U.S. patent application number 15/975912 was filed with the patent office on 2018-11-15 for guayule with increased rubber production and yield.
The applicant listed for this patent is Bridgestone Corporation, The United States of America, as represented by the Secretary of Agriculture, The United States of America, as represented by the Secretary of Agriculture. Invention is credited to Von Mark Cruz, David Dierig, Colleen M. McMahan, Dante Placido.
Application Number | 20180327770 15/975912 |
Document ID | / |
Family ID | 64096419 |
Filed Date | 2018-11-15 |
United States Patent
Application |
20180327770 |
Kind Code |
A1 |
McMahan; Colleen M. ; et
al. |
November 15, 2018 |
GUAYULE WITH INCREASED RUBBER PRODUCTION AND YIELD
Abstract
A reduction in the amount of functional PaAos in guayule results
in the production of increased amounts rubber compared to the
amount of rubber produced by wild-type guayule having a non-reduced
amount of functional PaAos. Further, the guayule with reduced
amount of functional PaAos are larger than wild-type guayule and
thus have larger rubber yield per acre than wild-type guayule.
Reduction of the amount of functional PaAos in guayule can be
caused by genetic alterations in PaAos. Guayule having PaAos with a
specific amino acid sequence produces more rubber than guayule with
PaAos having a different amino acid sequence. Thus, one can use the
sequence differences as a biomarker for selecting high rubber
producing guayule plants.
Inventors: |
McMahan; Colleen M.;
(SAUSALITO, CA) ; Placido; Dante; (OAKLAND,
CA) ; Dierig; David; (PHOENIX, AZ) ; Cruz; Von
Mark; (CASA GRANDE, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary of
Agriculture
Bridgestone Corporation |
Washington
Tokyo |
DC |
US
JP |
|
|
Family ID: |
64096419 |
Appl. No.: |
15/975912 |
Filed: |
May 10, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62504762 |
May 11, 2017 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/8261 20130101;
C08L 7/02 20130101; C12N 15/8218 20130101; C12Y 402/01092 20130101;
C07K 14/415 20130101; C12N 2310/11 20130101; C12N 2310/14 20130101;
C12N 15/1137 20130101; C12N 9/88 20130101; C12N 15/8243 20130101;
C12N 15/8251 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Claims
1. An altered guayule, parts and progeny thereof, that produces
more rubber than amount of rubber produced by a non-altered guayule
comprising a mutation in PaAos; wherein said mutation is selected
from the group consisting of (i) an alteration in a PaAos codon
encoding an amino acid to a stop codon, (ii) an alteration of
PaAos' translation initiation codon to another codon, (iii) an
alteration in PaAos ribosome binding site's sequence, (iv) an
alteration in one or more PaAos splice site codons, (v) a deletion
of part or all of said PaAos' sequence, (vi) an insertion of DNA
into PaAos, and (vii) an alteration in one or more PaAos DNA codon
sequences to encode a non-conservative amino acid; wherein said
mutation reduces said altered PaAos' functionality compared to
amount of PaAos functionality in said non-altered guayule; wherein
said reduced PaAos functionality causes said altered guayule to
produce an increased amount of rubber compared to said amount of
rubber produced by said non-altered guayule.
2. The altered guayule of claim 1; wherein said alteration in one
or more PaAos DNA codon sequences to encode a non-conservative
amino acid occurs at amino acids located at 318, 332, 336, 339,
359, 408, 411, and 459 with said PaAos sequence.
3. The altered guayule of claim 2; wherein said non-conservative
amino acid substitution excludes at least one of N318, V408 and
W459.
4. An altered cell of said altered guayule of claim 1, wherein said
altered cell comprises said mutation in PaAos.
5. An altered germplasm of said altered guayule of claim 1, wherein
said altered germplasm comprises said mutation in PaAos.
6. An altered seed of said altered guayule of claim 1, wherein said
altered seed comprises said mutation in PaAos.
7. A method for producing an altered guayule that comprises a
mutated PaAos and produces an increased amount of rubber compared
to amount of rubber produced by a non-altered guayule, said method
comprising exposing a non-altered guayule cell or seed to a mutagen
to produce a mutated guayule cell or seed with said mutated PaAos;
selecting said mutated guayule cell or seed comprising said mutated
PaAos, wherein said mutated PaAos encodes an altered PaAos having
reduced functionality compared to a non-altered PaAos'
functionality; and growing said selected mutated guayule cell or
seed comprising said mutated PaAos to produce an altered guayule
that produces said altered PaAos with reduced functionality and
said increased amount of rubber compared to said amount of rubber
produced by said non-altered guayule.
8. The method of claim 7, wherein said mutated PaAos comprises at
least one of (i) an alteration in a PaAos codon encoding an amino
acid to a stop codon, (ii) an alteration of PaAos' translation
initiation codon to another codon, (iii) an alteration in PaAos
ribosome binding site's sequence, (iv) an alteration in one or more
PaAos splice site codons, (v) a deletion of part or all of said
PaAos' sequence, (vi) an insertion of DNA into PaAos, and (vii) an
alteration in one or more PaAos DNA codon sequences to encode a
non-conservative amino acid.
9. The method of claim 8, wherein said alteration in one or more
PaAos DNA codon sequences to encode a non-conservative amino acid
occurs at amino acids located at 318, 332, 336, 339, 359, 408, 411,
and 459 with said PaAos sequence.
10. An altered cell of said altered guayule produced according to
said method of claim 7.
11. An altered germplasm of said altered guayule produced according
to said method of claim 7.
12. An altered seed of said altered guayule produced according to
said method of claim 7, wherein said altered seed comprises said
mutated PaAos.
13. A method of producing a population of high rubber producing
guayule plants or seeds comprising PaAos with low functionality,
said method comprising: genotyping a first population of guayule
plants or seeds, said first population of said guayule plants or
seeds comprising said PaAos with low functionality; selecting from
said first population one or more guayule plants or seeds
comprising said PaAos with low functionality based said genotyping;
and producing from said selected one or more guayule plants or
seeds comprising said PaAos with low functionality a second
population of guayule plants or seeds comprising said PaAos with
low functionality.
14. The method of claim 13, wherein said PaAos with low
functionality comprises at least one amino acid selected from group
of N318, V408, W459, conservative amino acids substitutions
thereof, and non-conservative amino acid substitutions at S332,
E336, R339, S359, and S411.
15. A method of identifying a high rubber producing guayule
comprising detecting PaAos with low functionality in a test
guayule, wherein when said test guayule contains PaAos with amino
acids N318, V408 and W459, then said test guayule is a high rubber
producing guayule.
16. The method of claim 15, wherein said detecting step comprises
contacting said PaAos from said test guayule with monoclonal
antibodies that bind to PaAos with amino acids N318, V408 and W459;
and determining if said monoclonal antibodies binds to said test
guayule PaAos.
17. The method of claim 15, wherein said detecting step comprises
obtaining nucleic acids from said test guayule; performing a PCR
assay with said obtained nucleic acids, primer sets having SEQ ID
NOs: 22 and 23 and SEQ ID NOs: 24 and 25, and a label; determining
if an amplicon is generated, wherein when said amplicon is
produced, then said test guayule contains a PaAos that encodes a
PaAos having amino acids N318, V408 and W459, and said test guayule
is a high rubber producing guayule.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This patent application claims priority to U.S. Patent
Application 62/504,762 filed on May 11, 2017, contents of which are
expressly incorporated by reference herein.
BACKGROUND OF THE INVENTION
Sequence Listing
[0002] The Sequence Listing submitted via EFS-Web as ASCII
compliant text file format (.txt) filed on May 10, 2018, named
"SequenceListing_ST25", (created on May 9, 2018, 24 KB), is
incorporated herein by reference. This Sequence Listing serves as
paper copy of the Sequence Listing required by 37 C.F.R. .sctn.
1.821(c) and the Sequence Listing in computer-readable form (CRF)
required by 37 C.F.R. .sctn. 1.821(e). A statement under 37 C.F.R.
.sctn. 1.821(f) is not necessary.
Field of the Invention
[0003] This invention relates to altered guayule plants that grow
larger and produce more rubber than non-altered guayule plants,
when grown under the same conditions. The altered guayule contain
the cDNA sequence of Parthenium argentatum Allene oxide synthase
(PaAos) in the reverse complement orientation under control of a
heterologous promoter which reduces the production of PaAos via
RNAi. Other types of genetic alternations can be made in guayule to
reduce the functionality of PaAos. Kits for identifying such
altered guayule, methods for identify the altered guayule, and
methods of increasing rubber yield in guayule via reducing PaAos
translation are also included.
Description of Related Art
[0004] Natural rubber is synthesized by more than 2,500 plant
species (Cornish, et al., J. Nat. Rubber Research 8:275-285 (1993);
Cornish, K., Phytochemistry 57:1123-1134 (2001)). Rubber is
produced by these plants as a secondary metabolite with no clear
indication of its function in plant cells. Possible reasons on why
these species synthesize rubber are to defend themselves against
pathogens and insect attacks, repair tissue damages caused by
mechanical wounding and protect cell damage induced by
environmental stresses (Demel, et al., Biochim. Biophys. Acta.
1375:36-42 (1998); Tangpakdee and Tanaka, J. Rubber Res. 1:14
(1998); Vereyken, et al., Biochim. Biophys. Acta, 1510:307-320
(2001); Kim, et al., Plant Cell Physiol., 412-414 (2003) and
references therein; Konno, K., Phytochemistry, 1510-1530 (2011);
and Sarkar, J., Rubber Science, 228-237 (2013)). According to a
2014 market report, the rubber that these plants produce accounted
for $16.5 billion in trade worldwide
(rubberworld.com/RWmarket_report.asp). Even more so, the end
products made from natural rubber, including tires for the
transportation industry, sports equipment, medical devices, and
more, are indispensable in our everyday life. The Hevea tree is the
main source of natural rubber but concerns exist as it is limited
geographically to tropical climates, mainly in Southeast Asia, is
susceptible to diseases, and produces rubber that causes allergic
reactions. Clearly, an alternative source for the production of
natural rubber is very important to reduce economic risk and
safeguard human health.
[0005] One plant known to be a promising source of natural rubber
is guayule (Parthenium argentatum, Gray), a desert shrub native to
the southwestern United States and northern Mexico (Mooibroek and
Cornish, Appl. Microbio. and Biochem. 53:355-365 (2000); van Beilen
and Poirier, Critical Reviews Biotech. 27:217-231 (2007)). The
majority of rubber synthesis in guayule occurs during the cold
season. Guayule synthesizes rubber within subcellular organelles
called rubber particles (Archer and Audley, Bot. J. Linnean Soc.
94:181-196 (1987)) stored in the parenchyma cells of stembark
tissues (Gilliland, M. v., Protoplasma, 169-177 (1984)); Macrae, S.
G., Plant Physiol., 1027-1032 (1986)). Natural rubber synthesis is
initiated by the action of allylic pyrophosphates initiators
(Cornish and Siler, J. Plant Physiol., 301-305 (1995)), usually
farnesyl pyrophosphate (FPP). Then, the monomer
isopentenyl-pyrophosphate (IPP), produced by the mevalonic acid
pathway (MEV) in the cytosol and the methylerythritol phosphate
(MEP) pathway in the plastid (Mooibroek and Cornish (2000); van
Beilen and Poirier, TRENDS in Biotech., 522-529 (2007)) elongates
the rubber chain. Rubber synthesis is mediated by rubber
transferases requiring magnesium ions as cofactor (Da Costa, et
al., Phytochemistry 67(15): 1621-1628 (2006)).
[0006] The proposed model for the structure of rubber particles
consists mostly of hydrophobic cis-polyisoprene units (natural
rubber) encapsulated inside a protein and phospholipid surface
monolayer (Nawamawat, et al., Colloids and Surfaces A:
Physicochemical and Engineering Aspects, 390:157-166 (2011);
Sansatsadeekul, et al., J. Biosci. and Bioeng, 111:628-634 (2011)).
The phospholipids serve to stabilize and solubilize the otherwise
insoluble (rubber) product. Guayule rubber particles include
several proteins (Whalen, et al., Development of crops to produce
industrially useful natural rubber. Chapter 23 in Isoprenoid
Synthesis in Plants and Microorganisms: New Concepts and
Experimental Approaches, Bach and Rohmer (eds.), DOI
10.1007/978-1-4614-4063-5_23, Springer Science+Business Media NY,
329-345 (2013)) of which Aos has been found to be the most abundant
(Backhaus, et al., Phytochemistry 30:2493-2497 (1991)). Aos is
well-known as an enzyme in the jasmonic acid biosynthetic pathway
(Harms, et al., Plant Cell, 1645-1654 (1995); Wang, et al., Plant
Mol. Biol., 783-793 (1999); Schaller, F., J. Exper. Botany, 11-23
(2001)). The role of Aos in rubber biosynthesis, and the reason for
the abundance of Aos protein on guayule rubber particle surfaces,
is not known (Whalen, et al. (2013)).
[0007] The need exists for a method to increase rubber production
in altered guayule compared to rubber production amounts in
non-altered guayule in order to improve the commercial
attractiveness of using guayule rubber as a replacement of
synthetic rubber and Hevea rubber. Further, a need exists for
increasing the rubber yield per acre obtained from altered guayule
compared to the rubber yield per acre obtained from non-altered
guayule. This greater rubber yield results from the altered guayule
being larger in size than non-altered guayule of similar age. A
need also exists for altered guayule that produce more rubber than
the amount of rubber produced by non-altered guayule. A need also
exists for altered guayule that have a larger size than similarly
aged non-altered guayule because the altered guayule that are
larger than the non-altered guayule will possess more tissue for
storage of rubber and thus generate greater rubber yield per acre
than the rubber yield per acre of wild-type guayule. And a need
exists for biomarkers which distinguish between low rubber
producing and high rubber producing guayules.
BRIEF SUMMARY OF THE INVENTION
[0008] It is an object of this invention to have altered guayule,
parts of altered guayule, and progeny of the altered guayule, the
altered guayule containing a mutation that causes the altered
guayule to produce more rubber than the amount of rubber produced
by a non-altered guayule. It is a further object of this invention
that the mutation in the altered guayule can be one or more of (i)
an alteration in a Parthenium argentatum Allene oxide synthase
(PaAos) codon encoding an amino acid to a stop codon, (ii) an
alteration of PaAos' translation initiation codon to another codon,
(iii) an alteration in PaAos ribosome binding site's sequence, (iv)
an alteration in one or more PaAos splice site codons, (v) a
deletion of part or all of PaAos' sequence, (vi) an insertion of
DNA into PaAos, and (vii) an alteration in one or more PaAos DNA
codon sequences to encode a non-conservative amino acid. It is an
object of this invention that each of these mutations reduces the
altered PaAos' functionality compared to the amount of PaAos
functionality in a non-altered guayule and that the reduced PaAos
functionality causes the altered guayule to produce an increased
amount of rubber compared to the amount of rubber produced by the
non-altered guayule. It is another object of this invention that
the alteration of one or more PaAos DNA codon sequences to encode a
non-conservative amino acid occurs at amino acids located at 318,
332, 336, 339, 359, 408, 411, and 459 with PaAos' sequence (see SEQ
ID NO: 10, 13, and 15). It is a further object of this invention
that the amino acids being changed to non-conservative amino acids
are D318, S332, E336, R339, S359, I408, S411, and/or L459. Another
object of this invention is that the non-conservative amino acid
substitutions are not N318, V408 and/or W459. It is another object
of this invention to have an altered cell, germplasm, and an
altered seed of the altered guayule, each containing the
mutation.
[0009] It is an object of this invention to have a method of
producing an altered guayule that contains a mutated PaAos and
produces more rubber compared to the amount of rubber produced by a
non-altered guayule. It is another object of the invention that the
method involves exposing a non-altered guayule cell or seed to a
mutagen to produce a mutated guayule cell or seed with the mutated
PaAos, selecting one or more of the mutated guayule cells or seeds
containing the mutated PaAos which encodes an altered PaAos with
reduced functionality compared to a non-altered PaAos's
functionality, and growing the selected mutated guayule cell or
seed containing the mutated PaAos to produce an altered guayule
that produces the altered PaAos with reduced functionality and an
increased amount of rubber compared to the amount of rubber
produced by the non-altered guayule. It is another object of this
invention that the mutated PaAos contains at least one of (i) an
alteration of a PaAos codon encoding an amino acid to a stop codon,
(ii) an alteration of PaAos' translation initiation codon to
another codon, (iii) an alteration of PaAos ribosome binding site's
sequence, (iv) an alteration of one or more PaAos splice site
codons, (v) a deletion of part or all of PaAos' sequence, (vi) an
insertion of DNA into PaAos, and (vii) an alteration of one or more
PaAos codon sequences to encode a non-conservative amino acid. It
is another object of this invention that the alteration of one or
more PaAos codon sequences to encode a non-conservative amino acid
occurs at amino acids located at 318, 332, 336, 339, 359, 408, 411,
and 459 with PaAos' sequence (see SEQ ID NO: 10, 13, and 15).
Another object of this invention is that the non-conservative amino
acid substitutions are not N318, V408 and/or W459. It is another
object of this invention to have an altered cell, germplasm, and an
altered seed of the altered guayule produced by this method and
each containing the mutation.
[0010] It is an object of this invention to have a method for
producing a population of high rubber producing guayule plants or
seeds which contain PaAos with low functionality. It is an object
of that method involves genotyping a first population of guayule
plants or seeds that contain PaAos with low functionality,
selecting from the first population one or more guayule plants or
seeds containing PaAos with low functionality based the genotyping,
and producing from the selected one or more guayule plants or seeds
containing PaAos with low functionality a second population of
guayule plants or seeds containing PaAos with low functionality. It
is another object of this invention PaAos with low functionality
contains at least one amino acid selected from group of N318, V408,
W459, conservative amino acids substitutions thereof, and
non-conservative amino acid substitutions at S332, E336, R339,
S359, and S411.
[0011] It is an object of this invention to have a method of
identifying a high rubber producing guayule by detecting the
presence of PaAos having amino acids N318, V408 and/or W459, or
conservative amino acid substitution at one or more of these
positions and/or having non-conservative amino acid substitutions
at S332, E336, R339, S359, and S411 in a test guayule. It is
further object of this invention that the method involves
contacting the PaAos from the test guayule with monoclonal
antibodies that binds to PaAos having the above mentioned amino
acids, and determining if the monoclonal antibodies bind to the
PaAos from the test guayule, where binding indicates the presence
of the high rubber producing PaAos and no binding indicates the
present of the low rubber producing PaAos. It is another object of
this invention that the invention involves obtaining nucleic acids
from the test guayule, performing a PCR assay with the obtained
nucleic acids, a label, and primer sets SEQ ID NOs: 22 and 23, and
SEQ ID NOs: 24 and 25, or similar sequences that encode
conservative amino acid substitutions, and determining if an
amplicon is generated; such that when an amplicon is produced, then
the test guayule contains a PaAos nucleic acid sequence that
encodes PaAos having amino acids N318, V408 and/or W459, or one or
more of the conservative amino acid substitution at these
positions, and/or non-conservative amino acid substitutions at
S332, E336, R339, S359, and S411, and the test guayule is a high
rubber producing guayule.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0012] FIG. 1 shows the design of the plasmids used in Example 1
for the overexpression of PaAos (pND6-Aos), silencing of PaAos
(pND6-AosiL), and for the control plasmid (pND6). Each expression
vector features the NPTII gene to confer kanamycin resistance for
selection, and the control plasmid contains the GUS
(.beta.-glucuronidase) reporter gene instead of PaAos or a portion
of PaAos in reverse complementary orientation. Thus, one can use a
histochemical GUS staining assay (the chromogenic substrate X-Gluc
(C.sub.14H.sub.13BrCINO.sub.7)) as a visual indicator of
transformed tissues with the negative control plasmid.
[0013] FIG. 2 shows the primers used in the qRT-PCR, PCR reaction
conditions, and the expected amplicon product size to determine RNA
expression in the genetically altered guayule. Amplicon (mRNA
product) PaAos.sub.OE is derived from plants transformed with
pND6-Aos (Aos overexpression). Amplicon (mRNA product)
PaAos.sub.RNAi is derived from plants transformed with pND6-AosiL
(Aos silencing). Amplicon (mRNA product) 18S is from 18S ribosome
RNA. Amplicon (mRNA product) PaAos is for wild-type guayule plants
with intact PaAos gene.
[0014] FIG. 3 provides size and weight measurements of the four
types of guayule plants grown under different conditions in growth
chambers. Plants are initially transferred to soil from tissue
culture media and grown under greenhouse conditions for one month.
Following, plants are moved to controlled-temperature growth
chamber conditions under 27.degree. C. (16 h)/25.degree. C. (8 h)
and at 27.degree. C. (16 h)/10.degree. C. (8 h). The four type of
guayule plants are wild-type (G7-11.1 and G7-11.2), guayule
transformed with the empty expression vector pND6 (pND6-10,
pND6-12, pND6-35), guayule transformed with pND6-AosiL for
silencing PaAos via RNAi (pND6-AosiL.sub.7-2, pND6-AosiL.sub.8-1,
pND6-AosiL.sub.9-16, pND6-AosiL.sub.12-1,), and guayule transformed
with pND6-Aos to overexpress PaAos (pND6-Aos.sub.4-1,
pND6-Aos.sub.4-2, pND6-Aos.sub.5-1, pND6-Aos.sub.7-1,) at 2 months
old. G7-11 is a breeder's nomenclature for what later became the
USDA publicly released guayule Germplasm line AZ-2 (Reg. No. GP-9;
PI 599676). The biomass of the shoot (leaves plus stems) and root
are weighed in 2 months old guayule plants grown in growth chambers
under 27.degree. C. (16 h)/25.degree. C. (8 h) and at 27.degree. C.
(16 h)/10.degree. C. (8 h). The asterisks, (*), (**) and (***),
indicate significant difference in comparison to (non-altered)
G7-11 at p>0.05, 0.005 and 0.0005, respectively.
[0015] FIG. 4 shows SPAD values indicating leaf chlorophyll
concentration ("SPAD units") for wild-type guayule (G7-11), guayule
transformed with the empty expression vector (pND6); guayule
transformed with pND6-Aos (Oe), and guayule transformed with
pND6-AosiL (RNAi) grown at 27.degree. C. (16 h)/25.degree. C. (8 h)
or 27.degree. C. (16 h)/10.degree. C. (8 h).
[0016] FIG. 5 shows the number of branches and stem diameter of 2
months old genetically altered guayule (pND6-AosiL and pND6-Aos
lines), non-altered guayule (G7-11) and empty vector control
guayule (pND6) plants grown in growth chambers at 27.degree. C. (16
h)/25.degree. C. (8 h) or at 27.degree. C. (16 h)/10.degree. C. (8
h). pND6-AosiL genotypes have larger number of stems than the
non-altered and empty vector controls. Additionally, the mature
stembark tissues in pND6-AosiL have significantly thicker diameter
(ranging from 35% to 54%) under both 27.degree. C. (16
h)/25.degree. C. (8 h) and 27.degree. C. (16 h)/10.degree. C. (8
h).
[0017] FIG. 6 shows results from gel permeation chromatography for
elution of cyclohexane extractables from transformed and
non-altered guayule plant lines. The natural rubber molecular
weight is calculated using Astra software for three pND6-AosiL
transformed guayule plants, three pND6-Aos transformed guayule
plants, two pND6 transformed guayule plants, and non-altered
guayule G7-11. The error bars represent 3 different plants with 3
technical replicates.
[0018] FIG. 7 shows the relative expression of PaAos in guayule
line G7-11, guayule line W6 549 ("W6549"), and guayule line PI
478652 ("478652").
[0019] FIG. 8A, FIG. 8B, and FIG. 8C show single nucleotide
polymorphisms (SNPs) in PaAos coding sequence for guayule cultivars
W6 549 ("W6549"; SEQ ID NO: 12), G7-11 (SEQ ID NO: 9), and PI
478652 ("478652"; SEQ ID NO: 14). The SNPs are contained in
boxes.
[0020] FIG. 9 shows an alignment of PaAos amino acid sequences
obtained from guayule cultivars W6 549 ("W6549"; SEQ ID NO: 13), PI
478652 ("478652"; SEQ ID NO: 15) and G7-11 (SEQ ID NO: 10). The
boxes highlight the different amino acids in the cultivars.
DETAILED DESCRIPTION OF THE INVENTION
[0021] This invention involves the discovery that a reduction in
the amount of functional PaAos in guayule results in an increase in
the amount of rubber produced. Further, different cultivars of
guayule, having different DNA and amino acid sequences for PaAos
and PaAos, respectively, produce different amounts of rubber. As
such, one can distinguish between guayule cultivars that are "high"
rubber producers and "low" rubber producers based on the
differences in the DNA and/or amino acid sequence of PaAos and/or
PaAos. Because single nucleotide polymorphisms (SNPs) exist in
these different cultivars, one can use primers and PCR techniques
to determine if any particular guayule is a "high" rubber producing
guayule or a "low" rubber producing guayule. Alternatively, one can
use antibodies that bind to the different PaAos proteins to
determine if any particular guayule is a "high" rubber producer or
a "low" rubber producer. As discussed below, guayule cultivar W6
549 has the lowest average rubber content (%) and guayule cultivar
PI 478652 has the highest average rubber content, among the tested
cultivars. PaAos from both cultivars have slight differences in DNA
sequences which, along with the differences in the amino acid
sequences, can be used to determine if any particular cultivar is a
high or low rubber producer. Any guayule that produces PaAos with
conservative amino acid changes to SEQ ID NO: 15 is a high rubber
producing guayule; any guayule that produces PaAos with
conservative amino acid changes to SEQ ID NO: 13 is a low rubber
producing guayule. Thus, one can screen plants for PaAos with
conservative sequences to SEQ ID NO: 15 via DNA or protein assays.
Alternatively, one screening for guayule with PaAos with the
similar (or lower) level of functionality as guayule cultivar PI
478652's PaAos would identify a high rubber producing guayule;
whereas screening for guayule with PaAos with a higher level of
functionality would identify a low rubber producing guayule.
[0022] Because changes in PaAos functionality changes the amount of
rubber produced by guayule, this invention also involves increasing
a guayule's rubber production by reducing the amount of functional
PaAos present in the genetically altered guayule (compared to the
amount of functional PaAos present in non-altered guayule). In one
embodiment, the genetically altered guayule has a mutation in
PaAos, such as, a null mutation which results in (i) no protein is
produced, (ii) a truncated protein is produced which has no
functionality, or (iii) a full-length protein is produced which has
no functionality. Other mutations simply reduce the functionality
(activity) of PaAos. Non-limiting examples of mutations that reduce
or eliminate PaAos functionality include (i) changing a codon
encoding an amino acid to a stop codon (see Table 1 supra for the
sequence of stop codons), (ii) changing the translation initiation
codon (ATG) to any other codon to disrupt protein translation,
(iii) changing a ribosome binding site's sequence to disrupt
protein translation, (iv) changing one or more splice site codons
to alter protein sequence, (v) deleting some or all of the gene's
DNA sequence, (vi) inserting DNA into the gene, and (vii) changing
one or more DNA codon sequences to encode non-conservative amino
acids. Within SEQ ID NO: 9, nucleotides 34-36 encode ATG, the
translation initiation codon for G7-11 guayule. Similarly,
nucleotides 1-3 of SEQ ID NOs: 12 and 14 encode ATG, the
translation initiation codon for cultivars PI 478652 and W6549,
respectfully. Thus, a change in the nucleotide sequence of the
equivalent codon in any other guayule would have the same result.
Within PaAos, non-conservative amino acid substitutions at D318,
S332, E336, R339, S359, I408, S411, and/or L459 (to name a few)
result in PaAos with reduced functionality. One alters guayule DNA
using the methods described herein and assesses changes in PaAos
functionality via the methods described herein (e.g., assessing the
amount of rubber produced by the altered guayule) or using methods
known to one of ordinary skill in the art. One can utilize SNPs,
antibodies, and other methods to identify the guayule that encode
the altered amino acids. When no functional PaAos is produced or
when PaAos with reduced functionality is produced, then it is
understood that the altered guayule produces "a reduced amount of
functional PaAos". Such altered guayule producing a reduced amount
of functional PaAos is another embodiment of this invention.
[0023] Because this invention involves production of genetically
altered plants and involves recombinant DNA techniques, the
following definitions are provided to assist in describing this
invention. The terms "isolated", "purified", or "biologically pure"
as used herein, refer to material that is substantially or
essentially free from components that normally accompany the
material in its native state or when the material is produced. In
an exemplary embodiment, purity and homogeneity are determined
using analytical chemistry techniques such as polyacrylamide gel
electrophoresis or high performance liquid chromatography. A
nucleic acid or particular bacteria that are the predominant
species present in a preparation is substantially purified. In an
exemplary embodiment, the term "purified" denotes that a nucleic
acid or protein that gives rise to essentially one band in an
electrophoretic gel. Typically, isolated nucleic acids or proteins
have a level of purity expressed as a range. The lower end of the
range of purity for the component is about 60%, about 70% or about
80% and the upper end of the range of purity is about 70%, about
80%, about 90% or more than about 90%.
[0024] The term "gene" refers to a DNA sequence involved in
producing a RNA or polypeptide or precursor thereof. The
polypeptide or RNA is encoded by a full-length coding sequence
(cds) or by intron-interrupted portions of the coding sequence,
such as exon sequences. In one embodiment of this invention, the
gene involved is Parthenium argentatum allene oxide synthase (PaAos
or Aos). PaAos cDNA and amino acid sequence is found in GenBank
accession number X78166.2 which is cultivar G7-11
(wild-type/non-altered) (USDA publicly released guayule Germplasm
line AZ-2 (Reg. No. GP-9; PI 599676)). The cDNA sequence is in SEQ
ID NO: 9; the protein sequence is in SEQ ID NO: 10. SEQ ID NO: 12
is the cDNA sequence for PaAos and SEQ ID NO: 13 is the amino acid
sequence for PaAos in guayule W6549 cultivar. SEQ ID NO: 14 is the
cDNA sequence for PaAos and SEQ ID NO: 15 is the amino acid
sequence for PaAos in guayule 478652 cultivar. A molecular marker
uses SNPs within PaAos to differentiate the cultivars with
differences in PaAos amino acid sequences which result in PaAos
with different functionalities.
[0025] The term "nucleic acid" as used herein, refers to a polymer
of ribonucleotides or deoxyribonucleotides. Typically, "nucleic
acid" polymers occur in either single- or double-stranded form, but
are also known to form structures comprising three or more strands.
The term "nucleic acid" includes naturally occurring nucleic acid
polymers as well as nucleic acids comprising known nucleotide
analogs or modified backbone residues or linkages, which are
synthetic, naturally occurring, and non-naturally occurring, which
have similar binding properties as the reference nucleic acid, and
which are metabolized in a manner similar to the reference
nucleotides. Exemplary analogs include, without limitation,
phosphorothioates, phosphoramidates, methyl phosphonates,
chiral-methyl phosphonates, 2-O-methyl ribonucleotides,
peptide-nucleic acids (PNAs). "DNA", "RNA", "polynucleotides",
"polynucleotide sequence", "oligonucleotide", "nucleotide",
"nucleic acid", "nucleic acid molecule", "nucleic acid sequence",
"nucleic acid fragment", and "isolated nucleic acid fragment" are
used interchangeably herein.
[0026] For nucleic acids, sizes are given in either kilobases (kb)
or base pairs (bp), or nucleotides (nt). Estimates are typically
derived from agarose or acrylamide gel electrophoresis, from
sequenced nucleic acids, or from published DNA sequences. For
proteins, sizes are given in kiloDaltons (kDa) or amino acid
residue numbers. Proteins sizes are estimated from gel
electrophoresis, from sequenced proteins, from derived amino acid
sequences, or from published protein sequences.
[0027] Unless otherwise indicated, a particular nucleic acid
sequence for each amino acid substitution (alteration) also
implicitly encompasses conservatively modified variants thereof
(e.g., degenerate codon substitutions), the complementary (or
complement) sequence, and the reverse complement sequence, as well
as the sequence explicitly indicated. Specifically, degenerate
codon substitutions may be achieved by generating sequences in
which the third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (see e.g.,
Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J.
Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell.
Probes 8:91-98(1994)). Because of the degeneracy of nucleic acid
codons, one can use various different polynucleotides to encode
identical polypeptides. Table 1, infra, contains information about
which nucleic acid codons encode which amino acids and is useful
for determining the possible nucleotide substitutions that are
included in this invention.
TABLE-US-00001 TABLE 1 Amino acid Nucleic acid codons Ala/A GCT,
GCC, GCA, GCG Arg/R CGT, CGC, CGA, CGG, AGA, AGG Asn/N AAT, AAC
Asp/D GAT, GAC Cys/C TGT, TGC Gln/Q CAA, CAG Glu/E GAA, GAG Gly/G
GGT, GGC, GGA, GGG His/H CAT, CAC Ile/I ATT, ATC, ATA Leu/L TTA,
TTG, CTT, CTC, CTA, CTG Lys/K AAA, AAG Met/M ATG Phe/F TTT, TTC
Pro/P CCT, CCC, CCA, CCG Ser/S TCT, TCC, TCA, TCG, AGT, AGC Thr/T
ACT, ACC, ACA, ACG Trp/W TGG Tyr/Y TAT, TAC Val/V GTT, GTC, GTA,
GTG Stop TAA, TGA, TAG
[0028] In addition to the degenerate nature of the nucleotide
codons which encode amino acids, alterations in a polynucleotide
that result in the production of a chemically equivalent amino acid
at a given site, but do not affect the functional properties of the
encoded polypeptide, are well known in the art. "Conservative amino
acid substitutions" are those substitutions that are predicted to
interfere least with the properties of the reference polypeptide.
In other words, conservative amino acid substitutions substantially
conserve the structure and the function of the reference protein.
Thus, a codon for the amino acid alanine, a hydrophobic amino acid,
may be substituted by a codon encoding another less hydrophobic
residue, such as glycine, or a more hydrophobic residue, such as
valine, leucine, or isoleucine. Similarly, changes which result in
substitution of one negatively charged residue for another, such as
aspartic acid for glutamic acid, or one positively charged residue
for another, such as lysine for arginine or histidine, is also
expected to produce a functionally equivalent protein or
polypeptide. Table 2 provides a list of exemplary conservative
amino acid substitutions. Conservative amino acid substitutions
generally maintain (a) the structure of the polypeptide backbone in
the area of the substitution, for example, as a beta sheet or alpha
helical conformation, (b) the charge or hydrophobicity of the
molecule at the site of the substitution, and/or (c) the bulk of
the side chain. In another embodiment, groups of amino acids that
are conservative substitutions for each other are (i) alanine (Ala
or A), serine (Ser or S), and threonine (Thr or T); (ii) aspartic
acid (Asp or D) and glutamic acid (Glu or E); (iii) asparagine (Asn
or N) and glutamine (Gln or Q); (iv) arginine (Arg or R) and lysine
(Lys or K); (v) isoleucine (Ile or I), leucine (Leu or L),
methionine (Met or M), and valine (Val or V); and (vi)
phenylalanine (Phe or F), tyrosine (Tyr or Y), and tryptophan (Trp
or W). See, Creighton, Proteins, W.H. Freeman and Co. (1984),
contents of which are expressly incorporated herein. In yet another
embodiment, amino acid(s) that are conservative substitutes for one
amino acid are grouped by the following characteristics: aliphatic
amino acids (alanine, glycine, isoleucine, leucine, and valine);
hydroxyl or sulfur containing amino acids (cysteine, serine,
methionine, and threonine); cyclic (proline); aromatic
(phenylalanine, tryptophan, and tyrosine); basic (arginine,
histidine, and lysine); acidic (aspartate and glutamate); and
uncharged (asparagine and glutamine). As discussed below, wild-type
guayule can be "high" rubber producers or "low" rubber producers.
In both types of guayule, there are several amino acid changes in
PaAos that be used to distinguish the "high" and "low" rubber
producers. As such, one may change DNA encoding one, two, or more
of the amino acids to change a "low" rubber producing guayule into
a "high" rubber producing guayule. Alternatively, one may change
the DNA to encode an amino acid that is a conservative substitute
(per Table 2) of the one, two, or more amino acids different in the
"high" rubber producing guayule. Further, because the "high" rubber
producing guayule discussed below have several amino acids
different in PaAos than the "low" rubber producing guayule, the
invention also includes any conservative amino acids changes that
can be made in these one, two, or more amino acids.
TABLE-US-00002 TABLE 2 Amino Acid Conservative Substitute Ala Gly,
Ser Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln
Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile
Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Ile, Leu Phe His, Leu,
Met, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe,
Trp Val Ile, Leu, Thr
[0029] The term "primer" refers to an oligonucleotide which may act
as a point of initiation of DNA extension. A primer may occur
naturally, as in a purified restriction digest, or may be produced
synthetically.
[0030] A primer is selected to be "substantially complementary" to
a strand of specific sequence of the template. A primer must be
sufficiently complementary to hybridize with a template strand for
primer elongation to occur. A primer sequence need not reflect the
exact sequence of the template. For example, a non-complementary
nucleotide fragment may be attached to the 5' end of the primer,
with the remainder of the primer sequence being substantially
complementary to the strand. Non-complementary bases or longer
sequences can be interspersed into the primer, provided that the
primer sequence is sufficiently complementary with the sequence of
the template to hybridize and thereby form a template primer
complex for synthesis of the extension product of the primer.
[0031] Oligonucleotides and polynucleotides that are not
commercially available can be chemically synthesized e.g.,
according to the solid phase phosphoramidite triester method first
described by Beaucage and Caruthers, Tetrahedron Letts.
22:1859-1862 (1981), or using an automated synthesizer, as
described in Van Devanter et al., Nucleic Acids Res. 12:6159-6168
(1984). Other methods for synthesizing oligonucleotides and
polynucleotides are known in the art. Purification of
oligonucleotides is by either native acrylamide gel electrophoresis
or by anion-exchange HPLC as described in Pearson & Reanier, J.
Chrom. 255:137-149 (1983).
[0032] The terms "identical" or percent "identity", in the context
of two or more polynucleotides or polypeptide sequences, refer to
two or more sequences or sub-sequences that are the same or have a
specified percentage of nucleotides or amino acids (respectively)
that are the same (e.g., 80%, 85% identity, 90% identity, 99%, or
100% identity), when compared and aligned for maximum
correspondence over a designated region as measured using a
sequence comparison algorithm or by manual alignment and visual
inspection.
[0033] The phrase "high percent identical" or "high percent
identity", in the context of two polynucleotides or polypeptides,
refers to two or more sequences or sub-sequences that have at least
about 80%, identity, at least about 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100% nucleotide or amino acid identity, when compared and aligned
for maximum correspondence, as measured using a sequence comparison
algorithm or by visual inspection. In an exemplary embodiment, a
high percent identity exists over a region of the sequences that is
at least about 16 nucleotides or amino acids in length. In another
exemplary embodiment, a high percent identity exists over a region
of the sequences that is at least about 50 nucleotides or amino
acids in length. In still another exemplary embodiment, a high
percent identity exists over a region of the sequences that is at
least about 100 nucleotides or amino acids or more in length. In
one exemplary embodiment, the sequences are high percent identical
over the entire length of the polynucleotide or polypeptide
sequences.
[0034] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters may be used, or
alternative parameters designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters. Methods of alignment of sequences for
comparison are well-known in the art. Optimal alignment of
sequences for comparison is conducted, e.g., by the local homology
algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981),
by the homology alignment algorithm of Needleman & Wunsch, J.
Mol. Biol. 48:443 (1970), by the search for similarity method of
Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by
computerized implementations of various algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), and/or by
manual alignment and visual inspection (see, e.g., Ausubel et al.
(eds.), Current Protocols in Molecular Biology, 1995
supplement).
[0035] The "complement" of a particular polynucleotide sequence is
that nucleotide sequence which would be capable of forming a
double-stranded DNA or RNA molecule with the represented nucleotide
sequence, and which is derived from the represented nucleotide
sequence by replacing the nucleotides by their complementary
nucleotide according to Chargaff's rules (A< >T; G< >C)
and reading in the 5' to 3' direction, i.e., in opposite direction
of the represented nucleotide sequence (reverse complement).
[0036] In one embodiment of the invention, sense and antisense RNAs
and dsRNA can be separately expressed in-vitro or in-vivo. In-vivo
production of sense and antisense RNAs may use different chimeric
polynucleotide constructs using the same or different promoters or
using an expression vector containing two convergent promoters in
opposite orientation. The sense and antisense RNAs which are formed
(e.g., in the same host cells or synthesized) then combine to form
dsRNA. To be clear, whenever reference is made herein to a dsRNA
chimeric or fusion polynucleotide or a dsRNA molecule, that such
dsRNA formed (e.g., in plant cells) from sense and antisense RNA
produced separately is also included. Also, synthetically made
dsRNA and self-annealing RNA strands are included herein when the
sense and antisense strands are present together.
[0037] As used herein, the term "promoter" refers to a
polynucleotide that, in its native state, is located upstream or 5'
to a translational start codon of an open reading frame (or
protein-coding region) and that is involved in recognition and
binding of RNA polymerase and other proteins (trans-acting
transcription factors) to initiate transcription. A "plant
promoter" is a native or non-native promoter that is functional in
plant cells, even if the promoter is present in a microorganism
that infects plants or a microorganism that does not infect plants.
The promoters that are predominately functional in a specific
tissue or set of tissues are considered "tissue-specific
promoters". A plant promoter can be used as a 5' regulatory element
for modulating expression of a particularly desired polynucleotide
(heterologous polynucleotide) operably linked thereto. When
operably linked to a transcribable polynucleotide, a promoter
typically causes the transcribable polynucleotide to be transcribed
in a manner that is similar to that of which the promoter is
normally associated.
[0038] Plant promoters include promoters produced through the
manipulation of known promoters to produce artificial, chimeric, or
hybrid promoters. Such promoters can also combine cis-elements from
one or more promoters, for example, by adding a heterologous
regulatory element to an active promoter with its own partial or
complete regulatory elements. The term "cis-element" refers to a
cis-acting transcriptional regulatory element that confers an
aspect of the overall control of gene expression. A cis-element may
function to bind transcription factors, trans-acting protein
factors that regulate transcription. Some cis-elements bind more
than one transcription factor, and transcription factors may
interact with different affinities with more than one
cis-element.
[0039] The term "vector" refers to DNA, RNA, a protein, or
polypeptide that are to be introduced into a host cell or organism.
The polynucleotides, protein, and polypeptide which are to be
introduced into a host may be therapeutic or prophylactic in
nature; may encode or be an antigen; may be regulatory in nature;
etc. There are various types of vectors including viruses, viroids,
plasmids, bacteriophages, cosmids, and bacteria.
[0040] An expression vector is nucleic acid capable of replicating
in a selected host cell or organism. An expression vector can
replicate as an autonomous structure, or alternatively integrate,
in whole or in part, into the host cell chromosomes or the nucleic
acids of an organelle, or it is used as a shuttle for delivering
foreign DNA to cells, and thus replicate along with the host cell
genome. Thus, an expression vector are polynucleotides capable of
replicating in a selected host cell, organelle, or organism, e.g.,
a plasmid, virus, artificial chromosome, nucleic acid fragment, and
for which certain genes on the expression vector (including genes
of interest) are transcribed and translated into a polypeptide or
protein within the cell, organelle or organism; or any suitable
construct known in the art, which comprises an "expression
cassette". In contrast, as described in the examples herein, a
"cassette" is a polynucleotide containing a section of an
expression vector. The use of the cassettes assists in the assembly
of the expression vectors. An expression vector is a replicon, such
as plasmid, phage, virus, chimeric virus, or cosmid, and which
contains the desired polynucleotide sequence operably linked to the
expression control sequence(s).
[0041] A heterologous polynucleotide sequence is operably linked to
one or more transcription regulatory elements (e.g., promoter,
terminator and, optionally, enhancer) such that the transcription
regulatory elements control and regulate the transcription and/or
translation of that heterologous polynucleotide sequence. A
cassette has the heterologous polynucleotide operably linked to one
or more transcription regulatory elements. As used herein, the term
"operably linked" refers to a first polynucleotide, such as a
promoter, connected with a second transcribable polynucleotide,
such as a gene of interest, where the polynucleotides are arranged
such that the first polynucleotide affects the transcription of the
second polynucleotide. In some embodiments, the two polynucleotide
molecules are part of a single contiguous polynucleotide. In other
embodiments, the two polynucleotides are adjacent. For example, a
promoter is operably linked to a gene of interest if the promoter
regulates or mediates transcription of the gene of interest in a
cell. Similarly, a terminator is operably linked to the
polynucleotide of interest if the terminator regulates or mediates
transcription of the polynucleotide of interest, and in particular,
the termination of transcription. Constructs of the present
invention would typically contain a promoter operably linked to a
transcribable polynucleotide operably linked to a terminator.
[0042] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
organism, nucleic acid, protein or vector, has been altered by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so altered. Thus, for example, recombinant
cells may express genes/polynucleotides that are not found within
the native (non-recombinant or non-altered or wild-type) form of
the cell or express native genes in an otherwise abnormal
amount--over-expressed, under-expressed or not expressed at
all--compared to the non-altered cell or organism. In particular,
one alters the genomic DNA of a non-altered plant by molecular
biology techniques that are well-known to one of ordinary skill in
the art and generate a recombinant plant.
[0043] The terms "transgenic", "transformed", "transformation", and
"transfection" are similar in meaning to "recombinant"
"Transformation", "transgenic", and "transfection" refer to the
transfer of a polynucleotide into a host organism or into a cell.
Such a transfer of polynucleotides may result in genetically stable
inheritance of the polynucleotides or in the polynucleotides
remaining extra-chromosomally (not integrated into the chromosome
of the cell). Genetically stable inheritance may potentially
require the transgenic organism or cell to be subjected for a
period of time to one or more conditions which require the
transcription of some or all of the transferred polynucleotide in
order for the transgenic organism or cell to live and/or grow.
Polynucleotides that are transformed into a cell but are not
integrated into the host's chromosome remain as an expression
vector within the cell. One may need to grow the cell under certain
growth or environmental conditions in order for the expression
vector to remain in the cell or the cell's progeny. Further, for
expression to occur the organism or cell may need to be kept under
certain conditions. Genetically altered organisms or cells
containing the recombinant polynucleotide are referred to as
"transgenic" or "transformed" organisms or cells or simply as
"transformants", as well as recombinant organisms or cells.
[0044] A genetically altered organism is any organism with any
changes to its genetic material involving the invention described
herein, whether in the nucleus or cytoplasm (organelle). As such, a
genetically altered organism may be a recombinant or transformed
organism. A genetically altered organism may also be an organism
that was subjected to one or more mutagens or the progeny of an
organism that was subjected to one or more mutagens and has
mutations in its DNA caused by the one or more mutagens, as
compared to the wild-type organism (i.e., organism not subjected to
the mutagens) or the non-altered organism (i.e., one that contains
alterations that are not the subject matter of this invention).
Also, an organism that has been bred to incorporate a mutation into
its genetic material is a genetically altered organism.
[0045] The term "altered" means that a change occurred compared to
the "non-altered" item. However, a "non-altered" item could contain
changes that are induced by man, but those changes are not the
subject matter of the inventions described herein. For example, a
non-altered guayule contains none of the described genetic changes
nor has been treated with any of the described external substance,
but may contain pre-existing changes which are not part of this
invention. An altered guayule (which also is a genetically altered
guayule) may contain DNA mutations which change PaAos' amino acid
sequence, even if that sequence exists in a non-altered plant. Such
DNA mutations may be induced by a mutagen (EMS, UV light, other
radiation, etc.).
[0046] Transformation and generation of genetically altered
monocotyledonous and dicotyledonous plant cells is well known in
the art. See, e.g., Weising, et al., Ann. Rev. Genet. 22:421-477
(1988); U.S. Pat. No. 5,679,558; Agrobacterium Protocols, ed:
Gartland, Humana Press Inc. (1995); and Wang, et al. Acta Hort.
461:401-408 (1998). A method to generate genetically altered
guayule is described in U.S. Pat. No. 9,018,449 (Dong &
Cornish) and in Dong, et al., Plant Cell Reports 25:26-34 (2006). A
method to generate transplastomic guayule is provided in U.S.
Patent Application Publication 2014/0325699, contents of which are
expressly incorporated herein. The choice of method varies with the
type of plant to be transformed, the particular application and/or
the desired result. The appropriate transformation technique is
readily chosen by the skilled practitioner.
[0047] A polynucleotide encoding PaAos (SEQ ID NOs: 9, 12, and/or
14), the reverse complement of PaAos, or a portion thereof (e.g.,
SEQ ID NO: 11), operably linked to one or two appropriate
promoters, can be stably inserted in a conventional manner into the
genome (cytoplasmic genome or nucleic genome) of a single plant
cell, and the altered plant cell can be used in a conventional
manner to produce a genetically altered plant that produces the
dsRNA of this invention. In this regard, a disarmed Ti-plasmid,
containing the polynucleotide of this invention, in Agrobacterium
tumefaciens can be used to genetically alter the plant cell, and
thereafter, a genetically altered plant can be regenerated from the
genetically altered plant cell using the procedures described in
the art, for example, in EP 0 116 718, EP 0 270 822, WO 84/02913
and EP 0 242 246. Plant regeneration from cultured protoplasts is
described in Evans et al., Protoplasts Isolation and Culture, in
Handbook of Plant Cell Culture, pp. 124-176, MacMillan Publishing
Company, New York, 1983; and Binding, Regeneration of Plants, in
Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985.
Regeneration can also be obtained from plant callus, explants,
organs, or parts thereof. Such regeneration techniques are
described generally in Klee, et al., Ann. Rev. of Plant Phys.
38:467-486 (1987).
[0048] Preferred Ti-plasmid vectors each contain the
polynucleotides described herein between the border sequences, or
at least located to the left of the right border sequence, of the
T-DNA of the Ti-plasmid. Of course, other types of vectors may be
used to transform the plant cell, using procedures such as direct
gene transfer (as described, for example in EP 0 233 247), pollen
mediated transformation (as described, for example in EP 0 270 356,
WO 85/01856, and U.S. Pat. No. 4,684,611), plant RNA virus-mediated
transformation (as described, for example in EP 0 067 553 and U.S.
Pat. No. 4,407,956), liposome-mediated transformation (as
described, for example in U.S. Pat. No. 4,536,475), and other
methods such as the methods for transforming certain lines of corn
(e.g., U.S. Pat. No. 6,140,553; Fromm, et al., Bio/Technology
8:833-839 (1990); Gordon-Kamm, et al., The Plant Cell 2:603-618
(1990) and rice (Shimamoto, et al., Nature 338:274-276 (1989);
Datta et al., Bio/Technology 8:736-740 (1990)) and the method for
transforming monocots generally (WO 92/09696). For cotton
transformation, the method described in WO 2000/71733 can be used.
For soybean transformation, reference is made to methods known in
the art, e.g., Hinchee, et al. (Bio/Technology 6:915 (1988)) and
Christou, et al. (Trends Biotechnology 8:145 (1990)) or the method
of WO 00/42207.
[0049] The resulting genetically altered plant can be used in a
conventional plant breeding scheme to produce more genetically
altered plants with the same characteristics or to introduce the
polynucleotide into other varieties of the same or related plant
species. Seeds, which are obtained from the genetically altered
plants, contain the expression vector as a stable genomic insert.
Altered plants include plants having or derived from root stocks of
plants containing the expression vector. Hence, any non-altered
grafted plant parts inserted on a genetically altered plant or
plant part are included in the invention.
[0050] For a genetically altered plant that produces dsRNA, one
constructs an expression vector or cassette (made from DNA) that
encodes, at a minimum, a first promoter and the dsRNA sequence of
interest such that the promoter sequence is 5' (upstream) to and
operably linked to the dsRNA sequence. The expression vector or
cassette may optionally contain a second promoter (same as or
different from the first promoter) upstream and operably linked to
the reverse complementary sequence of the dsRNA sequence such that
two strands of RNA that are complementary to each other are
produced. Alternatively, the expression vector or cassette can
contain one promoter operably linked to both the dsRNA sequence
(sense strand) in question and the complement or reverse complement
of the dsRNA sequence (anti-sense strand) in question, such that
the transcribed RNA bends on itself and the two desires sequences
anneal. Alternatively, a second expression vector or cassette (made
from DNA) may encode, at a minimum, a second promoter (same as or
different from the promoter) operably linked to the reverse
complementary sequence of the dsRNA such that two strands of
complementary RNA are produced in the plant. The expression
vector(s) or cassette(s) is/are inserted in a plant cell genome
(nuclear or cytoplasmic). The promoter(s) used should be a
promoter(s) that is/are active in a plant and is/are heterologous
to PaAos (not normally driving the transcription of RNA of genomic
PaAos). Of course, the expression vector or cassette may have other
transcription regulatory elements, such as enhancers, terminators,
etc.
[0051] Promoters (and more specifically, heterologous promoters for
PaAos) that are active in plants are well-known in the field. Such
promoters may be constitutive, inducible, and/or tissue-specific.
Non-limiting examples of constitutive plant promoters include 35S
promoters of the cauliflower mosaic virus (CaMV) (e.g., of isolates
CM 1841 (Gardner, et al., Nucleic Acids Research 9:2871-2887
(1981)), CabbB-S (Franck, et al., Cell 21:285-294 (1980)) and
CabbB-JI (Hull and Howell, Virology 86:482-493 (1987))), ubiquitin
promoter (e.g., the maize ubiquitin promoter of Christensen, et
al., Plant Mol. Biol. 18:675-689 (1992)), gos2 promoter (de Pater,
et al., The Plant J. 2:834-844 (1992)), emu promoter (Last, et al.,
Theor. Appl. Genet. 81:581-588 (1990)), actin promoter (see, e.g.,
An, et al., The Plant J. 10:107 (1996)) and Zhang, et al., The
Plant Cell 3:1155-1165 (1991)); Cassava vein mosaic virus promoters
(see, e.g., WO 97/48819 and Verdaguer, et al., Plant Mol. Biol.
37:1055-1067 (1998)), the pPLEX series of promoters from
Subterranean Clover Stunt Virus (WO 96/06932, particularly the S4
or S7 promoter), alcohol dehydrogenase promoter (e.g., pAdh1S
(GenBank accession numbers X04049, X00581)), and the TR1' promoter
and the TR2' promoter which drive the expression of the 1' and 2'
genes, respectively, of the T-DNA (Velten, et al., EMBO J.
3:2723-2730 (1984)). Tissue-specific promoters are promoters that
direct a greater level of transcriptional expression in some cells
or tissues of the plant than in other cells or tissue. Non-limiting
examples of tissue-specific promoters include the
phosphoenolpyruvate carboxylase (PEP or PPC1) promoter (Pathirana,
et al., Plant J. 12:293-304 (1997), and Kausch, et al., Plant Mol.
Biol. 45(1):1-15 (2001)), chlorophyll A/B binding protein (CAB)
promoter (Bansal, et al., Proc. Natl. Acad. Sci. USA 89(8):3654-8
(1992)), small subunit of ribulose-1,5-bisphosphate carboxylase
(ssRBCS) promoter (Bansal, et al., Proc. Natl. Acad. Sci. USA
89(8):3654-8 (1992)), senescence activated promoter (SEE1) (Robson,
et al., Plant Biotechnol. J. 2(2):101-12 (2004)), and sorghum leaf
primoridia specific promoter (RS2) (GenBank Accession No.
E1979305.1). These promoters (PPC1, CAB, ssRBCS, SSE1, and RS2) are
all active in the aerial part of a plant. Further, the PPC1
promoter is a strong promoter for expression in vascular tissue.
Some examples of phloem specific promoters are the sucrose
synthase-1 promoters (CsSUS1p and CsSUS1p-2) (Singer et al., Planta
234:623-637 (2011)) and the phloem protein-2 promoter (CsPP2)
(Miyata et al., Plant Cell Report 31(11):2005-2013 (2012)) from
Citrus sinensis. Alternatively, a plant-expressible promoter may
also be a wound-inducible promoter, such as the promoter of the pea
cell wall invertase gene (Zhang, et al., Plant Physiol.
112:1111-1117 (1996)).
[0052] Other types of RNA polymerase promoters that may be used are
promoters from microorganisms, such as, but not limited to the
bacteriophage T7 RNA polymerase promoter, yeast Galactose (GAL1)
promoter, yeast glyceraldehyde-3-phosphate dehydrogenase (GAP)
promoter, yeast Alcohol Oxidase (AOX) promoter.
[0053] Other elements used to increase transcription expression in
plant cells include, but are not limited to, an intron (e.g., hsp70
intron) at the 5' end or 3' end of the chimeric gene, or in the
coding sequence of the chimeric dsRNA gene (such as, between the
region encoding the sense and antisense portion of the dsRNA),
promoter enhancer elements, duplicated or triplicated promoter
regions, 5' leader sequences different from the chimeric gene or
different from an endogenous (plant host) gene leader sequence, 3'
untranslated sequences different from the chimeric gene or
different from an endogenous (plant host) 3' untranslated
sequence.
[0054] The expression vector or cassette could contain suitable 3'
untranslated transcription regulation sequences (i.e., transcript
formation and polyadenylation sequences). Potential polyadenylation
and transcript formation sequences include those sequences in the
nopaline synthase gene (Depicker, et al., J. Molec. Appl. Genetics
1:561-573 (1982)), the octopine synthase gene (Gielen, et al., EMBO
J. 3:835-845 (1984)), the SCSV or the Malic enzyme terminators
(Schunmann, et al., Plant Functional Biology 30:453-460 (2003)),
and the T-DNA gene 7 (Velten and Schell, Nucleic Acids Research
13:6981-6998 (1985)).
[0055] The term "plant" includes whole plants, plant organs,
progeny of whole plants or plant organs, embryos, somatic embryos,
embryo-like structures, protocorms, protocorm-like bodies (PLBs),
and suspensions of plant cells. Plant organs comprise, e.g., shoot
vegetative organs/structures (e.g., leaves, stems and tubers),
roots, flowers and floral organs/structures (e.g., bracts, sepals,
petals, stamens, carpels, anthers and ovules), seed (including
embryo, endosperm, and seed coat) and fruit (the mature ovary),
plant tissue (e.g., vascular tissue, ground tissue, and the like)
and cells (e.g., guard cells, egg cells, trichomes and the like).
The class of plants that can be used in the method of the invention
is generally as broad as the class of higher and lower plants
amenable to the molecular biology and plant breeding techniques
described herein, specifically angiosperms (monocotyledonous
(monocots) and dicotyledonous (dicots) plants). It includes plants
of a variety of ploidy levels, including aneuploid, polyploid,
diploid, haploid and hemizygous. The genetically altered plants
described herein are guayule plants.
[0056] Rubber yield may be expressed as a product of rubber content
(% rubber) and biomass (dry weight/unit area). Thus, rubber yield
may be improved by increasing either biomass and/or rubber content.
The altered guayule described herein produce more rubber and have
higher rubber content than non-altered guayule, thereby increasing
the processing efficiency of the guayule shrub.
[0057] Various methods exist to create a mutation. These methods
are well-known to one of ordinary skill in the art. One method is
by transforming the plant with a plasmid containing 5' sequence and
3' sequence of the gene and allowing a cross-over event to occur,
thereby excising the DNA from the plant's genome that is between
the plasmid's 5' sequence and 3' sequence. Also, one can use
transposon-mediated mutation to delete or add DNA to PaAos which
would result in the encoded protein having a reduced functionality
compared to a non-altered PaAos. Two other methods involve using a
chemical mutagen (such as ethyl methanesulfonate (EMS)) or physical
agents (radiation, UV, or proton, for example) to generate genetic
mutations in plant cells and/or germplasm. Also, one may use TALEN
or CRISPR-Cas9 to mutate the sequence of the target gene (PaAos)
such that a desired mutation is generated. One of ordinary skill in
the art can also use targeted cleavage events to induce targeted
mutagenesis, induce targeted deletions of cellular DNA sequences,
and facilitate targeted recombination and integration at
predetermined chromosomal locations to generate one or more of the
null mutations discussed above or to reduce the mutated protein's
functionality. Nucleotide editing techniques are well-known and
described in Urnov, et al., Nature 435(7042):646-51 (2010); U.S.
Patent Publications 2003/0232410, 2005/0208489, 2005/0026157,
2005/0064474, 2006/0188987, 2009/0263900, 2009/0117617,
2010/0047805, 2011/0207221, 2011/0301073, 2011/089775,
2011/0239315, and 2011/0145940; and International Publication WO
2007/014275, the disclosures of which are incorporated by reference
in their entireties for all purposes. Cleavage occurs by using
specific nucleases such as engineered zinc finger nucleases (ZFN),
transcription-activator like effector nucleases (TALENs), or using
the CRISPR/Cas9 system with an engineered crRNA/tracr RNA (`single
guide RNA`) to guide specific cleavage. U.S. Patent Publication
2008/0182332 describes the use of non-canonical zinc finger
nucleases (ZFNs) for targeted modification of plant genomes; U.S.
Patent Publication 2009/0205083 describes ZFN-mediated targeted
modification of a plant EPSPS locus; U.S. Patent Publication
2010/0199389 describes targeted modification of a plant Zp15 locus
and U.S. Patent Publication No. 20110167521 describes targeted
modification of plant genes involved in fatty acid biosynthesis. In
addition, Moehle, et al., Proc. Natl. Acad. Sci. USA
104(9):3055-3060 (2007) describes using designed ZFNs for targeted
gene addition at a specified locus. U.S. Patent Publication
2011/0041195 describes methods of making homozygous diploid
organisms. Information on CRISPR/Cas9 system is found, e.g., at
en.wikipedia.org/wiki/CRISPR;
neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-gen-
ome-editing-a-new-era-in-molecular-biology; and Cong, et al.,
Science, 339:819-823 (2013). Sigma-Aldrich (St. Louis, Mo.) and
Origene Technologies, Inc. (Rockville, Md.) are among the companies
that sell CRISPR/Cas9 kits.
[0058] After using any of these various methods to induce genetic
alterations in a cell's genome, one induces the treated cells to
grow into plants and then screen the plants using the methods
described herein for PaAos having reduced or no functionality,
and/or for reduced amounts of PaAos or no PaAos (via reduction in
gene expression and/or mRNA translation and/or other mechanism),
and/or for elevated production of rubber (compared to amounts
present in non-altered plants). Thus, another embodiment of this
invention is the generation of altered guayule having a genetic
alteration in PaAos such that the altered guayule produces more
rubber than produced by non-altered guayule.
[0059] In another embodiment, one synergistically increases the
amount of rubber produced by exposing an altered guayule to cold
temperatures (between approximately 7.degree. C. and approximately
15.degree. C. or between approximately 10.degree. C. and
approximately 15.degree. C.; approximately 8 hours per day) for
approximately two weeks or more. The altered guayule may contain
one of more of the following alterations: (1) DNA encoding (i)
anti-sense RNA for PaAos, (ii) double-stranded RNA for PaAos, (iii)
a mutation within PaAos that encodes a PaAos with reduced or no
function; and/or (2) exogenously administered PaAos dsRNA. The
combination of any of the above alterations and exposure to cold
temperatures (between approximately 7.degree. C. and approximately
15.degree. C. or between approximately 10.degree. C. and
approximately 15.degree. C.; approximately 8 hours per day) for
approximately two weeks or more result in production of increased
amounts of rubber than produced by the non-altered plant exposed to
the same temperatures for the same time period.
[0060] Many techniques involving molecular biology discussed herein
are well-known to one of ordinary skill in the art and are
described in, e.g., Green and Sambrook, Molecular Cloning, A
Laboratory Manual 4th ed. 2012, Cold Spring Harbor Laboratory;
Ausubel et al. (eds.), Current Protocols in Molecular Biology,
1994--current, John Wiley & Sons; and Kriegler, Gene Transfer
and Expression: A Laboratory Manual (1993). Unless otherwise noted,
technical terms are used according to conventional usage.
Definitions of common terms in molecular biology maybe found in
e.g., Benjamin Lewin, Genes IX, Oxford University Press, 2007 (ISBN
0763740632); Krebs, et al. (eds.), The Encyclopedia of Molecular
Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and
Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a
Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0061] The terms "approximately" and "about" refer to a quantity,
level, value or amount that varies by as much as 30% in one
embodiment, or in another embodiment by as much as 20%, and in a
third embodiment by as much as 10% to a reference quantity, level,
value or amount. As used herein, the singular form "a", "an", and
"the" include plural references unless the context clearly dictates
otherwise. For example, the term "a bacterium" includes both a
single bacterium and a plurality of bacteria.
[0062] The term "nucleic acid consisting essentially of",
"polynucleotide consisting essentially of", and "RNA consisting
essentially of", and grammatical variations thereof, means a
polynucleotide that differs from a reference nucleic acid sequence
by 20 or fewer nucleotides and also perform the function of the
reference polynucleotide sequence. Such variants include sequences
which are shorter or longer than the reference nucleic acid
sequence, have different residues at particular positions, or a
combination thereof.
[0063] Having now generally described this invention, the same will
be better understood by reference to certain specific examples and
the accompanying drawings, which are included herein only to
further illustrate the invention and are not intended to limit the
scope of the invention as defined by the claims. The examples and
drawings describe at least one, but not all embodiments, of the
inventions claimed. Indeed, these inventions may be embodied in
many different forms and should not be construed as limited to the
embodiments set forth herein; rather, these embodiments are
provided so that this disclosure will satisfy applicable legal
requirements.
Example 1. Construction of Genetically Altered Guayule
[0064] To better understand the role of PaAos in rubber synthesis,
genetically altered P. argentatum plants are generated in which
either PaAos is over-expressed or PaAos is silenced by RNAi. The
various plasmids used to achieve the overexpression or silencing of
PaAos in guayule are shown in FIG. 1. To generate these plasmids,
the guayule Aos (PaAos) is amplified by PCR using genomic DNA as a
template. The primers used to amplify PaAos are designed from the
cDNA PaAos sequence published in NCBI database (GeneBank accession
no. X78166.2) and have the following sequences: forward primer
5'-cttaagaggtggtATGGACCCATCGTCTAAACCC-3' (SEQ ID NO: 1) and reverse
primer 5'-ggatccTCATATACTAGCTCTCTTCAGGG-3' (SEQ ID NO: 2). The
nucleotides in lower case and underlined in the forward primer are
the recognition nucleotides for restriction enzyme AflII; the
nucleotides in lower case and underlined in the reverse primer are
the recognition nucleotides for restriction enzyme BamHI. The PCR
cycle program is the following: 94.degree. C. for 2 minutes
(initial heating step) and PaAos is amplified at 40 cycles of
94.degree. C. for 30 seconds (denaturation), 71.degree. C. for 30
seconds (annealing) and 68.degree. C. for 1 minute (extension) and
an additional 5 minutes extension at 68.degree. C. The resulting
amplicon is purified and subcloned into pGEM T Easy vector
(Promega, Madison, Wis.) using manufacturer's recommended protocol
and sequenced to confirm the sequence of the plasmids. The cDNA
sequence is in SEQ ID NO: 9 and the amino acid sequence is in SEQ
ID NO: 10. Subsequently, the PaAos amplicon is cut using AflII and
BamHI. Plasmid pND6 has a Nos promoter driving the NPTII gene for
conferring kanamycin resistance and a potato ubiquitin promoter
(Garbarino and Belknap, Plant Mol. Biol. 24:119-127 (1994))
controlling the GUSplus gene (CambiaLabs, Canberra, Australia). See
FIG. 1. Plasmid pND6-Aos (FIG. 1) is generated by replacing the
GUSplus gene in pND6 with cDNA PaAos sequence (SEQ ID NO: 9).
Plasmid pND6-AosiL (FIG. 1) is generated by replacing the GUSplus
gene in pND6 with an inverted repeat of a partial cDNA PaAos
sequence (SEQ ID NO: 11) containing a loop sequence of the Bar gene
between the inverted repeat of the partial cDNA PaAos sequence; the
complete sequence replacing GUSplus is SEQ ID NO: 26. The plasmids
pND6, pND6-AosiL, and pND6-Aos are used to transform Agrobacterium
EHA101 competent cells using the protocol described in Hood, et
al., J. Bacteriol. 168:1291-1301 (1986).
[0065] The transformed Agrobacterium EHA101 either harboring pND6,
pND6-AosiL, or pND6-Aos are used to transform guayule G7-11 using
the protocols set forth below. See also Dong, et al. (2006) and
Dong, et al., Industrial Crops and Products 46:15-24 (2013). For
Agrobacterium transformation, the overnight Agrobacterium culture
are prepared by inoculating 50 .mu.L glycerol stock into a 50 mL
Falcon tube containing 5 mL LB medium plus 40 mg/L rifampcin and
200 mg/L spectinomycin, and shaking at 200 rpm at 28.degree. C. The
suspension then is centrifuged for 15 minutes at 1600.times.g at
room temperature. The supernatant is discarded, and the pellet is
re-suspended in 25 mL of inoculation solution ( 1/10 MS salts plus
BA (2 mg/L), NAA (0.5 mg/L), glucose (10 g), acetosyringone (200
.mu.M), pluronic F68 (0.05%), pH=5.2 (PhytoTechnology Labs, Shawnee
Mission, Kans.)). For leaf transformation, leaf sections are cut
from the plants in the Magenta boxes (Caisson Labs, Smithfield,
Utah). The adaxial side of each leaf is placed facing up in a Petri
dish containing 5 ml Agrobacterium suspension. The leaf is cut into
.about.10 mm strips and immediately placed in an empty Petri dish
in non-overlapping manner. When this Petri dish is full, all leaf
strips are blotted with the filter paper and placed into another
empty Petri dish. The Petri dish is sealed by parafilm and left in
the dark at room temperature. The co-cultivation is replaced by
this co-desiccation according to Cheng, et al., In Vitro Cell Dev.
Biol. Plant, 39, 595-604 (2003). After three days, leaf strips are
transferred to MSB1T (MS medium with BA (1 mg/L), sucrose (30 g/L),
phytagel (3 g/L), and timentin (400 mg/L)) (Cheng, et al., Plant
Cell Rep., 17(8):646-649 (1998)) for recovery at low light for 5
days. The leaf strips are then transferred to MSB1TK30 (MS medium
containing BA (1 mg/L), sucrose (30 g/L), phytagel (3 g/L),
timentin (250 mg/L), and kanamycin (30 mg/L)) for selection under
low light for two weeks. The leaf strips are then subcultured every
2 weeks under high light till green shoots emerged. Green shoots 10
mm and longer are transferred to 1/2MS10.1TK10 for rooting (same as
1/2MSI0 but with timentin (250 mg/L) and kanamycin (10 mg/L)).
After 2-4 weeks, the rooted plantlets are micropropogated and
subsequently transplanted into soil.
[0066] While the genetically altered guayule are still growing in
tissue culture under selection, the genetically altered plants are
screened for integration of the expression vectors, pND6-Aos (PaAos
in forward orientation; SEQ ID NO: 9), pND6-AosiL (PaAos in the
reverse orientation (a portion of reverse complement of PaAos is
SEQ ID NO: 11)), and pND6 (negative control). DNA is extracted from
genetically altered plants using Sigma Kit (Sigma-Aldrich, St.
Louis, Mo.). Approximately 150 mg leaf tissue (3 leaf tissues) are
cut from the plants grown in tissue-cultured, placed into 2 mL
tubes and snapped-frozen in liquid nitrogen. A bead is added to
pulverize the tissue into a fine powder at a frequency of 30/s for
1 minute using the mixer mill MM 400 tissue lyser (Verder
Scientific, Inc., Newtown, Pa.).
[0067] PCR is carried out in 50 .mu.L mixture containing Taq
2.times. Master Mix (New England Biolabs, Ipswich, Mass.), 200 ng
guayule genomic DNA or 20 pg plasmid DNA, and 100 ng of PaAos
specific primers; namely SEQ ID NOs: 1 and 2 for guayule
transformed with pND6-Aos; and SEQ ID NOs: 3 and 4 for guayule
transformed with pND6-AosiL. See FIG. 2. After heating the samples
to 94.degree. C. for 2 minutes, the reaction proceeds with 35
cycles of 94.degree. C. for 30 seconds, 71.degree. C. to amplify
the PaAos in the overexpression lines (pND6-Aos) for 30 seconds or
56.degree. C. for the PaAos in the RNAi lines (pND6-AosiL) for 30
seconds, and 68.degree. C. for 1 minute. A final elongation step is
carried out at 68.degree. C. for 5 minutes. PCR products are
separated by electrophoresis on a 1% (w/v) agarose gel. The band
for the overexpression lines is at .about.1.4 kbp, as expected; the
band for the RNAi lines is at .about.0.5 kbp as expected. The
genetically altered guayule plants harboring the empty plasmid
(pND6 (negative control)) are confirmed by GUS staining (Karcher,
S., ABLE 23:29-42 (2002)). Briefly, plant tissues are placed in a
50 mL tube containing GUS assay solution (1 mM X-Gluc
(5-bromo-4-chloro-3-indolyl) B-D-glucuronic acid in 50 mM
Na.sub.2HPO.sub.4, pH 7.0 and 0.1% Triton X-100). The reaction is
incubated at 37.degree. C. for 1 hour followed by washing for 30
minutes with 70% ethanol to extract the chlorophyll.
Example 2. Determination of RNA Expression Levels in Genetically
Altered Plants
[0068] Guayule containing intact PaAos (non-altered; G7-11) and
genetically altered guayule containing one of the plasmids (pND6,
pND6-Aos, or pND6-AosiL) are further screened to determine the RNA
level (see Table 3). Leaves from the various genetically modified
plants (which are grown in tissue culture) are collected and
snap-frozen in liquid nitrogen for RNA extraction. RNA is extracted
using TRIzol.RTM. according to manufacturer's recommended protocol
(Ambion, Pittsburgh, Pa.). RNA concentration is quantified with the
NanoDrop ND1000 (ThermoScientific, Wilminton, Del.). RNA cleanup is
performed using the RNeasy MinElute Cleanup kit according to
manufacturer's recommended protocol (Qiagen Inc., Valencia,
Calif.). The RNA is eluted with 30-50 .mu.L of RNase-free water
along with on-column DNase1 treatment.
[0069] Using the RNA isolated from the leaves of the genetically
altered plants, cDNA is generated using iScript cDNA synthesis kit
(Bio-Rad, Hercules, Calif.) according to the manufacturer's
recommended protocol for semi-quantitative PCR and real-time
quantitative PCR (qRT-PCR). An amount of 1 .mu.g of RNA is used in
the 20 mL reaction mixture. For qRT-PCR, 2 .mu.L of the diluted
cDNA (1:20) is used in a 15 .mu.L reaction mixture. In the qRT-PCR
volume, 7.5 mL of iQ SYBR.RTM. Green Supermix is used (Bio-Rad,
Hercules, Calif.). The qRT-PCR is run using the 7500 Fast Real-Time
PCR system (Applied Biosystem, Waltham, Mass.) with the following
thermal cycle: 95.degree. C. pre-incubation for 3 minutes;
amplification is performed for 40 cycles at 95.degree. C. for 15
seconds and at 60.degree. C. for 30 seconds; the dissociation stage
is set for 95.degree. C. for 15 seconds, at 60.degree. C. for 1
minute, and at 95.degree. C. for 15 seconds. Each qRT-PCR run is
performed with three independent tissue samples, each sample having
two technical replicates. The 18S gene (.about.200 bp) is used as
an internal control. The primers used for each sequence, PCR
reaction conditions, and the expected amplicon size are contained
in FIG. 2. Crossing point value, which is the point at which the
fluorescence crosses the threshold, and melting curve analyses are
noted. The melting curve data are collected for all genes to ensure
a single peak, indicating amplification of a specific region by a
pair of primers. The relative expression values are calculated
using the 2(-Delta C(T)) method (Livak and Schmittgen, Methods,
25:402-408 (2001)). See Table 3 below.
TABLE-US-00003 TABLE 3 P. argentatum Average Relative Genotypes
Expression of Aos G7-11 1.02 .+-. 0.2 pND6-10 1.14 .+-. 0.2 pND6-12
1.02 .+-. 0.3 pND6-29 1.04 .+-. 0.2 pND6-32 1.00 .+-. 0.3 pND6-33
1.11 .+-. 0.3 pND6-35 0.90 .+-. 0.2 pND6-41 0.91 .+-. 0.2
pND6-AosiL.sub.5-1 0.39 .+-. 0.1* pND6-AosiL.sub.7-2 0.49 .+-. 0.1*
pND6-AosiL.sub.8-1 0.53 .+-. 0.1* pND6-AosiL.sub.9-15 0.44 .+-.
0.1* pND6-AosiL.sub.9-16 0.37 .+-. 0.04* pND6-AosiL.sub.12-1 0.55
.+-. 0.1* pND6-AosiL.sub.12-3 0.48 .+-. 0.1* pND6-AosiL.sub.13-2
0.55 .+-. 0.05* pND6-AosiL.sub.15-3 0.36 .+-. 0.1*
pND6-AosiL.sub.15-4 0.48 .+-. 0.2* pND6-Aos.sub.4-1 2.15 .+-. 0.1**
pND6-Aos.sub.4-2 2.11 .+-. 0.3** pND6-Aos.sub.5-1 2.29 .+-. 0.4**
pND6-Aos.sub.7-1 2.40 .+-. 0.6** pND6-Aos.sub.7-3 2.11 .+-. 0.2**
pND6-Aos.sub.8-2 2.15 .+-. 0.2** pND6-Aos.sub.10-1 2.44 .+-. 0.7**
pND6-Aos.sub.10-2 2.12 .+-. 0.4** pND6-Aos.sub.11-5 2.30 .+-. 0.3**
pND6-Aos.sub.14-2 2.23 + 0.1** G7-11 = wild-type control; pND6 =
empty vector (pND6 without Aos); pND6-AosiL = PaAos is
knocked-down/silenced; pND6-Aos = PaAos is over-expressed Results
are average of three independent plants, each plant having three
technical replicates. * and ** indicate significant difference in
comparison to G7-11 guayule and/or pND6 (controls) at p > 0.05
and 0.005, respectively.
[0070] Next, to gain more insight as to where the PaAos is
spatially located, the expression pattern of PaAos in various
guayule tissues is analyzed using qRT-PCR. Total RNA is extracted
from leaves, petiole, stem, root, young flower, mature flower,
peduncle, stembark of 8-week-old tissue-cultured genetically
altered plants as well as 2-month-old greenhouse grown genetically
altered plants using the protocol described above. qRT-PCR is
performed as described above on these samples of total RNA. Primers
(SEQ ID NOs: 7 and 8 in FIG. 2) are designed to amplify .about.200
bp PCR product in PaAos coding sequence. The expression level for
each tissue are compared to the tissue cultured and greenhouse leaf
tissues, respectively. The 18S gene (.about.200 bp) (forward primer
is SEQ ID NO: 5 and reverse primer is SEQ ID NO: 6, described supra
and in FIG. 2) is used as an internal control. As shown in Table 4,
infra, the largest level of PaAos expression is present in the
stem, root and stembark tissues, suggesting that these tissues are
sites in which PaAos is functioning.
TABLE-US-00004 TABLE 4 Growth Conditions: MS Medium Greenhouse
Tissue Source Relative Expression Leaf 0.98 .+-. 0.1 1.04 .+-. 0.2
Petiole 0.31 .+-. 0.06 0.41 .+-. 0.1 Stem 2.27 .+-. 0.2 3.37 .+-.
0.4 Root 2.47 .+-. 0.2 3.74 .+-. 0.3 Young Flower no data 1.23 .+-.
0.4 Mature Flower no data 0.69 .+-. 0.2 Peduncle no data 0.25 .+-.
0.1 Bark no data 4.49 .+-. 1.2 The error bars represent tissues
collected from 3 individual plants.
Example 3. Rubber Quantification in Tissue
[0071] Rooted plantlets (genetically altered, empty vector
transformed (pND6 without PaAos), and wild-type control) from
transferred shoot tips are grown on half-strength MS medium
(PhytoTechnology Laboratories, Overland Park, Kans.) in Magenta
boxes (Caisson Labs, Smithfield, Utah) for 6 weeks. The top part of
the plantlets are separated from the medium and lyophilized for 48
hours. The dried tissues are placed in a 50 mL stainless steel
grinding jar containing grinding ball, frozen in liquid nitrogen
for 5 minutes and finely ground using the Retsch mixer mill MM 400
at a frequency of 30/second for 1 minute (Verder Scientific Inc.,
Newtown, Pa.). Three hundred milligrams (0.3 g) of pulverized
tissues are partitioned with Ottawa sand (Fisher Scientific, Fair
Lawn, N.J.) and loaded into 11 mL stainless steel extraction cells
(Dionex, Sunnyvale, Calif.). Three sequential extractions are
performed using the Accelerated Solvent Extractor (ASE 2000;
Dionex, Sunnyvale, Calif.): 1. Acetone: to remove resinous material
and the low molecular weight organic solubles; 2. Methanol: to
remove chlorophyll and other alcohol-soluble materials; 3.
Cyclohexane: to remove rubber. Natural rubber is quantified
gravimetrically. The percent (%) rubber is the amount (% dw) of
cyclohexane extract from 0.3 g dried tissue. The pND6-AosiL plants
have 1.5 to 2 times more rubber than G7-11, pND6 and pND6-Aos in
tissue-cultured environment (Table 5). In Table 5, the rubber
content is quantified from leaf and stems of the indicated guayule
genotypes grown in MS media.
TABLE-US-00005 TABLE 5 Rubber content of guayule plant shoots
determined by Accelerated Solvent Extraction P. argentatum
Genotypes Average Rubber Content (%) G7-11.1 1.01 .+-. .01 G7-11.2
1.11 .+-. .02 pND6-12 1.13 .+-. 0.2 pND6-33 1.10 .+-. 0.1 pND6-35
1.04 .+-. 0.2 pND6-AosiL.sub.5-1 1.8 .+-. 0.1* pND6-AosiL.sub.7-2
2.0 .+-. 0.3** pND6-AosiL.sub.8-1 2.1 .+-. 0.04***
pND6-AosiL.sub.8-2 1.7 .+-. 0.1** pND6-AosiL.sub.9-15 1.7 .+-.
0.02** pND6-AosiL.sub.9-16 2.3 .+-. 0.4* pND6-AosiL.sub.12-1 2.46
.+-. 0.3* pND6-AosiL.sub.12-3 1.62 .+-. 0.002*** pND6-Aos.sub.4-1
0.96 .+-. 0.2 pND6-Aos.sub.4-2 0.85 .+-. 0.1 pND6-Aos.sub.5-1 1.09
.+-. 0.1 pND6-Aos.sub.5-2 1.23 .+-. 0.1 pND6-Aos.sub.7-1 0.96 .+-.
.02 pND6-Aos.sub.8-2 1.23 .+-. 0.1 pND6-Aos.sub.11-5 1.23 .+-. 0.1
Note: The rubber content is quantified from shoots (leaves + stems)
of guayule genotypes grown in MS media. Error bars represent three
biological plants with three technical replicates each. *, ** and
*** indicate significant difference in comparison to G7-11 guayule
and/or pND6 (controls) at p > 0.05, 0.005 and 0.0005,
respectively.
[0072] Next, the genetically altered guayule plants are
transplanted into soil and grown for 2 months under 27.degree. C.
(16 h)/25.degree. C. (8 h) and 27.degree. C. (16 h)/10.degree. C.
(8 h) in growth chamber conditions, representing a microcosm of
what guayule plants experience in the field during winter. Under
these conditions, pND6-AosiL plants also exhibited elevated rubber
content, having up to 31% times more rubber in comparison with
G7-11, pND6 and pND6-Aos plants (Table 6). In Table 6, the rubber
content is quantified from shoots and roots of the indicated
guayule genotypes grown in soil. These plants are approximately 4
months old when rubber content is analyzed (tissue culture (approx.
1.5 months), greenhouse (approx. 1 month), and growth chamber
(approx. 2 months)).
TABLE-US-00006 TABLE 6 Rubber content of guayule plant tissue
determined by Accelerated Solvent Extraction Average Rubber Content
(%) Shoot Root P. argentatum 27.degree. C. (16 h)/ 27.degree. C.
(16 h)/ 27.degree. C. (16 h)/ 27.degree. C. (16 h)/ Genotypes
25.degree. C. (8 h) 10.degree. C. (8 h) 25.degree. C. (8 h)
10.degree. C. (8 h) G7-11.1 1.22 + 0.09 1.13 + 0.11 1.10 + 0.04
0.95 + 0.10 G7-11.2 1.06 + 0.12 1.37 + 0.09 0.63 + 0.12 0.71 + 0.09
pND6-12 1.04 + 0.18 1.31 + 0.06 0.65 + 0.07 0.72 + 0.16 pND6-33
0.90 + 0.06 1.14 + 0.12 0.62 + 0.08 0.82 + 0.10 pND6-35 1.18 + 0.08
1.27 + 0.10 1.09 + 0.06 0.94 + 0.03 pND6-AosiL.sub.7-2 1.49 +
0.07*** 1.86 + 0.11*** 0.56 + 0.03 1.26 + 0.09** pND6-AosiL.sub.8-1
1.48 + 0.05*** 1.91 + 0.07*** 0.78 + 0.14 1.19 + 0.04**
pND6-AosiL.sub.9-16 1.46 + 0.04*** 2.01 + 0.08*** 0.66 + 0.12 1.16
+ 0.02** pND6-AosiL.sub.12-1 1.55 + 0.07* 1.80 + 0.05** 1.26 +
0.04*** 1.52 + 0.07** pND6-Aos.sub.4-1 0.97 + 0.26 1.13 + 0.2 0.57
+ 0.05 0.79 + 0.09 pND6-Aos.sub.4-2 1.21 + 0.10 1.30 + 0.08 1.05 +
0.10 0.94 + 0.11 pND6-Aos.sub.5-1 0.98 + 0.30 1.04 + 0.12 0.54 +
0.11 0.62 + 0.10 pND6-Aos.sub.7-1 0.96 + 0.30 1.15 + 0.14 0.57 +
0.06 0.55 + 0.08 Note: The rubber content is quantified from shoots
and roots of guayule genotypes grown in soil. Plants are
transferred to soil from tissue culture and are grown in a growth
chamber environment. Error bars represent three biological plants
with three technical replicates each. *, ** and *** indicate
significant difference in comparison to G7-11 and/or pND6
(controls) at p > 0.05, 0.005 and 0.0005, respectively.
[0073] Because rubber is also accumulated in root tissue, the
rubber content in the root tissues is also quantified. For the root
rubber content, the consistent, significant difference is only
under 27.degree. C. (16 h)/10.degree. C. (8 h) in which pND6-AosiL
guayule have an increased in rubber content compared with the
controls and pND6-Aos (Table 6). The data in Table 6 demonstrate
that the combination of cold temperature and silencing PaAos is
synergistic, causing guayule to produce a greater amount of rubber
than guayule exposed to just cold temperature or to just PaAos
silencing. For example, cold treatment alone increased shoot rubber
content in the control (pND6-12) by 19%--from an average of 1.04%
to 1.24%. But cold treatment of the Aos-downregulated plants
(pND6-AosiL) increased rubber by 27%--from average 1.50% to 1.90%.
In root tissues, cold treatment increased the rubber content for
the control (pND6-12) by 5.1% (from 0.79 to 0.83% rubber), but cold
treatment of the Aos-downregulated plants (pND6-AosiL) increased
rubber by 57%--from average 0.82% to 1.28%. See Table 7, infra.
From the ASE results, the increased in rubber content is very
apparent in the pND6-AosiL genotypes.
TABLE-US-00007 TABLE 7 Average Rubber Content (%) Shoot Root
27.degree. C. 27.degree. C. 27.degree. C. 27.degree. C. P.
argentatum (16 h)/ (16 h)/ (16 h)/ (16 h)/ Genotypes 25.degree. C.
(8 h) 10.degree. C. (8 h) 25.degree. C. (8 h) 10.degree. C. (8 h)
pND6-12 1.04 1.24 0.79 0.83 pND6-AosiL.sub.7-2 1.50 1.90 0.82 1.28
pND6-Aos.sub.4-1 1.03 1.16 0.68 0.73
Example 4. Protein Detection in Rubber Particles
[0074] Guayule washed rubber particles (WRPs) are isolated from
genetically altered guayule lines (pND6-AosiL and pND6-Aos) and
non-altered guayule using the protocol set forth in Cornish and
Backhaus, Phytochemistry, 29: 3809-3813 (1990). Rubber particles
are extracted from non-altered and genetically altered 1 year old
greenhouse plants. First, .about.60 g to .about.70 g of stembark
tissues are peeled off from the plant, grounded with a blender
containing cold-extraction buffer, and further purified with
cold-washed buffer three times by centrifugation. The protein
extracts (1 mg) are run on an SDS-PAGE and detected with silver
staining. On the SDS-PAGE gel, endogenous Aos protein runs as
.about.53 kDa in the non-altered and overexpressed plants but not
in the RNAi lines. To determine the dry weight of the WRPs, 50
.mu.L of the protein extracts are aliquoted 3.times. on a weighing
paper, oven-dried over-night in a 60.degree. C. incubator and
weighed the next day. Generally, approximately 0.5 mg/.mu.L to
approximately 1.5 mg/.mu.L WRPs are extracted.
Example 5. Hormone Production
[0075] PaAos is an enzyme in the biosynthetic pathway that produces
several different plant hormones, including jasmonic acid, SA,
abscisic acid, gibberellin A.sub.20, gibberellin A.sub.1, and
gibberellin A.sub.3. As such, the amount of these hormones is
quantified in genetically altered (pND6-AosiL and pND6-Aos), empty
vector transformed (pND6 without PaAos; control), and wild-type
(G7-11, control) tissue-cultured guayule plants using the protocol
described in Pan et al., Nature Protocols 5:986-992 (2010). See
Table 8, infra. Briefly, leaves and stems are snap-frozen and
ground to powder with mortar and pestle. Solvent extraction
solution containing 2-propanol/H.sub.2O/concentrated HCl
(2:1:0.002; vol/vol/vol) and internal standards are added to
.about.50 mg of pre-weighed tissues. After solvent extraction,
sample concentration and re-dissolution, 50 .mu.L of the sample
solution is placed into the liquid chromatography-tandem
spectrometry (Agilent GC-MS 5977A; Agilent Technologies, Santa
Clara, Calif.) for hormone analysis. Three biological plants, with
three technical replicates of each plant, are used.
TABLE-US-00008 TABLE 8 Concentration (ng/gfw) P. argentatum
Jasmonic Salicylic Abscisic Gibberellin Gibberellin Gibberellin
Genotypes Acid Acid Acid A.sub.20 A.sub.1 A.sub.3 G7-11 5.36 .+-.
1.2 5.50 .+-. 0.8 11.01 .+-. 1.9 15.95 .+-. 0.7 9.95 .+-. 0.07 3.52
.+-. 0.2 pND6-12 1.57 .+-. 0.1 4.89 .+-. 0.6 7.05 .+-. 0.8 14.11
.+-. 1.2 12.39 .+-. 2.2 2.19 .+-. 0.01 pND6-33 4.76 .+-. 1.0 5.04
.+-. 0.1 7.24 .+-. 0.3 13.86 .+-. 2.6 12.70 .+-. 2.3 1.79 .+-. 0.09
pND6-35 1.96 .+-. 0.4 5.6 .+-. 0.5 7.10 .+-. 0.6 14.41 .+-. 1.2
14.65 .+-. 0.2 2.16 .+-. 0.3 pND6-AosiL.sub.7-2 0.57 .+-. 0.1**
9.51 .+-. 0.5* 4.71 .+-. 0.6* 9.63 .+-. 1.2* 5.13 .+-. 0.8* 0.86
.+-. 0.3* pND6-AosiL.sub.9-16 0.57 .+-. 0.01** 7.65 .+-. 0.2* 2.96
.+-. 0.3* 8.85 .+-. 2.1* 7.81 .+-. 0.2** 0.80 .+-. 0.3*
pND6-AosiL.sub.12-1 0.68 .+-. 0.05** 9.65 .+-. 1.1* 3.64 .+-. 0.5*
10.59 .+-. 0.2* 6.75 .+-. 1.1* 1.32 .+-. 0.07* pND6-Aos.sub.4-1
1.48 .+-. 0.2 4.03 .+-. 0.7 9.13 .+-. 1.71 15.49 .+-. 0.6 10.78
.+-. 0.1 1.93 .+-. 0.06 pND6-Aos.sub.4-2 3.25 .+-. 0.2 4.76 .+-.
0.6 13.9 .+-. 1.3 no data 14.63 .+-. 2.7 1.80 .+-. 0.1
pND6-Aos.sub.7-1 1.41 .+-. 0.3 5.50 .+-. 0.02 8.84 .+-. 0.3 13.64
.+-. 1.2 13.96 .+-. 0.01 1.84 .+-. 0.1 .+-. represent three
biological plants with three technical replicates each plant. * and
** indicate significant difference in comparison to G7-11 and/or
pND6 (controls) at p > 0.05 and 0.005, respectively.
[0076] As evident in Table 8, the amount of jasmonic acid, abscisic
acid and gibberellic acids are reduced in the pND6-AosiL guayule
compared to the amount in the controls (wild-type (G7-11) and empty
vector transformed plants) and pND6-Aos guayule. Conversely, the SA
content is elevated in pND6-AosiL compared to the controls and
pND6-Aos lines. These results suggest that knocking down PaAos
expression not only reduces production of jasmonic acid but also
affects the level of other hormones as well.
Example 6. Plant Architecture and Photosynthetic Rates
[0077] Three independent events from each of the overexpression
(pND6-Aos) and of the silenced (pND6-AosiL) lines; as well as two
pND6 and one wild-type (G7-11) controls are selected for further
studies. pND6-AosiL plants grown in greenhouse (data not shown) and
growth chamber conditions are bigger (see FIG. 3), have darker
green leaves (data not shown), and increased chlorophyll
measurement than the wild type and other genetically altered plants
(see FIG. 4). As demonstrated in FIG. 3, under 27.degree. C. (16
h)/25.degree. C. (8 h) and 27.degree. C. (16 h)/10.degree. C. (8 h)
environments, pND6-AosiL plants are significantly taller and wider
in both conditions. These plant architectural traits reflect the
fact that pND6-AosiL plants are larger and have more shoot and root
biomass.
[0078] pND6-AosiL genotypes have also a greater number of stems
than the wild-type and empty vector controls. Well-branched guayule
plants are an indicator of having increased rubber yield because of
the presence of more sink tissue available to store rubber.
Additionally, the mature stembark tissues in pND6-AosiL have
thicker diameter (ranging from approximately 35% to approximately
54%) under both 27.degree. C. (16 h)/25.degree. C. (8 h) and
27.degree. C. (16 h)/10.degree. C. (8 h) in comparison to the
controls and pND6-Aos. See FIG. 5.
[0079] Based on this observation, the photosynthetic rate of the
plants is measured using LI-COR 6400xt (LI-COR Biosciences,
Lincoln, Nebr.) to measure the photosynthetic rate. Measurements
are taken between 0900 to 1200 h. Fully expanded middle leaf are
clamped on the Li-Cor head. After the measured and set parameters
are stabilized, the reading is taken. The middle leaf position is
chosen because this position shows significant differences based on
chlorophyll meter measurements, FIG. 4. (SPAD-502; Minolta Camera
Ltd., Japan). The pND6-AosiL plants exhibit higher photosynthetic
rate (23-31%) in comparison to G7-11, pND6 and pND6-Aos plants
(Table 9, infra). Additional physiological measurements reveal that
pND6-AosiL stomatal limitation is one of the factors involved in
the higher photosynthetic rate as pND6-AosiL plants show higher
stomatal conductance and transpiration rate when compared to G7-11,
pND6 and pND6-Aos plants (Table 9, infra). Furthermore, chlorophyll
fluorescence measurements clearly show PSII and ETR parameters used
for elucidating the efficiency of PSII are significantly higher
than G7-11, pND6 and pND6-Aos plants (Table 9, infra) in both
27.degree. C. (16 h)/25.degree. C. (8 h) and 27.degree. C. (16
h)/10.degree. C. (8 h) treatment conditions. Having higher PSII and
ETR indicate that amount of light energy absorbed and carbon
assimilated is available more in pND6-AosiL plants to convert into
energy for the plant to use (i.e., growth and development as well
as rubber production) compared to the controls and pND6-Aos plants.
Meanwhile, the NPQ measurements for the pND6-AosiL plants are lower
under the 27.degree. C. (16 h)/25.degree. C. (8 h) condition and
higher under the 27.degree. C. (16 h)/10.degree. C. (8 h) treatment
in comparison with the controls and pND6-Aos plants. Having higher
NPQ suggests that pND6-AosiL have improved heat dissipation ability
compared to G7-11, pND6 and pND6-Aos plants which could help
prevent lipid or other cell membrane damage under environmental
stress.
TABLE-US-00009 TABLE 9 P. argentatum Genotypes Pn g .PHI.PSII ETR
NPQ 27.degree. C. (16 h)/25.degree. C. (8 h) G7-11 5.75 .+-. 0.8
0.093 .+-. 0.03 2.33 .+-. 0.6 0.146 .+-. 0.015 115.73 .+-. 11.3
1.97 .+-. 0.2 pND6-10 6.29 .+-. 0.7 0.110 .+-. 0.03 2.74 .+-. 0.6
0.141 .+-. 0.015 111.1 .+-. 11.6 1.74 .+-. 0.1 pND6-12 6.20 .+-.
0.8 0.109 .+-. 0.02 2.66 .+-. 0.5 0.145 .+-. 0.015 114.4 .+-. 12.1
1.90 .+-. 0.2 pND6-AosiL.sub.7-1 8.56 .+-. 0.6*** 0.147 .+-.
0.01*** 3.41 .+-. 0.3*** 0.196 .+-. 0.027* 165.2 .+-. 10.7*** 1.33
.+-. 0.2*** pND6-AosiL.sub.8-1 8.40 .+-. 0.6*** 0.155 .+-. 0.02***
3.67 .+-. 0.4*** 0.180 .+-. 0.008* 141.4 .+-. 6.2** 1.27 .+-.
0.2*** pND6-AosiL.sub.9-16 7.96 .+-. 0.5*** 0.162 .+-. 0.03*** 3.57
.+-. 0.5*** 0.186 .+-. 0.018* 134.4 .+-. 3.3** 1.19 .+-. 0.3***
pND6-Aos.sub.4-1 5.62 .+-. 0.9 0.110 .+-. 0.04 2.59 .+-. 0.7 0.133
.+-. 0.008 104.7 .+-. 6.7 1.92 .+-. 0.3 pND6-Aos.sub.5-1 5.70 .+-.
0.8 0.110 .+-. 0.04 2.60 .+-. 0.7 0.133 .+-. 0.008 104.7 .+-. 6.7
1.95 .+-. 0.3 pND6-Aos.sub.7-1 5.87 .+-. 0.9 0.113 .+-. 0.04 2.65
.+-. 0.7 0.137 .+-. 0.008 107.7 .+-. 6.3 2.07 .+-. 0.4 27.degree.
C. (16 h)/10.degree. C. (8 h) G7-11 2.23 .+-. 0.5 0.054 .+-. 0.02
1.31 .+-. 0.4 0.070 .+-. 0.007 55.4 .+-. 5.4 1.58 .+-. 0.2 pND6-10
2.04 .+-. 0.4 0.057 .+-. 0.02 1.55 .+-. 0.3 0.064 .+-. 0.006 50.7
.+-. 4.9 1.56 .+-. 0.2 pND6-12 2.28 .+-. 0.4 0.065 .+-. 0.03 1.67
.+-. 0.7 0.066 .+-. 0.003 51.6 .+-. 2.7 1.51 .+-. 0.3
pND6-AosiL.sub.7-1 4.14 .+-. 0.4*** 0.104 .+-. 0.02*** 2.54 .+-.
0.4*** 0.104 .+-. 0.011*** 81.0 .+-. 8.2*** 2.29 .+-. 0.3**
pND6-AosiL.sub.8-1 4.15 .+-. 0.4*** 0.112 .+-. 0.05*** 2.71 .+-.
0.8*** 0.102 .+-. 0.013*** 77.0 .+-. 4.5*** 2.33 .+-. 0.2**
pND6-AosiL.sub.9-16 4.14 .+-. 0.6*** 0.101 .+-. 0.03*** 2.60 .+-.
0.5*** 0.101 .+-. 0.009*** 79.4 .+-. 6.9*** 1.95 .+-. 0.1**
pND6-Aos.sub.4-1 2.27 .+-. 0.5 0.059 .+-. 0.02 1.51 .+-. 0.3 0.065
.+-. 0.011 60.4 .+-. 5.5 1.35 .+-. 0.3 pND6-Aos.sub.5-1 2.97 .+-.
0.3 0.069 .+-. 0.01 1.81 .+-. 0.2 0.077 .+-. 0.007 58.5 .+-. 6.3
1.25 .+-. 0.2 pND6-Aos.sub.7-1 2.45 .+-. 0.6 0.063 .+-. 0.01 1.93
.+-. 0.3 0.065 .+-. 0.010 50.1 .+-. 5.8 1.44 .+-. 0.2 Pn = net
photosynthetic rate; g = stomatal conductance; = Transpiration
rate; .PHI.PSII = Efficiency of Photosystem II; ETR = Electron
Transport Rate; NPQ = Non-photochemical quenching *, ** and
***indicate significant difference in comparison to G7-11 and/or
pND6 (controls) at p > 0.05, 0.005 and 0.0005, respectively.
Example 7. Quality of Natural Rubber from pND6-AOSiL Plants
[0080] The length of the polymer chain, a.k.a. rubber molecular
weight, is the primary determinant of quality in natural rubber.
Gel permeation chromatography (GPC) is used to measure the
molecular weight of rubber from guayule tissue culture plants'
extracts. Cyclohexane extractables collected from ASE (see Example
3 and Table 5 supra) are re-suspended in approximately 3 mL of
tetrahydrofuran (THF) overnight with gentle shaking (Multi-Purpose
Rotator. Thermo Scientific, Waltham. Mass.). The solution is
syringe-filtered through a 1.6 .mu.m glass microfiber GF/A filter
(Whatman GE Healthcare, Piscataway, N.J.), then injected into a
Hewlett Packard 1100 series HPLC (1.0 mL/min flow rate, 50 .mu.L
injection volume, THF continuous phase) and size exclusion
separated by two Agilent PL gel 10 .mu.m Mixed-B columns in series
(35.degree. C.) (Santa Clara, Calif.). The resulting chromatograms
are used to calculate the rubber molecular weight shown in FIG. 6
(using Astra software (Wyatt Technology Corp., Santa Barbara,
Calif.)). The molecular weight of natural rubber from three
pND6-AosiL transformed guayule plants (silenced) is greater than
from wild-type guayule line G7-11, two negative control pND6
transformed guayule plants and three pND6-Aos transformed guayule
plants (overexpressed) indicating better quality rubber in the
PaAos silenced guayule plants. In FIG. 6, the asterisks (*) and
(**) above the three pND6-AosiL transformed guayule plant lines
indicate significant difference in comparison to the negative
control pND6 transformed guayule plant lines at p>0.05 and
p>0.005, respectively.
Example 8. PaAos SNPs Change Protein's Functionality
[0081] Eight different guayule cultivars grown in tissue culture
are evaluated for their rubber content and expression of PaAos
gene. The combination of ASE method (see Example 3 supra) and
qRT-PCR (see the protocols and primers discussed in Example 2,
supra, and FIG. 2) are used to compare rubber content to the level
of PaAos gene expression. First, seeds from guayule lines (PI
478648, W6 549, PI 478651, PI 478652, PI 478653, PI 478654, PI
478655, and PI 478662) are obtained from a public germplasm bank
(USDA-ARS National Plant Germplasm System, Parlier, Calif.). The
seeds are germinated and plants grown in tissue culture medium for
8 weeks. The natural rubber content is determined by standard
methods, as described previously (ASE). The rubber content varied
significantly between lines, from 0.95% to 1.73% (Table 10).
Cultivar (line) W6 549 has the lowest average rubber content (%)
and cultivar PI 478652 has the largest average rubber content (%)
(see Table 10).
TABLE-US-00010 TABLE 10 P. argentatum Germplasm Genotypes name
Average Rubber Content (%) PI 478648 11635 1.38 .+-. 0.08 W6 549
CAL 7 0.95 .+-. 0.03 PI 478651 11701 1.40 .+-. 0.13 PI 478652 12229
1.73 .+-. 0.28 PI 478653 12231 1.36 .+-. 0.11 PI 478654 N396 1.20
.+-. 0.05 PI 478655 N565 0.98 .+-. 0.01 PI 478662 A48118 1.07 .+-.
0.06
[0082] PaAos gene expression for cultivars W6 549 and PI 478652 are
determined by standard methods (qRT-PCR, see Example 2, supra).
Shoot tissues (leaf and stem) are collected into 2 mL tubes and are
snap-frozen in liquid nitrogen and then hand pulverized (mortar and
pestle). RNA is extracted using the TRIzol.RTM. method (Ambion,
Pittsburgh, Pa.) using manufacturer's recommended protocol. The RNA
concentration is quantified with the NanoDrop ND1000
(ThermoScientific, Wilmington, Del.). An RNA cleanup is performed
using RNeasy MinElute Cleanup kit using manufacturer's recommended
protocol (Qiagen Inc., Valencia, Calif.). The RNA is eluted with
30-50 tit of RNase-free water along with on-column DNase1
treatment.
[0083] An iScript cDNA synthesis kit (Bio-Rad, Hercules, Calif.) is
used to synthesize complementary DNA (cDNA) from the isolated RNA.
An amount of 1 .mu.g of RNA is used in the 20 mL reaction mixture.
For semi-qRT-PCR, 2 .mu.L of the diluted cDNA (1:20) is used in a
50 .mu.L reaction mixture of One Taq Quick-Load 2.times. Master Mix
with Standard Buffer (New England Biolabs, Inc., Ipswich, Mass.)
with the following forward and reverse primers (10 .mu.M)
5'-ATGGACCCATCGTCTAAACCC-3' (SEQ ID NO: 16) and
5'-TCATATACTAGCTCTCTTCAGG-3' (SEQ ID NO: 17), respectively. The
semi-qRT-PCR is run using Eppendorf thermocyler (ThermoFisher
Scientific, Waltham, Mass.) with the following thermal cycle:
94.degree. C. pre-incubation for 30 seconds; amplification for 35
cycles at 94.degree. C. for 30 seconds, annealing at 58.degree. C.
for 1.5 minutes, and extension at 68.degree. C. for 1 minute. The
final extension time is for 5 minutes at 68.degree. C. Each
semi-qRT-PCR run is performed with three independent tissue
samples. The 18S gene (.about.200 bp) is used as an internal
control using SEQ ID NO: 5 as forward primer and SEQ ID NO: 6 as
reverse primer. PCR products (.about.1.4 Kbp) are separated by
electrophoresis on a 1% (w/v) agarose gel. Results clearly
demonstrate that W6 549 ("W6549") cultivar has increased PaAos
expression compared to PI 478652 ("478652") cultivar (see FIG. 7).
These results suggest that the rubber content is inversely
correlated with the PaAos gene expression.
[0084] PaAos coding sequence in the two lines, W6 549 cultivar (low
rubber producer) and PI 478652 cultivar (high rubber producer) are
determined by PCR, and the sequences are compared to G7-11 cultivar
sequence. Extraction of the cDNA from the agarose gel is performed
with QIAquick Gel Extraction kit (Qiagen, Germantown, Md.) using
manufacturer's recommended protocol. The 1.4 kb band visualized
with ethidium bromide is excised from the gel with a clean razor
blade. After determining the weight of the gel slice, 300 .mu.l of
Buffer QG pH 7.5 is added for every 100 mg of gel slice with the
DNA fragment size of 100 bp-4 kb. To bind the DNA, 30 .mu.l QIAEX
II beads are added per 5 .mu.g of DNA. The resuspended gel is
dissolved by incubation at 50.degree. C. for 10 minutes with
vigorous vortexing every 2 minutes. Each sample is centrifuged at
16,110.times.g in a conventional table top microcentrifuge for 30
seconds. After centrifugation, each sample rests at room
temperature for five minutes. The supernatant is discarded, and the
pellet is washed with cold 750 .mu.l Buffer PE twice. The pellet is
air dried until it turned solid white. The DNA is resuspended with
50 .mu.l of 10 mM of Tris-Cl pH 8.5. The dissolved DNA pellet
stands at room temperature for 1 minute prior to centrifugation.
The supernatant is collected as the purified cDNA product. To
confirm the integrity of the sequence, three independent PCR
products are sent to Elim Biopharmaceuticals (Hayward, Calif.) for
analysis. The sequence alignment is performed using softwares MEGA
6.06 (Tamura, et al., Mol. Bio. and Evol., 30:2725-2729 (2013)) and
T-Coffee (Notredame, et al., J. Mol. Biol., 302:205-217 (2000)). As
evident in FIGS. 8A-8C, a few SNPs exist which give rise to changes
in the amino acid sequences (see FIG. 9). In particular, the amino
acids at positions 318, 408 and 459 are D, I and L in W6 549
("W6549") cultivar while PI 478652 ("478652") cultivar has N, V and
W, respectively (FIG. 9). These differences in three amino acids
result in different PaAos functionality which result in different
amounts of rubber being produced.
[0085] By reducing PaAos' functionality, one increases rubber
production in guayule. As discussed previously, reducing the amount
of PaAos by silencing PaAos expression or translation increases
rubber production. Null mutations (such as, but not limited to,
insertions that disrupt translation of a functional protein,
changing slice site recognition nucleotide(s), and changing ATG
initiation codon) alter the production of functional PaAos which
result in an increase in rubber production. Alternations in PaAos'
DNA sequence that result in specific amino acid changes within
PaAos also increase rubber production. For example, DNA alterations
that change PaAos sequence from D318, I408 and/or L459 (present in
W6 549 cultivar, low rubber producer) to N318, V408 and W459
(present in PI 478652 cultivar, high rubber producer) (or any other
non-conservative amino acid for D318, I408, and/or L459) result in
an increase in rubber production because of a decrease in PaAos
functionality. In addition, altering PaAos' DNA sequence encoding
S332, E336, R339, S359, and/or S411 to a sequence encoding
non-conservative amino acids results in reducing PaAos'
functionality and thus increasing rubber production. See, Pan, et
al., J. of Bio. Chem., 273(29):18139-18145 (1998), contents of
which are expressly incorporated by reference.
[0086] Based on these results from these assays, one can screen for
the presence of particular SNPs in the PaAos gene in various
guayule varieties at the seedling stage to determine if a
particular guayule variety is a high rubber producer or low rubber
producer. To screen guayule, one obtains a tissue sample from the
guayule to be screened, isolates the sample's mRNA or total RNA,
and conduct a PCR assay (regular PCR or RT-PCR or qRT-PCR) using
PaAos primers that surround the nucleotides encoding amino acids
N318, V408 and W459 to identify guayule plants encoding these amino
acids which indicate that the guayule produces more rubber than a
guayule not having these amino acids within PaAos. Guayule
seedlings (plants that are between 2-4 weeks and 8-10 weeks
post-germination) are screened. Alternatively, guayule plants that
are approximately 2 or 3 months old can be screened. While any
plant tissue can be used to conduct the SNP analysis, bark and
leaves may be easier to sample than other tissue (such as roots).
Using the two primer pairs 5'-CCTACTCGACGCCAAGAG-3' (forward, SEQ
ID NO: 18) and 5'-TTCAGCTGAGCATGTCTAGGT-3' (reverse, SEQ ID NO: 19)
and 5'-GGCATTGTTGAAGTACATATGG-3' (forward, SEQ ID NO: 20) and
5'-CCAAAGGAGACTCGCCTAATT-3' (SEQ ID NO: 21), one determines if the
seedling or plant contains D318, I408 and/or L459 (similar to G7-11
and W6 549 cultivars) and thus produces less rubber than a "high
rubber producer" plant. Alternatively, using the two primer pairs
5'-CCTACTCGACGCCAAAAGC-3' (forward, SEQ ID NO: 22) and
5'-CTTAAGTTGAGCATGTCTAGGTT-3' (reverse, SEQ ID NO: 23) and
5'-GGCATTGTTGAAGTACGTATGG-3' (forward, SEQ ID NO: 24) and
5'-CCCAAGGAGACTCGCCTA-3' (reverse, SEQ ID NO: 25), one determines
if the seedling or plant contains N318, V408 and/or W459 (similar
to PI 478652 cultivar) and thus produces more rubber than a "low
rubber producer" plant. Furthermore, guayule containing PaAos with
S332, E336, R339, S359, and/or S411, in combination with one or
more of D318, I408 and L459, are also low rubber producing plants.
Primers are designed to cover the SNPs for these amino acids which
are used to identify low rubber producing guayule. Similarly,
guayule containing PaAos with non-conservative amino acids to D318,
S332, E336, R339, S359, I408, S411, or L459, or a combination
thereof, are high rubber producing plants, and primers are designed
to cover the SNPs for these amino acids which are used to identify
high rubber producing plants. After performing the PCR assay, one
isolates the amplicon(s) and sequences the amplicon(s) to determine
the presence or absence of the indicated SNPs. Other techniques are
known to one of ordinary skill in the art for identifying amplicons
with the indicated SNPs.
[0087] Alternatively, an ELISA or other type of antibody assay can
distinguish between PaAos containing N318, V408 and/or W459 (PI
478652 cultivar (high producer)) and PaAos containing D318, I408
and/or L459 (W6 549 cultivar (low producer)), with or without one
or more of S332, E336, R339, S359, and S411. An ELISA using a
monoclonal antibody (mAb) that is specific for PaAos containing
N318, V408 and/or W459, with or without one or more
non-conservative amino acids substituted for S332, E336, R339,
S359, and S411, would identify high rubber producing plants.
Alternatively, an ELISA using a mAb that is specific for PaAos
containing D318, I408 and/or L459 with or without one or more amino
acids S332, E336, R339, S359, and S411, would identify low rubber
producing plants. Protein isolated from tissue sample, as described
above, can be contacted with the mAb(s) in the ELISA which then
changes color to indicate the presence of PaAos having the
particular amino acids and structure to which the mAb binds.
[0088] Guayule encoding PaAos with conservative amino acid
substitutions for N318, V408 and/or W459 (and optionally with
non-conservative amino acid substitutions for S332, E336, R339,
S359, and/or S411) are high rubber producing guayule. Similarly,
guayule encoding PaAos with conservative amino acid substitutions
for D318, I408 and/or L459 (and optionally with or without
conservative amino acid substitutions for S332, E336, R339, S359,
and/or S411) are low rubber producing guayule. See Table 2 and
preceding paragraph for information about conservative and
non-conservative amino acid substitutions, and Table 1 for DNA
codons for amino acids.
[0089] One can generate DNA mutations within guayule seed's genome
using EMS, UV light, protons, or other known mutagens to create
altered guayule seeds. Then one germinates the seeds into seedlings
and screen the seedlings for PaAos mutations which reduce PaAos'
functionality. In one embodiment, the above described primers are
used to determine if the indicated SNPs are present in the altered
guayule seedling so that one does not need to grow the altered
guayule for years before determining if the altered guayule is
likely a high rubber producer or a low rubber producer. One can
screen for D318, S332, E336, R339, S359, I408, S411, and/or L459
and conservative amino acids to determine if the altered seedling
is a low rubber producer; or screen for non-conservative amino acid
substitutions to determine if the altered seedling is a high rubber
producer.
[0090] The foregoing detailed description and certain
representative embodiments and details of the invention have been
presented for purposes of illustration and description of the
invention. It is not intended to be exhaustive or to limit the
invention to the precise forms disclosed. It will be apparent to
practitioners skilled in the art that modifications and variations
may be made therein without departing from the scope of the
invention. All references cited herein are incorporated by
reference.
Sequence CWU 1
1
26134DNAArtificial Sequenceprimer, chemical synthesis 1cttaagaggt
ggtatggacc catcgtctaa accc 34229DNAArtificial Sequenceprimer,
chemical synthesis 2ggatcctcat atactagctc tcttcaggg
29324DNAArtificial Sequenceprimer, chemical synthesis 3atgagcccag
aacgacgccc ggcc 24424DNAArtificial Sequenceprimer, chemical
synthesis 4gatctcggtg acgggcagga ccgg 24520DNAArtificial
Sequenceprimer, chemical synthesis 5caacaaaccc cgacttctgg
20620DNAArtificial Sequenceprimer, chemical synthesis 6cacccgtcac
caccatagta 20720DNAArtificial Sequenceprimer, chemical synthesis
7aacccggaag aaaccaaact 20820DNAArtificial Sequenceprimer, chemical
synthesis 8cgcaaccgac tggaaataat 2091422DNAParthenium argentatum
9atggacccat cgtctaaacc cctccgtgaa atccccggct cttatggcat tcctttcttt
60caaccgataa aagaccggtt ggagtatttt tacgggaccg gaggtcgaga cgagtacttc
120cggtcccgca tgcaaaaata ccaatccacg gtatttcgag ccaacatgcc
accgggccct 180ttcgtaagca gcaacccgaa ggtaatcgtc ctactcgacg
ccaaaagctt tccgatactc 240tttgatgtat ccaaagtcga gaagaaagat
ttgttcaccg gaacttacat gccgtcaacc 300aaactcactg gcggctatcg
cgtactctcg tacctcgacc catccgaacc tagacatgct 360caacttaaga
acctcttgtt cttcatgctt aaaaattcaa gcaaccgagt cattcctcag
420ttcgaaacca cttacaccga actctttgaa ggtcttgaag ccgagctagc
caaaaacggg 480aaagccgcgt tcaacgatgt tggtgaacaa gcggctttcc
ggtttttggg cagggcttat 540tttaactcga acccggaaga aaccaaacta
ggaactagtg cgcctacgtt aattagctcg 600tgggtgttat ttaatcttgc
ccccacgctc gacctcggac ttccgtggtt cttgcaggaa 660cctcttctac
acactttccg actgccggcg ttcctgatta agagtactta caacaaactt
720tacgattatt tccagtcggt tgcgactccg gttatggaac aagcagaaaa
attaggggtt 780ccgaaggatg aagctgtgca caatatctta ttcgcggttt
gcttcaatac ttttggtggt 840gttaagatcc tcttcccgaa tacactcaaa
tggatcggac ttgctggtga gaatttgcat 900acccaattgg cggaagagat
tagaggtgct ataaaatcat acggggacgg taacgtgacg 960ctggaagcaa
tcgagcagat gccgttgacg aagtcagtgg tgtacgagtc cctcaggatt
1020gaaccaccag tgcctccgca atatggaaaa gccaaaagca actttaccat
agagtcacac 1080gacgccactt tcgaagtcaa aaaaggagaa atgttattcg
ggtaccaacc gtttgcaacc 1140aaggacccaa aagtatttga ccgacctgag
gaatatgtcc ctgatcggtt cgttggggat 1200ggcgaggcat tgttgaagta
cgtatggtgg tctaatgggc cggagacaga gagtccgaca 1260gttgaaaata
aacaatgtgc cggaaaagac tttgtcgtgc ttataacgag gttgtttgtc
1320attgaacttt tccggcgata tgactctttt gaaatcgaat taggcgagtc
tcctttgggt 1380gcagctgtca cacttacgtt cctgaagaga gctagtatat ga
142210473PRTParthenium argentatum 10Met Asp Pro Ser Ser Lys Pro Leu
Arg Glu Ile Pro Gly Ser Tyr Gly 1 5 10 15 Ile Pro Phe Phe Gln Pro
Ile Lys Asp Arg Leu Glu Tyr Phe Tyr Gly 20 25 30 Thr Gly Gly Arg
Asp Glu Tyr Phe Arg Ser Arg Met Gln Lys Tyr Gln 35 40 45 Ser Thr
Val Phe Arg Ala Asn Met Pro Pro Gly Pro Phe Val Ser Ser 50 55 60
Asn Pro Lys Val Ile Val Leu Leu Asp Ala Lys Ser Phe Pro Ile Leu 65
70 75 80 Phe Asp Val Ser Lys Val Glu Lys Lys Asp Leu Phe Thr Gly
Thr Tyr 85 90 95 Met Pro Ser Thr Lys Leu Thr Gly Gly Tyr Arg Val
Leu Ser Tyr Leu 100 105 110 Asp Pro Ser Glu Pro Arg His Ala Gln Leu
Lys Asn Leu Leu Phe Phe 115 120 125 Met Leu Lys Asn Ser Ser Asn Arg
Val Ile Pro Gln Phe Glu Thr Thr 130 135 140 Tyr Thr Glu Leu Phe Glu
Gly Leu Glu Ala Glu Leu Ala Lys Asn Gly 145 150 155 160 Lys Ala Ala
Phe Asn Asp Val Gly Glu Gln Ala Ala Phe Arg Phe Leu 165 170 175 Gly
Arg Ala Tyr Phe Asn Ser Asn Pro Glu Glu Thr Lys Leu Gly Thr 180 185
190 Ser Ala Pro Thr Leu Ile Ser Ser Trp Val Leu Phe Asn Leu Ala Pro
195 200 205 Thr Leu Asp Leu Gly Leu Pro Trp Phe Leu Gln Glu Pro Leu
Leu His 210 215 220 Thr Phe Arg Leu Pro Ala Phe Leu Ile Lys Ser Thr
Tyr Asn Lys Leu 225 230 235 240 Tyr Asp Tyr Phe Gln Ser Val Ala Thr
Pro Val Met Glu Gln Ala Glu 245 250 255 Lys Leu Gly Val Pro Lys Asp
Glu Ala Val His Asn Ile Leu Phe Ala 260 265 270 Val Cys Phe Asn Thr
Phe Gly Gly Val Lys Ile Leu Phe Pro Asn Thr 275 280 285 Leu Lys Trp
Ile Gly Leu Ala Gly Glu Asn Leu His Thr Gln Leu Ala 290 295 300 Glu
Glu Ile Arg Gly Ala Ile Lys Ser Tyr Gly Asp Gly Asn Val Thr 305 310
315 320 Leu Glu Ala Ile Glu Gln Met Pro Leu Thr Lys Ser Val Val Tyr
Glu 325 330 335 Ser Leu Arg Ile Glu Pro Pro Val Pro Pro Gln Tyr Gly
Lys Ala Lys 340 345 350 Ser Asn Phe Thr Ile Glu Ser His Asp Ala Thr
Phe Glu Val Lys Lys 355 360 365 Gly Glu Met Leu Phe Gly Tyr Gln Pro
Phe Ala Thr Lys Asp Pro Lys 370 375 380 Val Phe Asp Arg Pro Glu Glu
Tyr Val Pro Asp Arg Phe Val Gly Asp 385 390 395 400 Gly Glu Ala Leu
Leu Lys Tyr Val Trp Trp Ser Asn Gly Pro Glu Thr 405 410 415 Glu Ser
Pro Thr Val Glu Asn Lys Gln Cys Ala Gly Lys Asp Phe Val 420 425 430
Val Leu Ile Thr Arg Leu Phe Val Ile Glu Leu Phe Arg Arg Tyr Asp 435
440 445 Ser Phe Glu Ile Glu Leu Gly Glu Ser Pro Leu Gly Ala Ala Val
Thr 450 455 460 Leu Thr Phe Leu Lys Arg Ala Ser Ile 465 470
11313DNAParthenium argentatum 11cgccggaaaa gttcaatcac aaacaacctc
gttataagca cggcaaagtc ttttccggca 60cattgtttat tttcaactga tcggactctc
tgtctccggc ccattagacc accatacgta 120cttcaacaat gcctcgccat
ccccaacgag ccgatcaggg acatattcct caggtcggtc 180aaatactttt
gggtccttgg ttgcaaacgg ttggtacccg aataacattt ctcctttttt
240gacttcgaaa gtggcgtcgt gtgactctat ggtaaagttg cttttggctt
ttccatattg 300cggaggcact ggt 313121422DNAParthenium argentatum
12atggacccat cgtctaaacc cctccgtgaa atccccggct cttatggcat tcctttcttt
60caaccgataa aagaccgatt ggagtatttt tacgggaccg gaggtcgaga cgagtacttc
120cggtcccgca tgcaaaaata ccaatccacg gtatttcgag ccaacatgcc
accgggccct 180ttcgtaagca gcaacccgaa ggtcatcgtc ctactcgacg
ccaagagctt tccgatactc 240tttgatgtat ccaaagtcga gaagaaagat
ttgttcaccg gaacttacat gccgtcaacc 300aaactcactg gcggctaccg
cgtactctcg tacctcgacc catccgaacc tagacatgct 360cagctgaaga
acctcttgtt cttcatgctt aaaaattcaa gcaaccgagt cattcctcag
420ttcgaaacca cttacaccga actctttgaa ggtcttgaag ccgagctagc
caaaaacggg 480aaagccgcgt tcaacgatgt tggtgaacaa gcggctttcc
ggtttttggg cagggcttat 540tttaactcga acccggaaga aaccaaacta
ggaactagtg cgcctacgtt aattagctcg 600tgggtgttat ttaatcttgc
ccccacgctc gacctcggac ttccgtggtt cttgcaggaa 660cctcttctac
acactttccg actgccggcg ttcctgatta agagtactta caacaaactt
720tacgattatt tccagtcggt tgcgactccg gttatggaac aagcagaaaa
attaggggtt 780ccgaaggatg aagctgtgca caatatctta ttcgcggttt
gcttcaatac ttttggtggt 840gtaaagatcc tcttcccgaa tacactcaaa
tggatcggac ttgctggtga gaatttgcat 900acccaattgg cggaagagat
tagaggtgct ataaaatcat acggggacgg tgacgtgacg 960ctggaagcaa
tcgagcagat gccgttgacg aagtcagtgg tgtacgagtc cctcaggatt
1020gaaccaccag tgcctccgca atatggaaaa gccaaaagca actttaccat
agagtcacac 1080gacgccactt tcgaagtcaa aaaaggagaa atgttattcg
ggtaccaacc gtttgcaacc 1140aaggacccaa aagtatttga ccgacccgag
gaatatgtcc ctgatcggtt cgttggggat 1200ggcgaggcat tgttgaagta
catatggtgg tctaatgggc cggagacaga gagtccgaca 1260gttgaaaata
aacaatgtgc cggaaaagac tttgttgtgc ttataacgag gttgtttgtc
1320attgaacttt tccggcgata tgactccttt gaaatcgaat taggcgagtc
tcctttgggt 1380gcagctgtca cacttacgtc cctgaagaga gctagtatat ga
142213473PRTParthenium argentatum 13Met Asp Pro Ser Ser Lys Pro Leu
Arg Glu Ile Pro Gly Ser Tyr Gly 1 5 10 15 Ile Pro Phe Phe Gln Pro
Ile Lys Asp Arg Leu Glu Tyr Phe Tyr Gly 20 25 30 Thr Gly Gly Arg
Asp Glu Tyr Phe Arg Ser Arg Met Gln Lys Tyr Gln 35 40 45 Ser Thr
Val Phe Arg Ala Asn Met Pro Pro Gly Pro Phe Val Ser Ser 50 55 60
Asn Pro Lys Val Ile Val Leu Leu Asp Ala Lys Ser Phe Pro Ile Leu 65
70 75 80 Phe Asp Val Ser Lys Val Glu Lys Lys Asp Leu Phe Thr Gly
Thr Tyr 85 90 95 Met Pro Ser Thr Lys Leu Thr Gly Gly Tyr Arg Val
Leu Ser Tyr Leu 100 105 110 Asp Pro Ser Glu Pro Arg His Ala Gln Leu
Lys Asn Leu Leu Phe Phe 115 120 125 Met Leu Lys Asn Ser Ser Asn Arg
Val Ile Pro Gln Phe Glu Thr Thr 130 135 140 Tyr Thr Glu Leu Phe Glu
Gly Leu Glu Ala Glu Leu Ala Lys Asn Gly 145 150 155 160 Lys Ala Ala
Phe Asn Asp Val Gly Glu Gln Ala Ala Phe Arg Phe Leu 165 170 175 Gly
Arg Ala Tyr Phe Asn Ser Asn Pro Glu Glu Thr Lys Leu Gly Thr 180 185
190 Ser Ala Pro Thr Leu Ile Ser Ser Trp Val Leu Phe Asn Leu Ala Pro
195 200 205 Thr Leu Asp Leu Gly Leu Pro Trp Phe Leu Gln Glu Pro Leu
Leu His 210 215 220 Thr Phe Arg Leu Pro Ala Phe Leu Ile Lys Ser Thr
Tyr Asn Lys Leu 225 230 235 240 Tyr Asp Tyr Phe Gln Ser Val Ala Thr
Pro Val Met Glu Gln Ala Glu 245 250 255 Lys Leu Gly Val Pro Lys Asp
Glu Ala Val His Asn Ile Leu Phe Ala 260 265 270 Val Cys Phe Asn Thr
Phe Gly Gly Val Lys Ile Leu Phe Pro Asn Thr 275 280 285 Leu Lys Trp
Ile Gly Leu Ala Gly Glu Asn Leu His Thr Gln Leu Ala 290 295 300 Glu
Glu Ile Arg Gly Ala Ile Lys Ser Tyr Gly Asp Gly Asp Val Thr 305 310
315 320 Leu Glu Ala Ile Glu Gln Met Pro Leu Thr Lys Ser Val Val Tyr
Glu 325 330 335 Ser Leu Arg Ile Glu Pro Pro Val Pro Pro Gln Tyr Gly
Lys Ala Lys 340 345 350 Ser Asn Phe Thr Ile Glu Ser His Asp Ala Thr
Phe Glu Val Lys Lys 355 360 365 Gly Glu Met Leu Phe Gly Tyr Gln Pro
Phe Ala Thr Lys Asp Pro Lys 370 375 380 Val Phe Asp Arg Pro Glu Glu
Tyr Val Pro Asp Arg Phe Val Gly Asp 385 390 395 400 Gly Glu Ala Leu
Leu Lys Tyr Ile Trp Trp Ser Asn Gly Pro Glu Thr 405 410 415 Glu Ser
Pro Thr Val Glu Asn Lys Gln Cys Ala Gly Lys Asp Phe Val 420 425 430
Val Leu Ile Thr Arg Leu Phe Val Ile Glu Leu Phe Arg Arg Tyr Asp 435
440 445 Ser Phe Glu Ile Glu Leu Gly Glu Ser Pro Leu Gly Ala Ala Val
Thr 450 455 460 Leu Thr Ser Leu Lys Arg Ala Ser Ile 465 470
141422DNAParthenium argentatum 14atggacccat cgtctaaacc cctccgtgaa
atccccggct cttatggcat tcctttcttt 60caaccgataa aagaccgatt ggagtatttt
tacgggaccg gaggtcgaga cgagtacttc 120cggtcccgca tgcaaaaata
ccaatccacg gtatttcgag ccaacatgcc accgggccct 180ttcgtaagca
gcaacccgaa ggtcatcgtc ctactcgacg ccaaaagctt tccgatactc
240tttgatgtat ccaaagtcga gaagaaagat ttgttcaccg gaacttacat
gccgtcaacc 300aaactcactg gcggctaccg cgtactctcg tacctcgacc
catccgaacc tagacatgct 360caacttaaga acctcttgtt cttcatgctt
aaaaattcaa gcaaccgagt cattcctcag 420ttcgaaacca cttacaccga
actctttgaa ggtcttgaag ccgagctagc caaaaacggg 480aaagccgcgt
tcaacgatgt tggtgaacaa gcggctttcc ggtttttggg cagggcttat
540tttaactcga acccggaaga aaccaaacta ggaactagtg cgcctacgtt
aattagctcg 600tgggtgttat ttaatcttgc ccccacgctc gacctcggac
ttccgtggtt cttgcaggaa 660cctcttctac acactttccg actgccggcg
ttcctgatta agagtactta caacaaactt 720tacgattatt tccagtcggt
tgcgactccg gttatggaac aagcagaaaa attaggggtt 780ccgaaggatg
aagctgtgca caatatctta ttcgcggttt gcttcaatac ttttggtggt
840gttaagatcc tcttcccgaa tacactcaaa tggatcggac ttgctggtga
gaatttgcat 900acccaattgg cggaagagat tagaggtgct ataaaatcat
acggggacgg taacgtgacg 960ctggaagcaa tcgagcagat gccgttgacg
aagtcagtgg tgtacgagtc cctcaggatt 1020gaaccaccag tgcctccgca
atatggaaaa gccaaaagca actttaccat agagtcacac 1080gacgccactt
tcgaagtcaa aaaaggagaa atgttattcg ggtaccaacc gtttgcaacc
1140aaggacccaa aagtatttga ccgacctgag gaatatgtcc ctgatcggtt
cgttggggat 1200ggcgaggcat tgttgaagta cgtatggtgg tctaatgggc
cggagacaga gagtccgaca 1260gttgaaaata aacaatgtgc cggaaaagac
tttgtcgtgc ttataacgag gttgtttgtc 1320attgaacttt tccggcgata
tgactctttt gaaatcgaat taggcgagtc tccttggggt 1380gcagctgtca
cacttacgtc cctgaagaga gctagtatat ga 142215473PRTParthenium
argentatum 15Met Asp Pro Ser Ser Lys Pro Leu Arg Glu Ile Pro Gly
Ser Tyr Gly 1 5 10 15 Ile Pro Phe Phe Gln Pro Ile Lys Asp Arg Leu
Glu Tyr Phe Tyr Gly 20 25 30 Thr Gly Gly Arg Asp Glu Tyr Phe Arg
Ser Arg Met Gln Lys Tyr Gln 35 40 45 Ser Thr Val Phe Arg Ala Asn
Met Pro Pro Gly Pro Phe Val Ser Ser 50 55 60 Asn Pro Lys Val Ile
Val Leu Leu Asp Ala Lys Ser Phe Pro Ile Leu 65 70 75 80 Phe Asp Val
Ser Lys Val Glu Lys Lys Asp Leu Phe Thr Gly Thr Tyr 85 90 95 Met
Pro Ser Thr Lys Leu Thr Gly Gly Tyr Arg Val Leu Ser Tyr Leu 100 105
110 Asp Pro Ser Glu Pro Arg His Ala Gln Leu Lys Asn Leu Leu Phe Phe
115 120 125 Met Leu Lys Asn Ser Ser Asn Arg Val Ile Pro Gln Phe Glu
Thr Thr 130 135 140 Tyr Thr Glu Leu Phe Glu Gly Leu Glu Ala Glu Leu
Ala Lys Asn Gly 145 150 155 160 Lys Ala Ala Phe Asn Asp Val Gly Glu
Gln Ala Ala Phe Arg Phe Leu 165 170 175 Gly Arg Ala Tyr Phe Asn Ser
Asn Pro Glu Glu Thr Lys Leu Gly Thr 180 185 190 Ser Ala Pro Thr Leu
Ile Ser Ser Trp Val Leu Phe Asn Leu Ala Pro 195 200 205 Thr Leu Asp
Leu Gly Leu Pro Trp Phe Leu Gln Glu Pro Leu Leu His 210 215 220 Thr
Phe Arg Leu Pro Ala Phe Leu Ile Lys Ser Thr Tyr Asn Lys Leu 225 230
235 240 Tyr Asp Tyr Phe Gln Ser Val Ala Thr Pro Val Met Glu Gln Ala
Glu 245 250 255 Lys Leu Gly Val Pro Lys Asp Glu Ala Val His Asn Ile
Leu Phe Ala 260 265 270 Val Cys Phe Asn Thr Phe Gly Gly Val Lys Ile
Leu Phe Pro Asn Thr 275 280 285 Leu Lys Trp Ile Gly Leu Ala Gly Glu
Asn Leu His Thr Gln Leu Ala 290 295 300 Glu Glu Ile Arg Gly Ala Ile
Lys Ser Tyr Gly Asp Gly Asn Val Thr 305 310 315 320 Leu Glu Ala Ile
Glu Gln Met Pro Leu Thr Lys Ser Val Val Tyr Glu 325 330 335 Ser Leu
Arg Ile Glu Pro Pro Val Pro Pro Gln Tyr Gly Lys Ala Lys 340 345 350
Ser Asn Phe Thr Ile Glu Ser His Asp Ala Thr Phe Glu Val Lys Lys 355
360 365 Gly Glu Met Leu Phe Gly Tyr Gln Pro Phe Ala Thr Lys Asp Pro
Lys 370 375 380 Val Phe Asp Arg Pro Glu Glu Tyr Val Pro Asp Arg Phe
Val Gly Asp 385 390 395 400 Gly Glu Ala Leu Leu Lys Tyr Val Trp Trp
Ser Asn Gly Pro Glu Thr 405 410 415 Glu Ser Pro Thr Val Glu Asn Lys
Gln Cys Ala Gly Lys Asp Phe Val 420 425 430 Val Leu Ile Thr Arg Leu
Phe Val Ile Glu Leu Phe Arg Arg Tyr Asp 435 440 445 Ser Phe Glu Ile
Glu Leu Gly Glu Ser Pro Trp Gly Ala Ala Val Thr 450 455 460
Leu Thr Ser Leu Lys Arg Ala Ser Ile 465 470 1621DNAParthenium
argentatum 16atggacccat cgtctaaacc c 211722DNAParthenium argentatum
17tcatatacta gctctcttca gg 221818DNAParthenium argentatum
18cctactcgac gccaagag 181921DNAParthenium argentatum 19ttcagctgag
catgtctagg t 212022DNAParthenium argentatum 20ggcattgttg aagtacatat
gg 222121DNAParthenium argentatum 21ccaaaggaga ctcgcctaat t
212219DNAParthenium argentatum 22cctactcgac gccaaaagc
192323DNAParthenium argentatum 23cttaagttga gcatgtctag gtt
232422DNAParthenium argentatum 24ggcattgttg aagtacgtat gg
222518DNAParthenium argentatum 25cccaaggaga ctcgccta
18261257DNAArtificial Sequenceanti-sense
RNAmisc_feature(1161)..(1161)n is a, c, g, or t 26ccatggccgc
gggattcgcc ggaaaagttc aatcacaaac aacctcgtta taagcacggc 60aaagtctttt
ccggcacatt gtttattttc aactgatcgg actctctgtc tccggcccat
120tagaccacca tacgtacttc aacaatgcct cgccatcccc aacgagccga
tcagggacat 180attcctcagg tcggtcaaat acttttgggt ccttggttgc
aaacggttgg tacccgaata 240acatttctcc ttttttgact tcgaaagtgg
cgtcgtgtga ctctatggta aagttgcttt 300tggcttttcc atattgcgga
ggcactggtg gatcccccgg gttaagagga gtccaccatg 360agcccagaac
gacgcccggc cgacatccgc cgtgccaccg aggcggacat gccggcggtc
420tgcaccatcg tcaaccacta catcgagaca agcacggtca acttccgtac
ggagccgcaa 480gaaccgcaag agtggacgga cgacctcgtc cgtctgcggg
agcgctatcc ctggctcgtc 540gccgaggtgg acggcgaggt cgccggcatc
gcctacgcgg gcccctggaa ggcacgcaac 600gcctacgact ggacggccga
gtcgaccgtg tacgtctccc cccgccacca gcggacggga 660ctgggctcca
cgctctacac ccacctgctg aagtccctcg aggcacaagg cttcaagagc
720gtggtcgctg tcatcgggct gcccaacgac ccgagcgtgc gcatgcacga
ggcgctcgga 780tatgcccccc gcggcatgct gcgggcggcc ggcttcaagc
acgggaactg gcatgacgtg 840ggtttctggc agctggactt cagcctgccg
gtaccgcccc gtccggtcct gcccgtcacc 900gagagatctc tagtgattgg
atccaccagt gcctccgcaa tatggaaaag ccaaaagcaa 960ctttaccata
gagtcacacg acgccacttt cgaagtcaaa aaaggagaaa tgttattcgg
1020gtaccaaccg tttgcaacca aggacccaaa agtatttgac cgacctgagg
aatatgtccc 1080tgatcggctc gttggggatg acgaggcatt gttgaagtac
gtatggtgat ctaatgggcc 1140ggagacacag agtccgatca nttgaaaata
aacaatgtgc cggaaaagac tttgccgtgc 1200ttataacgag gttgtttgtg
attgaacttt tccggcgccg cgggggagct cggatcc 1257
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