 1.
Example 2. Examination of Native Expression of Candidate Genes
[0404] Expression profiles of the candidate genes identified in
Example 1 are examined in tobacco trichomes. See FIG. 7. Trichomes
are isolated from mature leaves of tobacco cultivar `TN90` by
brushing a leaf with a brush dipped in liquid nitrogen. Trichomes
are collected into a receptacle containing liquid nitrogen for RNA
extraction. Total RNA is isolated using TRIzol.TM., and cDNA is
synthesized using methods known in the art. PCR is performed using
gene specific primers. Candidate genes for evaluation include
cyclase (SEQ ID NO: 17); phylloplanin (SEQ ID NO: 155);
premnaspirodiene oxygenase (PSO) (SEQ ID NO: 158); neomenthol
dehydrogenase (SEQ ID NO: 15); and cis-abienol synthase (SEQ ID NO:
4). CYP71D16 is a tobacco trichome-specific P450 hydroxylase gene,
used as a positive control for trichome gene expression. CYP71D16
shares approximately 98.5% homology with PSO.
Example 3. Examination of NMD and ABS Expression Profiles
[0405] Expression profiles of the candidate genes identified in
Example 1 are examined in flowers and leaves from tobacco cultivars
TN90 and K326 and N. benthamiana at various stages of tobacco plant
development.
[0406] Neomenthol dehydrogenase (NMD; SEQ ID NO: 15) and
cis-abienol (ABS) isoforms 1 (SEQ ID NO: 4) and 2 (SEQ ID NO: 5)
transcript abundance is performed using RNA sequencing at different
stages of plant development in TN90 and K326 tobacco cultivars.
[0407] Approximately 22 to 25 plants per variety are grown in two
rows. The experiment is triplicated with randomized design. Because
flowering time varies from plant to plant, all plants are topped
when at least 50% of the plants start to flower. Plants close to
uniform sizes are selected for sampling at different time points.
Assessed plant samples include flower (before topping) and leaf at
a variety of post topping stages (e.g., 1 day post-topping; 3 days
post-topping; 1 week post-topping; 2 weeks post-topping; 3 weeks
post-topping; and harvest stage). See FIGS. 8 and 9.
Example 4. Vector Constructs for Generating Modified Tobacco or
Cannabis Plants
[0408] Candidate genes identified in Example 1 encoding enzymes in
the cis-abienol biosynthetic pathway (e.g., geranylgeranyl
diphosphate synthase (GGPPS2) (SEQ ID NOs: 1 and 113),
8-hydroxy-copalyl diphosphate synthase (SEQ ID NOs: 3 and 115),
cis-abienol synthase isoform 1 (SEQ ID NO:4), and cis-abienol
synthase isoform 2 (SEQ ID NO: 5)) are cloned into constructs.
[0409] Separate transformation vectors comprising one each of SEQ
ID NOs: 1-21, 112-125, 155, 158, and 176-190 under the control of a
CaMV 35S promoter are constructed using methods known in the art
and vector pGWB551 (SEQ ID NO: 154). A representative vector
construct used to transform tobacco or Cannabis plants is depicted
in FIG. 10. A map of the vector backbone used to make the vector
constructs is depicted in FIG. 11.
[0410] Each vector construct generated is separately transformed
into tobacco or Cannabis cells in separate experiments, using
methods standard in the art. Briefly, vectors are introduced into
tobacco or Cannabis leaf discs via Agrobacterium-mediated
transformation. See, for example, Mayo et al., Nat. Protoc.,
1:1105-1111 (2006); and Horsch et al., Science, 227:1229-1231
(1985).
[0411] Representative images of calli from control and transgenic
tobacco lines expressing GFP in different backgrounds (Nb=N.
benthamiana; TN90=tobacco cultivar `TN90`) are provided in FIGS.
12-18. FIG. 12 depicts geranylgeranyl diphosphate synthase-TN90
(SEQ ID NOs: 1 and 43); FIG. 13 depicts geranylgeranyl diphosphate
synthase-Nb (SEQ ID NOs: 1 and 43); FIG. 14 depicts cembratrienol
synthase 2a-Nb (SEQ ID NOs: 17 and 59); FIG. 15 depicts neomenthol
dehydrogenase-Nb (SEQ ID NOs: 15 and 57); FIG. 16 depicts
cis-abienol synthase-TN90 (SEQ ID NOs: 4 and 46); FIG. 17 depicts
menthofuran synthase-Nb (SEQ ID NOs: 16 and 58); and FIG. 18
depicts GFP-TN90, a positive control.
Example 5. Regeneration of Modified Tobacco Plants
[0412] Tobacco genotypes (TN90 and K326) and N. benthamiana plants
are transformed with vector constructs as described in Example 4
are grown in Magenta.TM. GA-7 boxes and leaf discs are cut and
placed into Petri plates. Agrobacterium tumefaciens cells
comprising a transformation vector are collected by centrifuging a
20 mL cell suspension in a 50 mL centrifuge tube at 3500 RPM for 10
minutes. The supernatant is removed, and the Agrobacterium
tumefaciens cell pellet is re-suspended in 40 mL liquid
re-suspension medium. Tobacco leaves, avoiding the midrib, are cut
into eight 0.6 cm discs with a #15 razor blade and placed upside
down in a Petri plate. A thin layer of Murashige & Skoog (MS)
with B5 vitamin liquid re-suspension medium is added to the Petri
plate and the leaf discs are poked uniformly with a fine point
needle. About 25 mL of the Agrobacterium tumefaciens suspension is
added to the Petri plate and the leaf discs are incubated in the
suspension for 10 minutes.
[0413] Leaf discs are transferred to co-cultivation Petri plates
(1/2 MS medium) and discs are placed upside down in contact with
filter paper overlaid on the co-cultivation TOM medium (MS medium
with 30 g/L sucrose; 0.1 mg/L indole-3-acetic acid; and 1 mg/L
6-benzyl aminopurine (BAP)). The Petri plate is sealed with
parafilm prior to incubation in dim light (between 60 and 80 mE/ms)
with photoperiods of 18 hours light, 6 hours dark, at 24.degree. C.
for two days.
[0414] After incubation, leaf discs are transferred to
regeneration/selection TOM Hyg medium Petri plates (TOM medium plus
200 mg/L cefotaxime and 50 mg/L hygromycin). Leaf discs are
sub-cultured bi-weekly to fresh TOM K medium in dim light (between
60 and 80 mE/ms) with photoperiods of 18 hours light, 6 hours dark,
at 24.degree. C. until shoots become excisable. Shoots from leaves
are removed with forceps and inserted in MS rooting medium (MS
Gamborg B5, 3 g/L sucrose and 7 g/L dextrose) with 50 mg/L
hygromycin and 200 mg/L cefotaxime. Shoots on MS rooting medium
with 50 mg/L hygromycin and 200 mg/L cefotaxime are incubated under
light (approximately 60-80 mE/ms) with photoperiods of 18 hours
light, 6 hours dark, at 24.degree. C. to induce rooting.
[0415] When plantlets comprising both shoots and roots grow large
enough (e.g., over half the height of a Magenta.TM. GA-7 box), they
are transferred to Jiffy peat pellet for acclimatization in the
growth room. Once established, seedlings are transferred to a
greenhouse for further growth, breeding, and analysis.
Example 6. Confirming Expression of Terpene Biosynthesis Genes in
Modified Tobacco Plants
[0416] RNA is isolated from young leaves of modified tobacco plants
(To generation) generated in Example 5 during the vegetative stage
of growth. RNA is also isolated from control tobacco plants lacking
the recombinant nucleic acid constructs grown under comparable
conditions. Total RNA is isolated using TRIzol.TM., and cDNA is
synthesized using methods known in the art. Terpenoid gene
expression in different transgenic lines of each gene construct is
quantified via quantitative real-time PCR (qRT-PCR). Primers
specific for candidate genes are provided in Table 1 (SEQ ID NOs:
64-103).
[0417] Gene expression of NtNMD (SEQ ID NO: 15), NtaABS (SEQ ID NO:
4), and NtABS (SEQ ID NO: 5) in modified tobacco plants is
quantified using qRT-PCR and compared to NtNMD, NtaABS, and NtABS
expression in control tobacco plants. Relative gene expression of
cis-abienol synthase isoform 1 (aABS) (SEQ ID NO: 4) and
cis-abienol synthase isoform 2 (ABS) (SEQ ID NO: 5) are calculated
for T.sub.0 lines in transgenic tobacco plants. See FIGS. 19-20.
Relative gene expression of NtNMD (SEQ ID NO: 15) is calculated for
T.sub.0 lines in transgenic tobacco plants. See FIG. 21. Relative
expression of NtNMD (see FIG. 24) and NtaABS (see FIG. 25) are also
examined in T.sub.1 transgenic tobacco plants. Relative expression
is determined using 2.sup.(-.DELTA..DELTA.c.sup.(t).sup.)
methodology that is standard in the art, where native expression of
the overexpressed gene in wildtype plants are used as a baseline
(e.g., set to equal 1). See, for example, Livak and Schmittgen,
Methods, 25:402-408 (2001).
Example 7. Measuring Terpenoids in Modified Tobacco Plants
Overexpressing Candidate Genes
[0418] Quantitative metabolic profile analysis is performed using
T.sub.0 transgenic plants generated in Example 5 during the
vegetative growth stage. Leaf samples from young or old leaves,
separately, are ground in liquid nitrogen for solvent extraction of
terpenes. Ground leaves are mixed with 60:40 hexane: ethyl acetate
solvent mixture (v/v) supplemented with heptadecanol as an internal
standard and then incubated overnight in a shaker. The solvent
extract is concentrated in a refrigerated SpeedVac and placed into
a silica column. The column is washed with hexane and allowed to
flow through collection tubes. Samples are aliquoted from the flow
through and used for gas chromatography mass spectrometry (GC-MS)
metabolite analysis.
[0419] Elevated levels of neophytadiene are observed in transgenic
tobacco plants overexpressing NtNMD (SEQ TD NO: 15). See FIG. 22.
Elevated levels of neophytadiene are also observed in transgenic
tobacco plants overexpressing NtABS (SEQ ID NO: 5) and NtaABS (SEQ
ID NO: 4). See FIG. 23.
[0420] Neomenthol, as detected by GC-MS, hyperaccumulates in
transgenic tobacco plants overexpressing NtNMD (SEQ TD NO: 15),
compared to TN9 control plants. See Table 9.
TABLE-US-00009 TABLE 9 Accumulation of volatile aromas in NtNMD
transgenic tobacco plants Plant Ret.Time sample (minutes) Height
Name Description TN90 old 19.778 175137 Phytol acyclic diterpene
leaf alcohol 21.697 7959615 Thunbergol diterpene alcohol 27.779
192001 Squalene linear triterpene 30.686 1459330 Campesterol
phytosterols 30.882 3237771 Stigmasterol phytosterols TN90 21.696
2353386 Thunbergol diterpene alcohol young 30.685 515667
Campesterol phytosterols leaf 30.88 840605 Stigmasterol
phytosterols NtNMD 8.72 18116 D-Limonene cyclic monoterpene old
leaf 21.696 796720 Thunbergol diterpene alcohol 22.035 2387665
Phytol acyclic diterpene alcohol 30.685 1930116 Campesterol
phytosterols 30.882 3611696 Stigmasterol phytosterols 31.248
1538874 gamma-Sitosterol phytosterols 31.59 777680 beta-Amyrin
pentacyclic triterpenoid NtNMD 8.723 20209 D-Limonene cyclic
monoterpene young 22.224 310287 Neomenthol monoterpene leaf 22.53
539430 Caryophyllene bicyclic oxide sesquiterpene 22.607 651236
Phytol acyclic diterpene alcohol 30.688 2600761 Campesterol
phytosterols 30.884 4746148 Stigmasterol phytosterols 31.252
2730652 gamma-Sitosterol phytosterols 31.598 963961 alpha-Amyrin
pentacyclic triterpenoid
[0421] Enhanced volatile flavor compounds, as detected by GC-MS,
accumulate in transgenic tobacco plants overexpressing NtABS (SEQ
TD NO: 5) and NtaABS (SEQ TD NO: 4). See Table 10.
TABLE-US-00010 TABLE 10 Accumulation of volatile aromas in NtABS
and NtaABS transgenic tobacco plants Plant Ret.Time sample
(minutes) Height Name Description NtABS 8.781 4120018 Benzyl
alcohol aromatic alcohol young 10.107 1087817 Phenethyl alcohol
aromatic alcohol leaf 13.932 234315 Phytol acyclic diterpene
alcohol 19.785 1028557 Phytol acyclic diterpene alcohol 19.356
37060661 Neophytadiene sesquiterpenoids 21.709 2324963 Thunbergol
diterpene alcohol 23.862 2587845 Thunbergol diterpene alcohol
23.964 2979432 Thunbergol diterpene alcohol 27.788 2904632 Squalene
linear precursor to all triterpenes 28.394 2171239 Geranylgeraniol
diterpenoid 30.699 9012028 Campesterol phytosterols 30.899 18029891
Stigmasterol phytosterols NtABS 8.782 2608699 Benzyl alcohol
aromatic alcohol young 10.11 565512 Phenethyl alcohol aromatic
alcohol leaf 13.932 34195 Phytol acyclic diterpene alcohol 19.785
845134 Phytol acyclic diterpene alcohol 19.354 34282335
Neophytadiene sesquiterpenoids 27.787 2492163 Squalene linear
precursor to all triterpenes 30.698 7713903 Campesterol
phytosterols 30.898 15728977 Stigmasterol phytosterols NtaABS
21.701 784815 Thunbergol diterpene alcohol old leaf 22.041 1590365
Phytol acyclic diterpene alcohol 30.695 8699080 Campesterol
phytosterols 30.896 19240212 Stigmasterol phytosterols 10.108
288805 Phenethyl alcohol aromatic alcohol NtaABS 20.52 2179848
Squalane product of squalene young hydrogenisation leaf 21.708
3797521 Thunbergol diterpene alcohol 22.042 4400153 Phytol acyclic
diterpene alcohol product of squalene 24.24 2297502 Squalane
hydrogenisation 30.694 8477865 Campesterol phytosterols 30.894
16841470 Stigmasterol phytosterols 31.26 9109415 gamma-Sitosterol
phytosterols
Example 8. Generating Mutations in Candidate Genes
[0422] Mutations are produced in each of the genes identified in
Example 1 by specifically editing SEQ ID NOs: 1-42 112-139,
155-156, 158-159, and 161-190, separately, in the tobacco or
Cannabis genomes. Tobacco or Cannabis protoplasts are transfected
using polyethylene glycol (PEG) with plasmids encoding a CRISPR
protein or a CRISPR protein and specific guide RNA (gRNA) targeting
individual genes at desired positions.
[0423] Transfected protoplasts are then immobilized in 1% agarose
beads and subjected to tissue culture. When calli grow to
approximately 1 millimeter in diameter, they are spread on TOM2
plates. Calli are screened for mutations (e.g., insertions or
deletions (indels)) at the target positions using fragment
analysis. Candidates, showing size shifts compared to wildtype
control, are selected for further culture and the consequent shoots
are tested by fragment analysis again to confirm the presence of
mutations.
[0424] Modified tobacco or Cannabis plants (To generation) are
grown as described in Example 5. Then, terpenoid levels are
measured as described in Example 7.
Example 9. Knockdown of Candidate Genes Using Small RNA Molecules
in Tobacco and Cannabis
[0425] Reducing the expression of genes identified in Example 1 is
tested for their effect on the levels of specific terpenoids.
[0426] Separate transformation vectors comprising an artificial
miRNA designed to reduce the transcription or translation of one
each of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190
driven by CaMV 35S are constructed.
[0427] Modified tobacco plants (To generation) are grown as
described in Example 5. Then, terpenoid levels are measured as
described in Example 7.
Example 10. Expressing Terpenoid Biosynthesis Genes with
Trichome-Specific Promoters
[0428] Trichome-specific promoters are used to drive the expression
of genes involved in terpenoid biosynthesis. Constructs are
produced as described in Example 4, with each trichome-specific
promoter (e.g., SEQ ID NOs: 104-111 and 206-208, see Table 1)
driving the expression of neomenthol dehydrogenase (NtNMD; SEQ ID
NO: 15) or one of two isoforms of cis-abienol synthase
(NtaABS(Isoform 1; SEQ ID NO: 4) and NtABS(Isoform 2; SEQ ID NO: 5)
or a homolog thereof, for example, cis-abienol synthase (e.g. SEQ
ID NO: 119), in separate constructs.
[0429] Each of the constructs are separately transformed into
tobacco or Cannabis cells and modified tobacco or Cannabis plants
are regenerated as described in Example 5. Modified tobacco or
Cannabis plants are tested for terpenes as provided in Example 7.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220243215A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220243215A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References