U.S. patent application number 16/359961 was filed with the patent office on 2019-11-14 for methods and compositions for obtaining marker-free transgenic plants.
The applicant listed for this patent is MONSANTO TECHNOLOGY LLC. Invention is credited to Paul S. Chomet, Larry A. Gilbertson, Shihshieh Huang, Susan J. Johnson, Michael W. Petersen, David Walters, Xudong Ye.
Application Number | 20190345507 16/359961 |
Document ID | / |
Family ID | 38663159 |
Filed Date | 2019-11-14 |
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United States Patent
Application |
20190345507 |
Kind Code |
A1 |
Ye; Xudong ; et al. |
November 14, 2019 |
METHODS AND COMPOSITIONS FOR OBTAINING MARKER-FREE TRANSGENIC
PLANTS
Abstract
The invention provides methods and compositions for identifying
transgenic seed that contain a transgene of interest, but lack a
marker gene. Use of an identification sequence that results in a
detectable phenotype increases the efficiency of screening for seed
and plants in which transgene sequences not linked to a gene of
interest have segregated from the sequence encoding a gene of
interest.
Inventors: |
Ye; Xudong; (Chesterfield,
MO) ; Gilbertson; Larry A.; (Chesterfield, MO)
; Huang; Shihshieh; (Woodland, CA) ; Johnson;
Susan J.; (Creve Coeur, MO) ; Petersen; Michael
W.; (Sauk City, WI) ; Walters; David; (North
Stonington, CT) ; Chomet; Paul S.; (Mystic,
CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MONSANTO TECHNOLOGY LLC |
St. Louis |
MO |
US |
|
|
Family ID: |
38663159 |
Appl. No.: |
16/359961 |
Filed: |
March 20, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15376495 |
Dec 12, 2016 |
10240165 |
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16359961 |
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14455747 |
Aug 8, 2014 |
9540700 |
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15376495 |
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13558336 |
Jul 25, 2012 |
8829275 |
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14455747 |
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13271666 |
Oct 12, 2011 |
8237016 |
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13558336 |
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11747824 |
May 11, 2007 |
8076536 |
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13271666 |
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60799875 |
May 12, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/8222 20130101;
C12N 15/8265 20130101; C12N 15/8287 20130101; C12N 15/8205
20130101; C12N 15/8209 20130101; C12N 15/8263 20130101; C12N
15/8294 20130101; C12N 9/1085 20130101; C12N 15/8234 20130101; C12N
15/8261 20130101; C12N 15/8289 20130101; C12N 15/8231 20130101;
C12N 15/8267 20130101; C12N 15/8218 20130101; C12N 15/821 20130101;
C12N 15/829 20130101; C12N 15/8212 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Claims
1-48. (canceled)
49. A method of preparing marker free pollen comprising the steps
of: (a) obtaining a plant transformation construct comprising (i) a
gene of interest; and (ii) a marker gene physically linked to a DNA
cassette comprising an identification sequence that is operably
linked to a promoter functional in a pollen cell; wherein
expression of the DNA cassette affects pollen viability, pollen
development, or seed fertilization; (b) transforming a plant cell
with the transformation construct; and obtaining a transgenic plant
from the transgenic plant cell. (c) producing pollen comprising the
gene of interest and not comprising the identification
sequence.
50. The method of claim 49, further comprising pollinating a second
plant with pollen of the transgenic plant; and selecting a seed
comprising the gene of interest and not comprising the
identification sequence.
51. The method of claim 50, wherein selecting a seed further
comprises assaying the seed for the presence of the gene of
interest and selecting a seed that comprises the gene of
interest.
52. The method of claim 49, wherein the transformation construct is
expressed in pollen and seed tissue.
53. The method of claim 49, wherein the DNA cassette comprises DNA
encoding an antisense or sense RNA that silences an endogenous gene
to result in non-viable pollen.
54. The method of claim 53, wherein the DNA cassette comprises a
pair of inverted repeats of a DNA fragment homologous to the
endogenous gene.
55. The method of claim 54, wherein the inverted DNA fragment
repeat is embedded in an intron within the marker gene.
56. The method of claim 49, wherein the marker gene is a selectable
marker gene.
57. The method of claim 56, wherein the selectable marker gene
encodes a product selected from the group consisting of CP4 EPSPS,
phosphinothricin acetyltransferase, DMO, NptII, glyphosate acetyl
transferase, mutant acetolactate synthase, methotrexate resistant
DHFR, dalapon dehalogenase, PMI, Protox, hygromycin
phosphotransferase and 5-methyl tryptophan resistant anthranilate
synthase.
58. The method of claim 49, wherein the gene of interest and the
identification sequence are bounded by different T-DNA border
sequences.
59. The method of claim 49, wherein the DNA cassette comprises a
nucleic acid sequence operably linked to a promoter selected from
the group consisting of: an LAT52 promoter, an LAT59 promoter, a
maize waxy gene promoter, and a rice small subunit ADP-glucose
pyrophosphorylase promoter.
60. The method of claim 49, wherein the identification sequence
comprises a first transgene and a second transgene.
61. The method of claim 60, wherein the first transgene of the
identification sequence is expressed in pollen, and the second
transgene of the identification sequence is expressed in seeds.
62. The method of claim 49, wherein the identification sequence
produces a protein lethal to pollen.
63. The method of claim 49, wherein the identification sequence
produces a protein that inhibits pollen germination.
64. A DNA construct comprising (i) a gene of interest; and (ii) a
marker gene physically linked to a DNA cassette comprising an
identification sequence that is operably linked to a promoter
functional in a pollen cell; wherein expression of the DNA cassette
affects pollen viability, pollen development, or seed
fertilization.
65. A transgenic cell transformed with the construct of claim
64.
66. The cell of claim 65, wherein the cell is a plant cell.
67. A transgenic plant transformed with the construct of claim
64.
68. A transgenic plant co-transformed with a DNA construct
comprising a first DNA segment comprising left and right T-DNA
borders flanking a gene of interest operably linked to a promoter
functional in plants and a second DNA construct containing a second
DNA segment comprising a second set of left and right T-DNA borders
flanking a promoter functional in a pollen cell operably linked to
a DNA cassette, wherein expression of the DNA cassette affects
pollen viability, pollen development or seed fertilization of a
plant comprising the DNA cassette, and a marker gene operably
linked to a promoter functional in a plant cell.
69. A cell of the plant of claim 68.
70. A DNA construct comprising right and left T-DNA borders,
wherein a first DNA segment comprising a gene of interest operably
linked to a promoter functional in plants is located after the left
border and a second DNA segment comprising a DNA cassette, wherein
expression of the DNA cassette affects pollen viability, pollen
development or seed fertilization of a plant comprising the DNA
cassette, and a marker gene operably linked to a promoter
functional in plants is located after the right border.
71. A DNA construct comprising right and left T-DNA borders,
wherein a first DNA segment comprising a DNA cassette, wherein
expression of the DNA cassette affects pollen viability, pollen
development or seed fertilization of a plant comprising the DNA
cassette and a marker gene operably linked to a promoter functional
in plants is located after the right border, and a second DNA
segment comprising a gene of interest operably linked to a promoter
functional in plants is located after the left border.
72. A DNA construct comprising first and second right T-DNA
borders, wherein a first DNA segment comprising a gene of interest
operably linked to a promoter functional in plants is located after
the first right border and a second DNA segment comprising a DNA
cassette, wherein expression of the DNA cassette affects pollen
viability, pollen development or seed fertilization of a plant
comprising the DNA cassette, and a marker gene operably linked to a
promoter functional in plants is located after the second right
border.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 15/376,495, filed Dec. 12, 2016, now U.S. Pat. No. 10,240,165,
which is a divisional of U.S. application Ser. No. 14/455,747,
filed Aug. 8, 2014, now U.S. Pat. No. 9,540,700, which is a
divisional of U.S. application Ser. No. 13/558,336, filed Jul. 25,
2012, now U.S. Pat. No. 8,829,275, which is a divisional of U.S.
application Ser. No. 13/271,666, filed Oct. 12, 2011, now U.S. Pat.
No. 8,237,016, which is a divisional of U.S. application Ser. No.
11/747,824, filed May 11, 2007, now U.S. Pat. No. 8,076,536, which
claims the priority of U.S. Provisional Application Ser. No.
60/799,875, filed May 12, 2006, the entire disclosures of which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0002] The invention generally relates to transgenic plants. More
specifically, the invention relates to identification and removal
of unwanted or unnecessary DNA in transformed plants.
2. Description of Related Art
[0003] The identification of unnecessary or unwanted transgenic DNA
in transformed plants has been the subject of numerous
investigations and many different methods have been examined in
efforts to eliminate these transgenic sequences from such plants
(e.g. Hanson et al., 1999; Dale et al., 1991; Ebinuma et al., 1997;
Yoder et al., 1994; Kononov et. al., 1997; Hare and Chua, 2002;
Scutt et al., 2002; Puchta, 2003; de Vetten et al., 2003; Halpin,
2005; U.S. Published Appln. 20030110532; U.S. Published Appln.
20040237142; U.S. Pat. No. 6,458,594). In general, it is beneficial
to identify plants that do not include transgenic DNA not
contributing to an agronomically useful trait of the transgenic
plant.
[0004] Many methods for introducing transgenes in plants by
Agrobacterium-mediated transformation utilize a T-DNA (transferred
DNA) that incorporates a transgene and associated genetic elements,
and transfers these into the genome of a plant. Generally, the
transgene(s) is bordered by a right border DNA molecule (RB) and a
left border DNA molecule (LB), and is transferred into the plant
genome, integrating at one or more loci. It has been observed that
when a DNA construct contains more than one T-DNA, these T-DNAs and
the transgenes contained within may be integrated into the plant
genome at separate loci (Framond et al., 1986). This is referred to
as co-transformation.
[0005] The process of co-transformation can be achieved by delivery
of the T-DNAs with a mixture of Agrobacterium strains transformed
with plasmids carrying the separate T-DNAs. Co-transformation can
also be achieved by transforming one Agrobacterium strain with two
or more DNA constructs, each containing one T-DNA. An additional
method employs two T-DNAs on a single DNA vector and identifying
transgenic cells or plants that have integrated the T-DNAs at
different loci. In a non-Agrobacterium-mediated transformation
system, such as a physical method for introducing DNA including
bombardment with microprojectiles, two DNA molecules could be
integrated independently into the target genome, and then segregate
independently in a subsequent generation. Use of 2 T-DNA constructs
allowing for independent insertion of sequences and their genetic
segregation, has also been described (e.g. U.S. Pat. No. 5,731,179;
Zhou et al., 2003; Breitler et al., 2004; Sato et al., 2004). While
the foregoing has furthered the understanding in the art, there
remains a need for improved methods and compositions for obtaining
marker free plants to make product development more efficient.
Previously described screening processes have been highly labor
intensive, for instance requiring Southern blot or PCR.TM. analysis
following growth of R0 and/or R1 plant material.
[0006] U.S. Publication 20060041956 describes use of a visual
marker gene in conjunction with Agrobacterium-mediated
transformation. However, the publication does not describe any
method where such markers are linked to a selectable or screenable
marker gene and unlinked to a gene of interest. Thus, there remains
a great need in the art for methods and compositions that would
improve the ease and efficiency with which plants lacking marker
sequences and/or other transgenic DNA which is not agronomically
useful can be identified and eliminated.
SUMMARY OF THE INVENTION
[0007] In one aspect, the invention provides a method of preparing
marker-free seeds from a transgenic plant comprising the steps of:
a) obtaining seeds of a transgenic plant transformed with a first
DNA segment comprising a nucleic acid of interest and a second DNA
segment comprising a plant marker gene physically and/or
genetically linked to a DNA cassette that is operably linked to a
promoter functional in the seed, wherein the DNA cassette confers a
detectable phenotype to seeds that comprise the DNA cassette; b)
screening the seeds for the absence of the detectable phenotype;
and c) selecting at least a first seed that lacks the detectable
phenotype to obtain a seed that is free of the marker gene. In one
embodiment, step c) further comprises assaying the seed for the
presence of the nucleic acid of interest and selecting a seed that
comprises the nucleic acid of interest and lacks the selectable
marker gene. In certain embodiments, the marker gene is a
selectable or screenable marker gene.
[0008] In certain embodiments, the DNA cassette may be
translationally or transcriptionally fused to the selectable marker
gene; that is, it may encode an RNA that is translationally or
transcriptionally fused to the selectable marker gene. In a further
embodiment the DNA cassette comprises an antisense or sense DNA
fragment with at least 19 or 21 bp of homology to an endogenous
gene, for instance wherein the antisense or sense DNA fragment is
operably linked to a promoter functional in a seed. In yet another
embodiment, the DNA cassette comprises a pair of inverted repeats
of a DNA fragment, wherein each fragment is at least 19 or 21 bp in
size, and wherein the DNA fragment is homologous to an endogenous
gene, operably linked to a promoter functional in the seed. The
inverted DNA fragment repeat homologous to an endogenous gene may
also be embedded in an intron within the selectable marker gene. In
certain embodiments, the DNA cassette encodes a sense or antisense
RNA comprising at least 19 or 21 nucleotides wherein the DNA
fragment is homologous to an endogenous gene.
[0009] In a method of preparing marker-free seeds according to the
invention, seed selected may lack a screenable or screenable gene
and DNA cassette. Obtaining seeds of a transgenic plant may
comprise transforming or co-transforming the transgenic plant or a
progenitor thereof of any previous generation with first and second
DNA segments on separate DNA constructs. Obtaining seeds of a
transgenic plant may also comprise transforming the transgenic
plant or a progenitor thereof of any previous generation with a
single DNA construct comprising the first and second DNA segments.
First and second DNA segments may be bounded by different T-DNA
border sequences. In a method of the invention, a transgenic plant
may be produced by transforming the plant or a progenitor thereof
of any previous generation with a DNA construct comprising (i) the
first DNA segment flanked by left and right T-DNA borders, and (ii)
the second DNA segment flanked by a second set of left and right
T-DNA borders, wherein the second DNA segment further comprises a
selectable marker gene operably linked to a promoter functional in
the transgenic plant. The first and second DNA segments may or may
not be genetically linked in the transgenic plant.
[0010] Transgenic plants used according to the invention may be
produced by introducing first and second DNA segments into the
plant or a progenitor thereof of any previous generation by
transformation mediated by a bacterial strain selected from the
genus Agrobacterium, Rhizobium, Mesorhizobium, or Sinorhizobium.
The transgenic plants may also be produced, for example, by
microprojectile bombardment.
[0011] A selectable marker used with the invention may encode a
product selected from the group consisting of CP4 EPSPS, bar, DMO,
NptII, glyphosate acetyl transferase, mutant acetolactate synthase,
methotrexate resistant DHFR, dalapon dehalogenase, PMI, Protox,
hygromycin phosphotransferase and 5-methyl tryptophan resistant
anthranilate synthase. A DNA cassette sequence for use with the
invention may be selected, for example, from the group consisting
of crtB, gus, gfp, sacB, lux, an anthocyanin synthesis gene,
DefH9-iaaM, rolB, OsCDPK2, AP2, AFR2, ANT transcription factor,
LEC2, Snf-1, cobA, KAS4, splA, zein inverted repeats, B-peru, and
yeast ATP-PFK. The cassette may be operably linked to a promoter
functional in a tissue selected from an embryo, seed endosperm,
cotyledon, aleurone, and seed coat. The promoter may be, for
example, selected from the group consisting of a napin promoter, a
beta-phaseolin promoter, a beta-conglycinin subunit promoter, a
zein promoter, an Osgt-1 promoter, an oleosin promoter, a starch
synthase promoter, a globulin 1 promoter, a barley LTP2 promoter,
an alpha-amylase promoter, a chitinase promoter, a beta-glucanase
promoter, a cysteine proteinase promoter, a glutaredoxin promoter,
a HVA1 promoter, a serine carboxypeptidase II promoter, a catalase
promoter, an alpha-glucosidase promoter, a beta-amylase promoter, a
VP1 promoter, a USP promoter, USP88 promoter, USP99 promoter,
Lectin, and a bronze2 promoter. The detectable phenotype may be
assayed by detection of a catalytic activity. The detectable
phenotype may be selected from the group consisting of seed color,
seed opacity, seed germinability, seed size, seed viability, seed
shape, seed texture, and a defective or aborted seed. Screening of
seeds may be done by an automated seed sorting machine.
[0012] In another aspect, the invention provides a DNA construct
comprising (a) a first DNA segment comprising left and right T-DNA
borders flanking a gene of interest operably linked to a promoter
functional in plants, and (b) a second DNA segment comprising a
second set of left and right T-DNA borders flanking a promoter
functional in a seed operably linked to a DNA cassette that confers
a detectable phenotype in seeds comprising the DNA cassette and a
selectable marker gene operably linked to a promoter functional in
plants. The gene of interest may confer a trait selected from the
group consisting of herbicide tolerance, insect or pest resistance,
disease resistance, increased biomass, modified fatty acid
metabolism, modified carbohydrate metabolism, and modified
nutritional quality. In the construct, the DNA cassette and
selectable marker gene may be operably linked to the same promoter.
In one embodiment, the DNA cassette and the selectable marker gene
are operably linked to different promoters. In specific
embodiments, the selectable marker gene encodes a product selected
from the group consisting of CP4 EPSPS, phosphinothricin
acetyltransferase, DMO, NptII, glyphosate acetyl transferase,
mutant acetolactate synthase, methotrexate resistant DHFR, dalapon
dehalogenase, PMI, Protox, hygromycin phosphotransferase and
5-methyl tryptophan resistant anthranilate synthase. In another
embodiments, the DNA cassette is selected from the group consisting
of crtB, gus, gfp, sacB, lux, an anthocyanin synthesis gene,
DefH9-iaaM, rolB, OsCDPK2, AP2, AFR2, ANT transcription factor,
LEC2, Snf-1, cobA, KAS4, splA, zein inverted repeats, B-peru, and
yeast ATP-PFK. The DNA cassette may be operably linked to a
promoter functional in a tissue selected from the group consisting
of an embryo, seed endosperm, cotyledon, aleurone, and seed coat.
In one embodiment, the DNA cassette is operably linked to a
promoter selected from the group consisting of a napin promoter, a
beta-phaseolin promoter, a beta-conglycinin subunit promoter, a
zein promoter, an Osgt-1 promoter, an oleosin promoter, a starch
synthase promoter, a globulin 1 promoter, a barley LTP2 promoter,
an alpha-amylase promoter, a chitinase promoter, a beta-glucanase
promoter, a cysteine proteinase promoter, a glutaredoxin promoter,
a HVA1 promoter, a serine carboxypeptidase II promoter, a catalase
promoter, an alpha-glucosidase promoter, a beta-amylase promoter, a
VP1 promoter, a USP88 or USP99 promoter, and a bronze2
promoter.
[0013] In yet another aspect, the invention provides transgenic
cells and plants transformed with a construct provided herein. In
one embodiment, a transgenic plant is provided that is
co-transformed with a DNA construct containing a first DNA segment
comprising left and right T-DNA borders flanking a gene of interest
operably linked to a promoter functional in plants and a second DNA
construct containing a second DNA segment comprising a second set
of left and right T-DNA borders flanking a promoter functional in a
seed operably linked to a DNA cassette that confers a detectable
phenotype in seeds comprising the DNA cassette and a selectable
marker gene operably linked to a promoter functional in plants.
Cells of such a plant are also provided.
[0014] In still yet another aspect, the invention provides a DNA
construct comprising right and left T-DNA borders, wherein a first
DNA segment comprising a gene of interest operably linked to a
promoter functional in plants is located after the right border and
a second DNA segment comprising a DNA cassette that confers a
detectable phenotype to plant seeds that comprise the DNA cassette
and a marker gene, such as a selectable marker gene, operably
linked to a promoter functional in plants is located after the left
border.
[0015] In still yet another aspect, the invention provides a DNA
construct comprising right and left T-DNA borders, wherein a first
DNA segment comprising a DNA cassette that confers a detectable
phenotype to plant seeds that comprise the DNA cassette and a
selectable marker gene operably linked to a promoter functional in
plants is located after the right border and a second DNA segment
comprising a gene of interest operably linked to a promoter
functional in plants is located after the left border.
[0016] In still yet another aspect, the invention provides a DNA
construct containing two right T-DNA borders, wherein a first DNA
segment comprising a gene of interest operably linked to a promoter
functional in plants is located after one right border and a second
DNA segment comprising a DNA cassette that confers a detectable
phenotype to plant seeds that comprise the DNA cassette and a
selectable marker gene operably linked to a promoter functional in
plants located after the other right border.
[0017] In yet another aspect, the invention provides an isolated
nucleic acid sequence comprising SEQ ID NO:2, SEQ ID NO:3, or a
sequence with at least 70%, 75%, 85%, or 95% identity to SEQ ID
NO:2 or SEQ ID NO:3, and encoding a polypeptide with phytoene
synthase activity. In one embodiment, the invention also provides a
recombinant DNA construct comprising a nucleic acid sequence of SEQ
ID NO:2 or SEQ ID NO:3, or a recombinant DNA construct comprising a
sequence with at least 71%, 80%, 90%, 95%, 98%, or 99% identity to
SEQ ID NO:2 or SEQ ID NO:3, and encoding a polypeptide with
phytoene synthase activity, operably linked to a heterologous
promoter functional in a plant. A host cell comprising such a
sequence, wherein the cell is a bacterial cell or a plant cell is
another embodiment of the invention. In another embodiment, the
invention provides a transgenic plant or seed comprising SEQ ID
NO:2 or SEQ ID NO:3 SEQ ID NO:2, SEQ ID NO:3, or a sequence with at
least 71%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:2 or
SEQ ID NO:3, and encoding a polypeptide with phytoene synthase
activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The following drawings are part of the present specification
and are included to further demonstrate certain aspects of the
present invention. The invention may be better understood by
reference to the drawings in combination with the detailed
description of specific embodiments presented herein.
[0019] FIGS. 1A-1C. Schematic diagrams of: (A) pMON10338; (B)
pMON10339; and (C) pMON67465.
[0020] FIG. 2. CrtB expression in soybean tissues transformed with
pMON67465.
[0021] FIG. 3. CrtB expression in seed from event A33908.
[0022] FIG. 4. Expression of crtB, gus, and CP4/EPSPS in immature
R1 seed.
[0023] FIG. 5. Expression of crtB in mature R1 seed.
[0024] FIG. 6. GUS staining of pMON67465 seed.
[0025] FIG. 7. CP4 & CrtB PCR on GUS positive seeds.
[0026] FIG. 8. Comparison of linkage-Southern and screenable-marker
approaches for screening transgenic events.
[0027] FIG. 9. Schematic summary of DNA sequences transferred by
use of construct comprising a screenable gene linked to CP4
selectable marker genes for marker-free seeds. A) GOI located in
one T-DNA flanked with a RB and LB and physically linked to a
second T-DNA containing a screenable gene linked to a CP4
selectable marker gene in one construct used for
Agrobacterium-mediated transformation; B) One vector containing two
borders, the GOI is placed after a RB while the screenable and
selectable marker genes are placed after the second RB or after a
LB together with backbone; C) The GOI and screenable genes--DNAs
are separated in two vectors and transformed in either one
Agrobacterium cell or separate Agrobacterium cells; D) Possible
linkage of two DNA segments from the GOI and screenable and
selectable marker genes. Only the GOI alone will show normal seed
appearance, while cells containing the screenable gene show a
visible phenotype; E) Two separate DNA segments contain either the
GOI or screenable and selectable marker genes used for
non-bacterial mediated transformation.
[0028] FIG. 10. pMON67420 represents a GOI construct for
co-transformation.
[0029] FIG. 11. Plasmid pMON99575 containing Schizosaccharomyces
pombe ATP dependent phosphofructokinase driven by the seed-specific
zein promoter.
[0030] FIG. 12. Corn ear expressing seed-specific yeast ATP
dependent phosphofructokinase abolished normal kernel
development.
[0031] FIG. 13. Schematic diagram of dsRNA-encoding constructs used
to demonstrate that inverted repeats placed within an intron of a
marker gene result in a visible phenotype.
[0032] FIG. 14. Inverted repeats embedded in an intron give rise to
a visible phenotype.
[0033] FIG. 15. Silencing of a-zeins in corn kernels leads to a
visible phenotype.
[0034] FIG. 16. Schematic diagram of pMON83530 containing KAS4.
[0035] FIG. 17. Progeny of soybean seeds transformed with
pMON83530. Seeds on the left are shrunken due to expression of KAS4
and indicate the presence of selectable marker, while seeds on the
right are normal and marker-free.
[0036] FIG. 18. Schematic diagram of pMON107314 containing KAS4
useful as an identification sequence in a 2T DNA construct.
[0037] FIG. 19. Schematic diagram of pMON68581 containing a splA
gene useful as an identification gene.
[0038] FIG. 20. Progeny soybean seeds transformed with pMON68581.
Seeds on the right are shrunken due to the expression of splA and
indicate the presence of the screenable or selectable marker, while
seeds on the left are normal and marker-free.
[0039] FIG. 21. 2 T-DNA vector formats-schematic diagram.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The following is a detailed description of the invention
provided to aid those skilled in the art in practicing the present
invention. Those of ordinary skill in the art may make
modifications and variations in the embodiments described herein
without departing from the spirit or scope of the present
invention.
[0041] The invention overcomes deficiencies in the prior art by
providing constructs and methods allowing for efficiently
distinguishing seeds lacking an identification sequence and
marker-gene sequence, but including a gene or genes of interest
(GOI), from seeds that contain an identification gene sequence and
marker-gene sequence, based on a phenotype conferred by a
seed-expressed identification gene, sequence, or cassette. In
particular, the present invention provides, in one embodiment,
transformation constructs and methods for transformation of plant
cells which include: (i) a gene of interest; and (ii) an
identification sequence expressed in seed and physically linked to
a selectable or screenable marker gene that may be expressed in
various plant tissues, wherein the construct and/or transformation
method is designed so that the genetic elements of (i) and (ii) can
integrate independently into the plant genome, and thus genetically
segregate from each other.
[0042] The expression or lack thereof of the identification
sequence in seed tissues allows for direct identification of seeds
and plants that lack the seed-expressed identification sequence and
the physically linked selectable or screenable marker gene, while
allowing for choice of seed and plants still containing the
transgene of interest. Choosing seed including the gene of interest
and lacking marker-gene sequences at the seed level represents a
significant advance in that it avoids the need for previously
utilized screening methods that are comparatively cost and labor
intensive. Additionally, time may be saved as screening can be done
prior to or during germination without requiring growth of the next
generation of plants to a size permitting tissue harvest, as needed
for linkage-Southern analysis, for example.
[0043] In one embodiment, transformation of plant tissue is
performed by an Agrobacterium or other Rhizobia-mediated method
(See e.g., U.S. Provisional Patent Application Ser. No. 60/800,872,
filed May 16, 2006, entitled "Use of Non-Agrobacterium Bacterial
Species for Plant Transformation" and assigned attorney docket No.
MONS:100USP1, the entire disclosure of which is specifically
incorporated herein by reference), and the DNA sequences including
the identification sequence expressed in seed and the physically
linked selectable or screenable marker gene are present together on
a T-DNA or other sequence (e.g., vector backbone) that is
transferred into a plant cell (e.g. flanked by T-DNA RB and/or LB
sequences or without a border sequence). The identification
sequence and marker gene may be transferred to the plant physically
linked, while the gene of interest is present on a separate T-DNA
flanked by its own RB and LB sequences, or other sequence
transferred into a plant cell, and can be integrated at an
independent locus (e.g. FIG. 21). The selectable and/or screenable
marker permits identification of transformed plant tissues. Fertile
plants can be obtained and selfed or crossed in a breeding scheme
in order to follow segregation of phenotypes in the next
generation. Strategies for performing such breeding are well known
in the art, and may vary in details between different plants. Seed
expression, or lack of expression, of the identification sequence
permits facile identification of seed with respect to the presence
of marker and identification sequences. The gene of interest, for
example, may contain at least one plant expression cassette
encoding a trait selected from the group consisting of herbicide
tolerance, antibiotic resistance, insect resistance, disease
resistance, stress resistance (e.g., drought and cold), enhanced
nutrient use efficiency, enhanced nutritive content (e.g., amino
acid, protein, sugars, carbohydrates, fatty acids, and/or oil),
sterility systems, industrial enzymes (e.g., pharmaceuticals and
processing enzymes for bio-fuels) and enhanced yield.
[0044] The sequences that may be transferred into a plant cell
(e.g. T-DNAs) may be present on one transformation vector in a
bacterial strain being utilized for transformation. In another
embodiment, the sequences including the identification sequence and
plant selectable marker, and the sequence(s) comprising the gene(s)
of interest may be present on separate transformation vectors in
the bacterial strain. In yet another embodiment, the T-DNA
including the identification sequence and plant selectable marker
and the T-DNA comprising the gene of interest may be found in
separate bacterial cells or strains used together for
transformation.
[0045] In still another embodiment, DNA sequences including the (i)
gene of interest; and (ii) identification sequence expressed in
seed and physically linked to a selectable or screenable marker
gene may be introduced into a plant cell by a physical method such
as microprojectile bombardment. In such an embodiment, the DNA
sequences of (i) and (ii) can be located on separate DNA fragments
that may be mixed together prior to or during the coating of
microprojectiles with DNA. The DNA sequences may be present on a
single microprojectile, or they may be present on separate
microprojectiles that are mixed together prior to bombardment.
[0046] The phenotype conveyed by the identification sequence can be
achieved by ectopic overexpression of a heterogenous or endogenous
gene linked to a constitutive or seed-specific promoter, or by
downregulation of an endogenous gene using antisense RNA, RNA
interference or co-suppression technology. Examples of the
endogenous gene may include, but are not limited to, genes involved
in sugar/starch metabolism, protein metabolism, and fatty acid
metabolism.
[0047] In one embodiment, seed expression of the identification
sequence results in a detectable phenotype in seed of a transgenic
plant containing an identification sequence. In some embodiments
the phenotype may be detected by visual inspection, and may include
a change in seed color, opacity (or translucence), fluorescence,
texture, size, shape, germinability, viability, or generally any
component or property that is physically or biochemically assayable
and different from that found in the nontransgenic recipient
genotype. In certain embodiments, the identification sequence
includes a gusA, gfp (Pang et al., 1996), phytoene synthase, or
phytoene desaturase encoding gene, or an anthocyanin gene (P1, Lc,
B-Peru, C1, R, Rc, mybA or mybI (e.g. Selinger et al., 1998; Ludwig
et al., 1989; Himi et al., 2005; Kobayashi et al., 2002)). In a
particular embodiment, the identification gene comprises a crtB
gene encoding a phytoene synthase (U.S. Pat. Nos. 5,429,939;
6,429,356; U.S. Pat. No. 5,545,816), including a gene comprising a
crtB sequence codon-optimized for expression in a monocot plant,
such a corn plant. Another example of gene that could be used in
this regard is a gene involved in production of seed pigment.
[0048] In other embodiments, the phenotype is assayable by
detection of a catalytic activity. In yet other embodiments, the
phenotype is a tissue ablation phenotype, for instance a blockage
in the formation of pollen, egg, or seed tissue. Compositions and
methods that silence genes required for the production or viability
of gametes, reducing or preventing fertilizations that include the
marker gene, are also envisioned. For example, sequences could be
used that result in the silencing of genes required for pollen
development and viability. The pollen that are derived from meiotic
segregants carrying the marker gene would not develop or would be
inviable, thus preventing the transmission of the marker gene to
the progeny through the pollen. In outcross pollinations, all
progeny would be marker free. Use of sequences that result in
silencing of other endogenous genes (e.g. RNAi technologies
including miRNA) to result in a seed phenotype is also envisioned.
Such genes include, but are not limited to: genes encoding or
modifying expression of seed storage proteins such as zeins,
Opaque2, Waxy, and other genes encoding proteins involved in
carbohydrate, protein, and/or lipid accumulation in seeds.
[0049] Expression of an identification sequence that confers a
phenotype of nonviable pollen is also desirable because only the
pollen grains without the identification sequence will be capable
of fertilizing eggs thus increasing the yield of seeds free of the
identification sequence and the marker sequence when the transgenic
line carrying the identification sequence under the control of a
pollen-specific promoter is used as a male pollinator. The
identification sequence can produce a protein that is lethal to the
pollen or inhibitory to pollen germination. Alternatively,
expression of a pair of inverted repeats homologous to an essential
endogenous pollen gene can be used to silence the gene rendering
the pollen nonviable. Examples of pollen specific genes and
promoters are known to those skilled in the art and include for
instance LAT52 and LAT59 genes and promoters of tomato as described
(Eyal et al., 1995).
[0050] In another embodiment, the identification sequence can be
expressed in both the seed and the pollen for further enhancing the
selection of seeds with the gene of interest and eliminating the
seeds with the identification sequence and the marker gene. This
can be achieved by using an identification sequence comprised of
two transgenes; the one of which expresses in the seed and the
other expresses in the pollen. Alternatively, a promoter that can
express the same identification sequence in the pollen and seed can
also result in a detectable phenotype in both the pollen and the
seed. Examples of promoters that express in pollen and seed are the
promoters from the maize Waxy gene (zmGBS; Shure et al., 1983), and
the rice small subunit ADP-glucose pyrophosphorylase gene (osAGP;
Anderson et al., 1991). Pollen and seed expression patterns are
also described in Russell and Fromm, 1997.
[0051] The identification sequence may alternatively alter
carbohydrate, protein, lipid, or other products of cell or seed
metabolism so as to yield a detectable phenotype. In one
embodiment, the identification sequence allows for
endosperm-specific expression of a sacB gene encoding a
levansucrase or a yeast ATP-dependent phosphofructokinase (ATP-PFK)
which abolishes starch accumulation in seeds containing the
identification sequence and marker gene (Caimi et al., 1996; FIG.
12). The identification sequence can be a gene. The identification
sequence can further encode a transcriptional or translational
fusion (e.g. U.S. Pat. No. 6,307,123; U.S. Patent Publication
20060064772).
[0052] U.S. Pat. No. 6,307,123 relates to the construction of a
translational fusion between a selectable marker gene (nptII) and a
screenable marker gene (gfp). The method can be applied to produce
a fusion between an identification sequence described herein and a
selectable or a screenable marker described herein.
[0053] Another method that can be used to make a polypeptide fusion
is based on the Ubiquitin (Ub) processing pathway. This method can
be used to cleave a long polypeptide comprising two protein domains
into two separate active proteins. In this method, a single gene
cassette can encode two ORFs, where the two ORFs, e.g., for crtB
and EPSPS-CP4 are separated by the 14 C-terminal amino acids of Ub,
followed by a full-length Ub sequence. After translation in vivo,
endogenous de-ubiquitinating enzymes (DUBs) cleaves the polyprotein
into three separate units: 1) the N-terminal protein, which
comprises the identification sequence crtB terminating in the 14
C-terminal amino acids of Ub; 2) a Ub monomer; and 3) the
C-terminal polypeptide, which encodes a selectable marker
EPSPS-CP4. Such methods are known to those skilled in the art (e.g.
Walker et al, 2007).
[0054] A transcriptional fusion between an identification sequence
and a selectable or a screenable marker can be made by using
internal ribosome entry sites (IRES). For example, a transcript
could be made that encodes the ORF of crtB followed by the ORF of
ESPSP-CP4 with a functional IRES element positioned between them.
Several IRES are known to those skilled in the art (see e.g.,
Dinkova et al. 2005 and references therein; and U.S. Pat. No.
7,119,187, incorporated herein by reference).
[0055] The phenotype of the identification sequence in seed tissue
may also be detected by methods including visual, biochemical,
immunological and nucleic-acid based (e.g. PCR-based) methods,
among others. The identification sequence may confer a detectable
phenotype in seed tissue that may be distinguished from the
phenotype of the marker gene. The phenotype conferred by the
identification sequence can include altered seed germination. The
identification sequence may be expressed in one or more portions of
a seed (kernel), including the embryo, endosperm, cotyledon(s), and
seed coat (testa), such that a phenotype may be discerned.
[0056] The identification sequence may also cause a seedless
phenotype. To accomplish tissue ablation, in one embodiment, the
identification sequence directs ovule-specific expression of
defH9-iaaM or rolB in plants (e.g. Rotino et al. 1997, Carmi et al.
2003, GenBank AM422760, X64255, AE009418), which abolishes ovule
development and results in a seedless phenotype in marker and
identification sequence-containing ovaries. In yet another
embodiment, the identification sequence directs over expression of
OsCDPK2 in cereal crops disrupting seed development (Morello et al.
2000; e.g., GenBank Y13658).
[0057] Genetic elements may also be designed to suppress the
expression of an endogenous gene, resulting in the production of a
seed phenotype that permits distinguishing of seeds that contain
the marker gene from those that do not. The genetic elements of
this identification sequence are physically linked to the marker
gene, e.g. embedded within the marker DNA cassette, such that the
seed phenotype is linked to the presence of the marker, allowing
for the rapid identification of marker containing seeds.
[0058] RNAi may be used to silence one or more genes resulting in
an easily scored, preferably visible, seed phenotype. The DNA
sequences required for an RNAi-mediated seed phenotype are
positioned on the same T-DNA as the marker gene. Any progeny seed
that contains the marker gene would also display the seed phenotype
and would be easily identified. Such seeds would not need to be
grown and screened for the presence of the marker gene. Thus, only
seeds without the phenotype conferred by the identification
sequence are grown and/or screened for the presence of the GOI.
Depending on whether the seeds are from self-pollination or
outcrossing, this method reduces the number of seeds that need to
be planted and screened by at least 3.times. for selfing plant
species or 1.times. for outcrossing plant species.
[0059] A wide variety of compositions are known to those skilled in
the art that can be used to silence a target gene using RNAi
related pathways. One embodiment is to assemble a DNA cassette that
will transcribe an inverted repeat of sequences, to produce a
double-stranded RNA (dsRNA), typically at least about 19-21 bp in
length and corresponding to a portion of one or more genes targeted
for silencing. The dsRNA can be about 19-21 bp in length and
corresponding to a portion of one or more genes targeted for
silencing. This DNA cassette including an identification sequence
is positioned within the same T-DNA as the selectable marker gene.
Other methods to silence a gene known to those skilled in the art
include, but are not limited to: cosuppression, antisense,
expression of miRNAs (natural or engineered), expression of
trans-acting siRNAs, and expression of ribozymes. Any of these
methods may be used if the sequences required for the gene
silencing effect are positioned in the same T-DNA as the marker
gene.
[0060] The identification sequence may increase or decrease seed
size. In one embodiment, the identification sequence confers down
regulation of AP2 gene (e.g., GenBank U12546) by antisense RNA or
RNA interference or cosuppression technology (Jofuku et al., 2005),
which results in larger seeds containing the identification
sequence and selectable marker genes. The larger seed size may also
be achieved by ectopic expression of an AFR2 gene (Schruff et al.,
2006; e.g., GenBank Accessions NM_203251; NM_180913), or ANT
transcription factor (Mizukami and Fisher, 2000; e.g., GenBank
NM_202701, NM_119937, NM_180024, NM_101474, NM_202110). In another
embodiment, the identification sequence conveys down regulation of
a LEC2 (e.g., GenBank AF400123) or a Snf-1 (GenBank AB101657,
AB101656, AB101655) gene by antisense RNA or RNAi or cosuppression
technology, which leads to decreased seed size (Mendoza et al.,
2005; Radchuk et al., 2006). The changed seed size can be easily
sorted by weight, shape or sieving by manual and/or mechanical
means.
[0061] It is specifically contemplated that automated screening
techniques may be implemented with the current invention for the
identification of seeds having a particular detectable phenotype.
In this manner large numbers of seeds can be efficiently screened
and seeds lacking an identification sequence may be collected.
Automated techniques may be faster, less expensive and more
accurate than reliance upon human technicians. Such seed sorting
machines which could be used in this manner have been described.
For example, U.S. Pat. No. 4,946,046 describes an apparatus for
sorting seeds according to color. In this machine, seeds are sorted
according to color by placing the seeds in uniform rows of
indentations in a rotating drum and passing the seeds beneath a
digital imaging camera and a light source. Images are read by the
camera and are fed to a computer, which also receives information
from a drum speed sensor. The computer generates a signal which
causes a blast of air to blow through an opening in the bottom of
an indentation containing a colored seed to collect such seed.
Collected seeds are fed into a collection hopper, and the
non-colored seeds into a separate hopper.
[0062] By varying the wavelength of the light source used for
detection of colored seeds, as well as barrier filters placed
between the colored seed and the detection camera, potentially any
identification marker could be detected with this technique. For
example, to detect seeds expressing GFP, the excitation wavelength
is in the blue light UV spectrum, typically at about 395 nm.
Suitable light sources for UV emission are well known to those of
skill in the art, and include xenon or mercury lamps. Suitable
filter sets also are well known to those of skill in the art, and
include, for example, a BP450-490 exciter filter, an FT510
chromatic beam splitter, and a BP515-565 barrier filter (Carl
Zeiss, Inc., Thornwood, N.Y.). Such filter sets and emission
wavelengths are discussed in more detail in Heim and Tsien, 1996,
the disclosure of which is specifically incorporated herein by
reference in its entirety.
[0063] By use of constructs including one or more identification
sequence(s), the selective power can be extended to multiple
selectable and/or screenable genes and genes of interest.
Therefore, large numbers of transgenic seeds, representing a
variety of different transformation events, can be efficiently
screened and only those seeds having (or lacking) a desired set of
identification sequences may be selected.
[0064] A recombinant DNA vector may, for example, be a linear DNA
segment or a closed circular plasmid. The vector system may be a
single vector or plasmid or two or more vectors or plasmids that
together contain the total DNA to be introduced into the plant
genome. Nucleic acid molecules as set forth herein can, for
example, be suitably inserted into a vector under the control of a
suitable promoter that functions in a plant cell to drive
expression of a linked coding sequence or other DNA sequence. Many
vectors are available for this purpose, and selection of the
appropriate vector will depend mainly on the size of the nucleic
acid to be inserted into the vector and the particular host cell to
be transformed with the vector. Each vector contains various
components depending on its function and the particular vector and
plant cell with which it is used or is compatible.
[0065] A number of vectors suitable for stable transformation of
plant cells or for the establishment of transgenic plants are well
known, e.g., Gelvin et al. (1990). Typically, plant expression
vectors include, but are not limited to, one or more gene of
interest transcription units, each of which includes: a 5'
untranslated region, which includes sequences that control
transcription (e.g., cis-acting promoter sequences such as
enhancers, the transcription initiation start site, etc.) and
translation (e.g., a ribosome binding site) of an operably linked
protein-coding sequence ("open reading frame", ORF); a 3'
untranslated region that includes additional regulatory regions
from the 3' end of plant genes (Thornburg et al., 1987); An et al.,
1989), e.g., a 3' terminator region to increase mRNA stability.
Alternatively a plant expression vector may be designed for
expression of an mRNA molecule that may, for instance, alter plant
gene expression by an RNAi-mediated approach. In addition, such
constructs commonly include a selectable and/or screenable marker
transcription unit and optionally an origin of replication or other
sequences required for replication of the vector in a bacterial
host cell.
[0066] The constructs may also contain the plasmid backbone DNA
segments that provide replication function and antibiotic selection
in bacterial cells, for example, an Escherichia con origin of
replication such as ori322, a broad host range origin of
replication such as oriV or oriRi, and a coding region for a
selectable marker such as Spec/Strp that encodes for Tn7
aminoglycoside adenyltransferase (aadA) conferring resistance to
spectinomycin or streptomycin, or a gentamicin (Gm, Gent)
selectable marker gene. For plant transformation, the host
bacterial strain is often Agrobacterium tumefaciens ABI, C58,
LBA4404, EHA101, or EHA105 carrying a plasmid having a transfer
function for the expression unit. Other strains known to those
skilled in the art of plant transformation can function in the
present invention.
[0067] Plant expression vectors optionally include RNA processing
signals, e.g., introns, which may be positioned upstream or
downstream of a polypeptide-encoding sequence in the transgene. In
addition, the expression vectors may also include additional
regulatory sequences from the 3'-untranslated region of plant
genes. These 3' untranslated regions contain mRNA transcription
termination signals. Other movable elements contained in plant
expression vectors may include 5' leader sequences, transit signal
sequences, and coding sequences.
[0068] Expression and cloning vectors may contain a selection gene,
also referred to as a plant selectable marker. This gene encodes a
protein necessary for the survival or growth of transformed plant
cells grown in a selective culture regimen. Typical selection genes
encode proteins that confer resistance to selective agents such as
antibiotics including herbicides, or other toxins, e.g., neomycin,
methotrexate, dicamba, glufosinate, or glyphosate. Those cells that
are successfully transformed with a heterologous protein or
fragment thereof produce a protein conferring, e.g. drug resistance
and thus survive the selection regimen. Examples of various
selectable/screenable/scorable markers and genes encoding them are
disclosed in Miki and McHugh, 2004.
[0069] An expression vector for producing a mRNA can also contain
an inducible or tissue specific promoter that is recognized in the
host plant cell and is operably linked to the nucleic acid
encoding, the nucleic acid molecule, or fragment thereof, of
interest. Plant promoters are discussed below.
[0070] In one embodiment, the plant transformation vector that is
utilized includes an isolated and purified DNA molecule including a
heterologous seed-specific promoter operatively linked to one or
more nucleotide sequences of the present invention. In another
embodiment, the promoter is seed-expressed, but not seed-specific.
A plant transformation vector may contain sequences from one or
more genes, thus allowing production of more than mRNA in a plant
cell. One skilled in the art will readily appreciate that segments
of DNA can be combined into a single composite DNA segment for
expression in a transgenic plant.
[0071] Suitable methods for transformation of host cells for use
with the current invention are believed to include virtually any
method by which DNA can be introduced into a cell (see, for
example, Miki et al., 1993), such as by transformation of
protoplasts (U.S. Pat. No. 5,508,184; Omirulleh et al., 1993), by
desiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985),
by electroporation (U.S. Pat. No. 5,384,253), by agitation with
silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. No.
5,302,523; and U.S. Pat. No. 5,464,765), by Agrobacterium-mediated
transformation (U.S. Pat. Nos. 5,563,055; 5,591,616; 5,693,512;
5,824,877; 5,981,840; 6,384,301) and by acceleration of DNA coated
particles (U.S. Pat. Nos. 5,015,580; 5,550,318; 5,538,880;
6,160,208; 6,399,861; 6,403,865; Padgette et al. 1995), etc.
Through the application of techniques such as these, the cells of
virtually any species may be stably transformed. In the case of
multicellular species, the transgenic cells may be regenerated into
transgenic organisms.
[0072] The most widely utilized method for introducing an
expression vector into plants is based on the natural
transformation system of Agrobacterium (for example, Horsch et al.,
1985) The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,
respectively, carry genes responsible for genetic transformation of
the plant. Descriptions of Agrobacterium vector systems and methods
for Agrobacterium-mediated gene transfer are provided by numerous
references, including Gruber et al., 1993; Miki et al., 1993,
Moloney et al., 1989, and U.S. Pat. Nos. 4,940,838 and 5,464,763.
Other bacteria such as Sinorhizobium, Rhizobium, and Mesorhizobium
that interact with plants naturally can be modified to mediate gene
transfer to a number of diverse plants (Broothaerts et al., 2005).
These plant-associated symbiotic bacteria can be made competent for
gene transfer by acquisition of both a disarmed Ti plasmid and a
suitable binary vector. DNA sequences to be transferred via an
Agrobacterium-mediated transformation method include one or more
"border" sequences, such as right border (RB) and left border (LB)
sequences that usually define the extent of the transferred DNA
(T-DNA) containing one or more genes to be expressed in a plant
cell, and may further include an enhancer sequence such as an
overdrive sequence (Toro et al., 1989) or a plurality of overdrive
sequences as disclosed in U.S. Provisional Patent Application No.
60/831,814, incorporated herein by reference.
[0073] Techniques that may be particularly useful in the context of
cotton transformation are disclosed in U.S. Pat. Nos. 5,846.797,
5,159,135, 5,004,863, and 6,624,344. Techniques for transforming
Brassica plants in particular are disclosed, for example, in U.S.
Pat. No. 5,750,871; and techniques for transforming soybean are
disclosed in, for example, Zhang et al., 1999, U.S. Pat. No.
6,384,301, and U.S. Pat. No. 7,002,058. Techniques for transforming
corn are disclosed in WO9506722. Some non-limiting examples of
plants that may find use with the invention include alfalfa,
barley, beans, beet, broccoli, cabbage, carrot, canola,
cauliflower, celery, Chinese cabbage, corn, cotton, cucumber, dry
bean, eggplant, fennel, garden beans, gourd, leek, lettuce, melon,
oat, okra, onion, pea, pepper, pumpkin, peanut, potato, pumpkin,
radish, rice, sorghum, soybean, spinach, squash, sweet corn,
sugarbeet, sunflower, tomato, watermelon, and wheat.
[0074] A vector or construct may also include various regulatory
elements. The 5' non-translated leader sequence can be derived from
the promoter selected to express the heterologous gene sequence of
the DNA molecule of the present invention, and can be specifically
modified if desired so as to increase translation of mRNA. The 5'
non-translated regions can also be obtained from plant viral RNAs
(e.g. Tobacco mosaic virus, Tobacco etch virus, Maize dwarf mosaic
virus, Alfalfa mosaic virus) from suitable eukaryotic genes, plant
genes (wheat and maize chlorophyll a/b binding protein gene
leader), or from a synthetic gene sequence. The leader sequence
could also be derived from an unrelated promoter or coding
sequence. Leader sequences useful in context of the present
invention include the maize Hsp70 leader (U.S. Pat. No. 5,362,865
and U.S. Pat. No. 5,859,347, herein incorporated by reference in
their entirety.), and the TMV omega element (Gallie et al., 1989).
Examples of translation leader sequences include maize and petunia
heat shock protein leaders (U.S. Pat. No. 5,362,865), plant virus
coat protein leaders, plant rubisco leaders, GmHsp (U.S. Pat. No.
5,659,122), PhDnaK (U.S. Pat. No. 5,362,865), AtAnt1, TEV
(Carrington and Freed, 1990), AGRtunos (GenBank Accession V00087;
Bevan et al., 1983), OsAct1 (U.S. Pat. No. 5,641,876), OsTPI (U.S.
Pat. No. 7,132,528), and OsAct15 (US Publication No. 20060162010),
among others.
[0075] Intron sequences are known in the art to aid in the
expression of transgenes in monocot plant cells. Examples of
introns include the corn actin intron (U.S. Pat. No. 5,641,876),
the corn HSP70 intron (ZmHSP70; U.S. Pat. No. 5,859,347; U.S. Pat.
No. 5,424,412), and rice TPI intron (OsTPI; U.S. Pat. No.
7,132,528), and are of benefit in practicing this invention.
[0076] A vector may also include a transit peptide nucleic acid
sequence. Many chloroplast-localized proteins, including those
involved in carotenoid synthesis, are expressed from nuclear genes
as precursors and are targeted to the chloroplast by a chloroplast
transit peptide (CTP) that is removed after the import steps.
Examples of other such chloroplast proteins include the small
subunit (SSU) of Ribulose-1,5,-bisphosphate carboxylase, and the
light-harvesting complex protein I and protein II. It has been
demonstrated in vivo and in vitro that non-chloroplast proteins may
be targeted to the chloroplast by use of protein fusions with a CTP
and that a CTP sequence is sufficient to target a protein to the
chloroplast. Incorporation of a suitable chloroplast transit
peptide, such as the Arabidopsis thaliana (At) EPSPS CTP (Klee et
al., 1987), and the Petunia hybrida (Ph.) EPSPS CTP (della-Cioppa
et al., 1986) has been shown to target heterologous protein
sequences to chloroplasts in transgenic plants. Those skilled in
the art will recognize that various chimeric constructs can be
made, if needed, that utilize the functionality of a particular CTP
to import a given gene product into a chloroplast. Other CTPs that
may be useful in practicing the present invention include
PsRbcS-derived CTPs (Pisum sativum Rubisco small subunit CTP;
Coruzzi et al., 1984); AtRbcS CTP (Arabidopsis thaliana Rubisco
small subunit 1A CTP; CTP1; U.S. Pat. No. 5,728,925); AtShkG CTP
(CTP2; Klee et al., 1987); AtShkGZm CTP (CTP2synthetic; codon
optimized for monocot expression; SEQ ID NO:14 of WO04009761);
PhShkG CTP (Petunia hybrida EPSPS; CTP4; codon optimized for
monocot expression; Gasser et al., 1988); TaWaxy CTP (Triticum
aestivum granule-bound starch synthase CTPsynthetic, codon
optimized for corn expression: Clark et al., 1991): OsWaxy CTP
(Oryza sativa starch synthase CTP; Okagaki, 1992); NtRbcS CTP
(Nicotiana tabacum ribulose 1,5-bisphosphate carboxylase small
subunit chloroplast transit peptide; Mazur, et al., 1985); ZmAS CTP
(Zea mays anthranilate synthase alpha 2 subunit gene CTP; Gardiner
et al., 2004); and RgAS CTP (Ruta graveolens anthranilate synthase
CTP; Bohlmann, et al., 1995). Other transit peptides that may be
useful include maize cab-m7 signal sequence (PCT WO 97/41228) and
the pea (Pisum sativum) glutathione reductase signal sequence (PCT
WO 97/41228).
[0077] Termination of transcription may be accomplished by a 3'
non-translated DNA sequence operably linked to a recombinant
transgene (e.g. the gene of interest, the identification sequence
including a screenable gene, or the plant selectable marker
gene).
[0078] The 3' non-translated region of a recombinant DNA molecule
contains a polyadenylation signal that functions in plants to cause
the addition of adenylate nucleotides to the 3' end of the RNA. The
3' non-translated region can be obtained from various genes that
are expressed in plant cells. The nopaline synthase 3' untranslated
region (Fraley et al., 1983), is commonly used in this capacity.
Polyadenylation molecules from a Pisum sativum RbcS2 gene
(Ps.RbcS2-E9; Coruzzi et al., 1984), AGRtu.nos (Genbank Accession
E01312), E6 (Accession #U30508), rice glutelin (Okita et al.,
1989), and TaHsp17 (wheat low molecular weight heat shock protein
gene; Accession #X13431) in particular may be of benefit for use
with the invention.
[0079] For embodiments of the invention in which the use of a
constitutive promoter is desirable, any well-known constitutive
plant promoter may be used. Constitutive plant promoters include,
for example, the cauliflower mosaic virus (CaMV) 35S promoter,
which confers constitutive, high-level expression in most plant
tissues (see, e.g., Odell et al., 1985), including monocots (see,
e.g., Dekeyser et al., 1990); Terada et al., 1990); the nopaline
synthase promoter (An et al., 1988), the octopine synthase promoter
(Fromm et al., 1989), cauliflower mosaic virus 19S promoter,
figwort mosaic virus 35S promoter, rice actin 1 promoter, mannopine
synthase promoter, and a histone promoter.
[0080] For other embodiments of the invention, well-known plant
gene promoters that are regulated in response to environmental,
hormonal, chemical, and/or developmental signals may be used,
including promoters regulated by (1) heat (Callis et al., 1988),
(2) light (e.g., pea rbcS-3A promoter, Kuhlemeier et al., 1989;
maize rbcS promoter, Schaffner and Sheen, 1991; or chlorophyll
a/b-binding protein promoter, Simpson et al., 1985), (3) hormones,
such as abscisic acid (Marcotte et al., 1989), (4) wounding (e.g.,
wunl, Siebertz et al., 1989); or (5) chemicals such as methyl
jasmonate, salicylic acid, etc. It may also be advantageous to
employ (6) organ-specific promoters (e.g., Roshal et al., 1987;
Schernthaner et al., 1988; Bustos et al., 1989).
[0081] There are a wide variety of plant promoter sequences which
may be used to drive tissue-specific expression of polynucleotides
in transgenic plants. Indeed, in particular embodiments of the
invention, the promoter used is a seed specific promoter. The
promoter for .beta.-conglycinin (Chen et al., 1989) or other
seed-specific promoters such as the napin promoter, which are
regulated during plant seed maturation (Kridl et al., 1991;
Kohno-Murase et al., 1994), barley Hv.Per1 (Stacey et al., 1996),
phaseolin (Bustos et al., 1989), soybean trypsin inhibitor (Riggs
et al., 1989), ACP (Baerson et al., 1993), stearoyl-ACP desaturase
(Slocombe et al., 1994), soybean .alpha.' subunit of
.beta.-conglycinin (P-Gm7S, see for example, Chen et al., 1986),
Vicia faba USP (P-Vf.Usp, see for example, SEQ ID NO: 1, 2, and 3,
U.S. Appln. Pub. 20030229918), the globulin promoter (see for
example Belanger and Kriz, 1991), soybean alpha subunit of
.beta.-conglycinin (7S alpha; U.S. Pat. No. 6,825,398, incorporated
by reference) and Zea mays L3 oleosin promoter (P-Zm.L3, see, for
example, Hong et al., 1997; see also U.S. Pat. No. 6,433,252, the
disclosure of which is specifically incorporated herein by
reference).
[0082] The zeins are a group of storage proteins found in Zea mays
endosperm. Genomic clones for zein genes have been isolated
(Pedersen et al., 1982; U.S. Pat. No. 6,326,527), and the promoters
from these clones, including the 15 kDa, 16 kDa, 19 kDa, 22 kD, 27
kDa, and gamma genes, could also be used. Other promoters known to
function, for example, in Zea mays include the promoters for the
following genes: waxy (Russell and Fromm, 1997; Shure et al.,
1983), Brittle (Giroux et al., 1994), Shrunken 2, Branching enzymes
I and II, starch synthases, debranching enzymes, oleosins,
glutelins, and sucrose synthases. Another promoter for Zea mays
endosperm expression is the promoter for the glutelin gene from
rice, more particularly the Osgt-1 promoter (Zheng et al., 1993).
Examples of such promoters in rice include those promoters for the
ADPGPP subunits, the granule bound and other starch synthase, the
branching enzymes, the debranching enzymes, sucrose synthases (Yang
et al., 1990), and Betl1 (basal endosperm transfer layer) and
globulin1.
[0083] Examples of other promoters that may be useful with the
present invention are described in the U.S. Pat. No. 6,437,217
(maize RS81 promoter), U.S. Pat. No. 5,641,876 (rice actin
promoter; OsAct1), U.S. Pat. No. 6,426,446 (maize RS324 promoter),
U.S. Pat. No. 6,429,362 (maize PR-1 promoter), U.S. Pat. No.
6,232,526 (maize A3 promoter), U.S. Pat. No. 6,177,611
(constitutive maize promoters), U.S. Pat. Nos. 5,322,938,
5,352,605, 5,359,142 and 5,530,196 (35S promoter), U.S. Pat. No.
6,433,252 (maize L3 oleosin promoter), U.S. Pat. No. 6,429,357
(rice actin 2 promoter as well as a rice actin 2 intron), U.S. Pat.
No. 5,837,848 (root specific promoter), U.S. Pat. No. 6,294,714
(light inducible promoters), U.S. Pat. No. 6,140,078 (salt
inducible promoters), U.S. Pat. No. 6,252,138 (pathogen inducible
promoters), U.S. Pat. No. 6,175,060 (phosphorus deficiency
inducible promoters), U.S. Pat. No. 6,635,806 (gamma-coixin
promoter), and U.S. Pat. No. 7,151,204 (maize chloroplast aldolase
promoter). Additional promoters that may find use are a nopaline
synthase (NOS) promoter (Ebert et al., 1987), the octopine synthase
(OCS) promoter (which is carried on tumor-inducing plasmids of
Agrobacterium tumefaciens), the caulimovirus promoters such as the
cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., 1987),
the CaMV 35S promoter (Odell et al., 1985), the figwort mosaic
virus 35S-promoter (Walker et al., 1987), the sucrose synthase
promoter (Yang et al., 1990), the R gene complex promoter (Chandler
et al., 1989), and the chlorophyll a/b binding protein gene
promoter, etc. In the present invention, CaMV35S with enhancer
sequences (e35S; U.S. Pat. Nos. 5,322,938; 5,352,605; 5,359,142;
and 5,530,196), FMV35S (U.S. Pat. Nos. 6,051,753; 5,378,619),
peanut chlorotic streak caulimovirus (PC1SV; U.S. Pat. No.
5,850,019), At.Act 7 (Accession #U27811), At.ANT1 (US Patent
Application Publication 20060236420), FMV.35S-EF1a (U.S. Patent
Application Publication 20050022261), eIF4A10 (Accession #X79008)
and AGRtu.nos (GenBank Accession V00087; Depicker et al, 1982;
Bevan et al., 1983), rice cytosolic triose phosphate isomerase
(OsTPI; U.S. Pat. No. 7,132,528), and rice actin 15 gene (OsAct15;
U.S. Patent Application Publication 20060162010) promoters may be
of particular benefit. In some instances, e.g., OsTPI and OsAct 15,
a promoter may include a 5'UTR and/or a first intron. Other
promoters useful in the practice of the invention that are known by
one of skill in the art are also contemplated by the invention.
[0084] A plant expression vector may also include a screenable or
scorable marker gene cassette that may be used in the present
invention to monitor segregating cells or progeny for (loss of)
expression. Exemplary markers are known and include
.beta.-glucuronidase (GUS) that encodes an enzyme for various
chromogenic substrates (Jefferson et al., 1987a; Jefferson et al.,
1987b); an R-locus gene, that encodes a product that regulates the
production of anthocyanin pigments (red color) in plant tissues
(Dellaporta et al., 1988); a .beta.-lactamase gene (Sutcliffe et
al., 1978); a gene that encodes an enzyme for that various
chromogenic substrates are known (e.g., PADAC, a chromogenic
cephalosporin); a luciferase gene (Ow et al., 1986); a xy1E gene
(Zukowsky et al., 1983) that encodes a catechol dioxygenase that
can convert chromogenic catechols; an .alpha.-amylase gene (Ikatu
et al., 1990); a tyrosinase gene (Katz et al., 1983) that encodes
an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone
that in turn condenses to melanin; green fluorescence protein
(Elliot et al., 1999) and an a-galactosidase. A screenable or
scorable marker gene may encode the same gene product as an
identification sequence including a screenable or scorable gene, or
a different gene product. However, the identification sequence is
expressed in egg, pollen or seed tissues, while the screenable or
scorable marker gene is expressed during the process of identifying
transformed plant cells. The identification sequence may also be
expressed constitutively, but only convey a phenotype in egg,
pollen, or seed tissues.
[0085] Transgenic plants may be regenerated from a transformed
plant cell by methods well known in the field of plant cell
culture. A transgenic plant formed using Agrobacterium
transformation methods typically contains a single simple
recombinant DNA sequence inserted into one chromosome and is
referred to as a transgenic event. Such transgenic plants can be
referred to as being heterozygous for the inserted exogenous
sequence. A transgenic plant homozygous with respect to a transgene
can be obtained by sexually mating (selfing) an independent
segregant transgenic plant that contains a single exogenous gene
sequence to itself, for example an F0 plant, to produce F1 seed.
One fourth of the F1 seed produced will be homozygous with respect
to the transgene. Germinating F1 seed results in plants that can be
tested for zygosity, typically using a SNP assay or a thermal
amplification assay that allows for the distinction between
heterozygotes and homozygotes (i.e., a zygosity assay).
[0086] A number of identification sequences may be used, for
instance genes whose expression may result in a visible phenotype,
including use of gus, gfp, and luc (see, e.g., Ow et al., 1986; WO
97/41228 and U.S. Pat. No. 6,583,338; e.g., M26194; M15077). A
levansucrase gene, sacB (Caimi et al., 1996; e.g., X02730) leading
to a "shrunken" seed phenotype, or a pyrophosphatase gene
(Hajirezaei et al., 1999) leading to inhibition of germination, may
also be employed. Genes encoding phytoene synthase (crtB) are known
in the art, including those from Erwinia uredovora (e.g. Misawa et
al., 1990; Sandmann and Misawa, 1992; U.S. Pat. Nos. 5,429,939;
6,429,356), and Pantoea/Enterobacter agglomerans (e.g. GenBank
M38423; M87280), among others. Seed-specific expression of crtB
that results in orange coloration has been described (Shewmaker et
al., 1999; U.S. Pat. No. 6,429,356).
[0087] Most transgenes producing pleiotropic seed phenotypes may be
used as a visible label gene linked to a selectable marker to
identify marker-free gene of interest positive seeds. The visible
phenotype may be produced by ectopic overexpression of a transgene
or result from down regulation of endogenous metabolic pathway
genes by antisense RNA, RNA interference or co-suppression
technology.
[0088] The following definitions and methods are provided to better
define the present invention and to guide those of ordinary skill
in the art in the practice of the present invention. Unless
otherwise noted, terms are to be understood according to
conventional usage by those of ordinary skill in the relevant art.
Definitions of common terms in molecular biology may also be found
in Rieger et al. (1991); and Lewin (1994). The nomenclature for DNA
bases as set forth at 37 CFR .sctn. 1.822 is used.
[0089] "CP4", "aroA:CP4", "AGRTU.aroA:CP4", "CP4 EPSPS" and "EPSPS
CP4" refer to the EPSP synthase gene or protein purified from
Agrobacterium tumefaciens (AGRTU) strain CP4 that when expressed in
plants confers tolerance to glyphosate and glyphosate containing
herbicide formulations (U.S. Pat. No. 5,633,435, herein
incorporated by reference in its entirety). The gene sequence may
be native or modified for enhanced expression in plants.
[0090] A DNA "segment" refers to a region of DNA sequence of a DNA
construct. A DNA segment may be within, between, or flanking the
T-DNA molecules found in a construct used for
Agrobacterium-mediated plant cell transformation. For instance, a
DNA segment may contain genetic elements for replication of
plasmids in bacteria or other various elements and expression
cassettes of the DNA construct designed for use in plant cell
transformation. Thus, a "DNA cassette" may comprise a DNA segment,
including element(s) for expression of the DNA sequence in a
cell.
[0091] A "fusion protein" refers to a translational fusion
expressed as a single unit, yet producing a gene product conferring
the phenotypes of the protein encoded by the non-fused starting
gene sequences.
[0092] An "isolated" nucleic acid is substantially separated or
purified away from other nucleic acid sequences in the cell of the
organism in which the nucleic acid naturally occurs, i.e., other
chromosomal and extrachromosomal DNA and RNA, by conventional
nucleic acid-purification methods. The term also embraces
recombinant nucleic acids and chemically synthesized nucleic
acids.
[0093] The term "glyphosate resistance gene" refers to any gene
that, when expressed as a transgene in a plant, confers the ability
to tolerate levels of the herbicide glyphosate that would otherwise
damage or kill the plant. Any glyphosate tolerance gene known to
the skilled individual are suitable for use in the practice of the
present invention. Glyphosate (including any herbicidally active
form of N-phosphonomethylglycine and any salt thereof) inhibits the
enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). A
variety of native and variant EPSPS enzymes have been expressed in
transgenic plants in order to confer glyphosate tolerance, any of
which can be used in the invention. Examples of some of these
EPSPSs include those described and/or isolated in accordance with
U.S. Pat. No. 4,940,835, U.S. Pat. No. 4,971,908, U.S. Pat. No.
5,145,783, U.S. Pat. No. 5,188,642, U.S. Pat. No. 5,310,667, and
U.S. Pat. No. 6,803,501. They can also be derived from a
structurally distinct class of non-homologous EPSPS genes, such as
the class II EPSPS genes isolated from Agrobacterium sp. strain CP4
(AGRTU.aroA:CP4).
[0094] The term "identification sequence" refers to a nucleic acid
that encodes a product conferring a detectable phenotype such as a
change in seed or gamete color, opacity or translucence,
fluorescence, texture, size, shape, germinability, or viability, or
other product of cell or seed metabolism. The identification
sequence may include a nucleotide sequence (e.g. a gene fragment)
that may confer a phenotype via down regulation of the expression
of another gene, such as via an RNAi-mediated process. In certain
embodiments, the identification sequence includes a screenable gene
such as a gusA, gfp, or crtB gene. In a particular embodiment, the
identification sequence includes a crtB gene encoding a phytoene
synthase from Erwinia herbicola (Pantoea agglomerans; GenBank
M38423, incorporated herein by reference; and U.S. Pat. Nos.
5,429,939, 6,429,356). The identification sequence is physically
linked to a plant selectable, screenable, and/or scorable marker
gene, such as one encoding antibiotic resistance or herbicide
tolerance. The identification sequence can confer a detectable
(e.g. screenable or selectable) phenotype in seed.
[0095] A first nucleic-acid sequence is "operably" connected or
"linked" with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is operably
linked to a protein-coding sequence if the promoter affects the
transcription or expression of the coding sequence. Generally,
operably linked DNA sequences are contiguous and, where necessary
to join two protein-coding regions, are in the same reading
frame.
[0096] The term "plant" encompasses any higher plant and progeny
thereof, including monocots (e.g., lily, corn, rice, wheat, barley,
etc.), dicots (e.g., soybean, cotton, tomato, canola, potato,
Arabidopsis, tobacco, etc.), gymnosperms (pines, firs, cedars,
etc.) and includes parts of plants, including reproductive units of
a plant (e.g., seeds, bulbs, tubers, or other parts or tissues from
that the plant can be reproduced), fruit, flowers, etc.
[0097] A "recombinant" nucleic acid is made by an artificial
combination of two otherwise separated segments of sequence, e.g.,
by chemical synthesis or by the manipulation of isolated segments
of nucleic acids by genetic engineering techniques.
[0098] The terms "DNA construct" or "DNA vector" refers to any
plasmid, cosmid, virus, autonomously replicating sequence, phage,
or other circular single-stranded or double-stranded DNA or RNA
derived from any source that includes one or more DNA sequences,
such as promoters, protein-coding sequences, 3' untranslated
regions, etc., that have been linked in a functionally operative
manner by recombinant DNA techniques. Recombinant DNA vectors for
plant transformation are commonly double-stranded circular plasmids
capable of replication in a bacterial cell. Conventional
compositions and methods for making and using recombinant nucleic
acid constructs are well known, e.g. Sambrook et al., 1989; and
Ausubel et al., 1992 (with periodic updates), and Clark et al.
(1997), among others.
[0099] The term "promoter" or "promoter region" refers to a nucleic
acid sequence, usually found upstream (5') to a coding sequence
that controls expression of the coding sequence by controlling
production of messenger RNA (mRNA) by providing the recognition
site for RNA polymerase and/or other factors necessary for start of
transcription at the correct site. As contemplated herein, a
promoter or promoter region includes variations of promoters
derived by means of ligation to various regulatory sequences,
random or controlled mutagenesis, and addition or duplication of
enhancer sequences. A promoter region is responsible for driving
the transcription of coding sequences under their control when
introduced into a host as part of a suitable recombinant vector, as
demonstrated by its ability to produce mRNA.
[0100] "Regeneration" refers to the process of growing a plant from
a plant cell (e.g., plant protoplast or explant).
[0101] "Selectable marker" refers to a nucleic acid sequence whose
expression confers a phenotype facilitating identification of cells
containing the nucleic acid sequence. Selectable markers include
those that confer resistance to toxic chemicals (e.g. antibiotic
resistance), or impart a visually distinguishing characteristic
(e.g. color changes or fluorescence).
[0102] Useful dominant plant selectable marker genes include genes
encoding antibiotic resistance genes (e.g. resistance to
hygromycin, imidazolinone, kanamycin, bleomycin, G418, streptomycin
or spectinomycin); and herbicide resistance genes (e.g.
phosphinothricin acetyltransferase, modified ALS, BAR, modified
class I EPSPSs, class II EPSPSs, DMOs), among others.
[0103] Included within the terms "scorable marker genes" or
"screenable marker genes" are genes that encode a secretable marker
whose secretion can be detected as a means of identifying or
selecting for transformed cells. Examples include markers that
encode a secretable antigen that can be identified by antibody
interaction, or even secretable enzymes that can be detected
catalytically. Secretable proteins fall into a number of classes,
including small, diffusible proteins that are detectable, (e.g., by
ELISA), small active enzymes that are detectable in extracellular
solution (e.g. .alpha.-amylase, .beta.-lactamase, phosphinothricin
acetyltransferase), or proteins that are inserted or trapped in the
cell wall (such as proteins that include a leader sequence such as
that found in the expression unit of extension or tobacco PR-S).
Other possible selectable and/or screenable marker genes will be
apparent to those of skill in the art.
[0104] "T-DNA" refers to a DNA molecule that integrates into a
plant genome via an Agrobacterium or other Rhizobia-mediated
transformation method. At least one end of the T-DNA molecule is
flanked by at least one border region of the T-DNA from an
Agrobacterium Ti or Ri plasmid. These border regions are generally
referred to as the Right border (RB) and Left border (LB) regions
and exist as variations in nucleotide sequence and length depending
on their source (e.g. nopaline or octopine producing strains of
Agrobacterium). The border regions commonly used in DNA constructs
designed for transferring transgenes into plants are often several
hundred polynucleotides in length and include a nick site where
virD2 endonuclease derived from Ti or Ri helper plasmid digests the
DNA and covalently attaches to the 5' end after T-strand formation
to guide the T-strand integration into the genome of a plant. The
T-DNA molecule(s) generally contain one or more plant expression
cassettes.
[0105] The term "transgene" refers to any nucleic acid sequence
nonnative to a cell or organism transformed into said cell or
organism. "Transgene" may also refer to any endogenous sequence
which is ectopically expressed by modifying coding sequence or
regulatory sequences. "Transgene" also encompasses the component
parts of a native plant gene modified by insertion of a nonnative
or native nucleic acid sequence by directed recombination.
EXAMPLES
[0106] Those of skill in the art will appreciate the many
advantages of the methods and compositions provided by the present
invention. The following examples are included to demonstrate the
preferred embodiments of the invention. It should be appreciated by
those of skill in the art that the techniques disclosed in the
examples that follow represent techniques discovered by the
inventors to function well in the practice of the invention, and
thus can be considered to constitute preferred modes for its
practice. However, those of skill in the art should, in light of
the present disclosure, appreciate that many changes can be made in
the specific embodiments that are disclosed and still obtain a like
or similar result without departing from the spirit and scope of
the invention. All references cited herein are incorporated herein
by reference to the extent that they supplement, explain, provide a
background for, or teach methodology, techniques, or compositions
employed herein.
Example 1
Preparation of 2T-DNA Vectors with an Identification Sequence and
Marker Gene, and a Gene-of-Interest
[0107] Two T-DNA plant expression vectors, pMON67465, pMON101338
and pMON101339 (FIG. 1), were constructed according to standard
molecular cloning procedure (Sambrook et al., 1989). One T-DNA
includes a CaMV 35S promoter operably linked to an nptII gene
encoding resistance to kanamycin and a CaMV 35S promoter operably
linked to a GUS reporter gene. The other T-DNA comprises a
napin:ctp-crtB: napin 3' cassette and a 35S:ctp:CP4 EPSPS cassette,
that confers glyphosate resistance. The crtB in pMON67465 with oriV
replication origin was driven by a 1.8 kb seed-specific napin
promoter and 1 kb napin terminator from Brassica napus. The crtB in
pMON101338 with oriV replicon and in pMON101339 with pRi
replication origin was driven by a shorter version of napin
promoter (1 kb) and terminator (0.3 kb). The crtB gene, encoding a
phytoene synthase from Erwinia sp. confers orange color to soybean
seed without affecting transformation frequency (FIG. 3).
Example 2
Transformation and Regeneration of Soy Explants with pMON67465
[0108] Soybean (cv. A3244) tissues were transformed with pMON67465
(FIG. 1) via an
[0109] Agrobacterium-mediated method, essentially as previously
described (U.S. Pat. No. 6,384,301, herein incorporated by
reference). Briefly, hand excised soy meristem explants were
co-cultivated with Agrobacterium for 2-4 days at 23.degree. C.,
transferred onto WPM solid medium with 75 .mu.M glyphosate
selection in a PLANTCON and cultured at 28.degree. C. under 16/8
light/dark period. After two weeks, the explants were transferred
to fresh WPM medium and cultured until shoot harvest. After 2
months, shoots with true trifolia were cut and cultured onto BRM
rooting medium for 2-4 weeks. The rooted plantlets were grown in
greenhouse for seed maturation. Among 40 events analyzed, 72.5%
displayed co-transformation with both T-DNAs. 45% of the 40 events
contained both nptII and gus genes. Event A33908, containing T-DNAs
from pMON67465, was further analyzed.
[0110] Additional events with plasmid pMON67465 were obtained by
re-transformation of the plasmid into the same cultivar A3244 and
13 transgenic lines were obtained with transformation frequency of
0.22%. Seven out of 13 lines are gene of interest-positive and
marker free after analyzing normal appearance seeds via PCR for
presence or absence of a gus or CP4 marker gene (FIG. 6). The
orange seeds from R0 plants were also analyzed by PCR for the
presence of the crtB gene and CP4 marker gene. All orange seeds but
three were found to be positive for both crtB and CP4 (FIG. 7),
whereas none of normal appearance seeds (FIG. 7, white cells)
contained CP4 or crtB genes, which indicated the crtB phenotype is
tightly linked to the CP4 selectable marker gene.
[0111] Thus, use of a 2 T-DNA construct with an identification
sequence including a screenable gene linked to a selectable marker
gene allows for more efficient screening and selection of
transgenic events containing a gene of interest while lacking
sequences encoding a selectable marker. An exemplary comparison
between a linkage-Southern based approach ("Standard 2T") and a
label-based (i.e., identification sequence based) screen for
identifying progeny seed in which a gene of interest and a
selectable marker have independently segregated is found in FIG. 8.
Use of the identification sequence approach allows for screening
more transgenic events and more progeny of each event in order to
identify progeny useful for further analysis.
Example 3
CrtB/GUS/CP4 Expression in Soy Event A33908 and R1 Progeny Seed
[0112] Visual inspection of tissues from event A33908 (FIG. 2)
indicated that stems and young unfolded leaves displayed an orange
cast. Leaf and root tissue was otherwise phenotypically normal in
CrtB-expressing plants. Seed coats from seed of the R0 plant (i.e.
the R1 generation) displayed slight CrtB expression, while the
cotyledons of A33908-derived seed displayed a distinct orange color
and some with a wrinkled phenotype (FIG. 3).
[0113] Twelve immature R1 seeds from event A33908 were dissected
from the seed coat and subjected to CP4-EPSPS ELISA, and CrtB and
GUS-visual analyses (FIG. 4). Segregation of the CrtB, GUS, and
CP4-EPSPS phenotypes was evident. 9/12 seed were positive in all
three assays. 1/12 seed was CrtB and CP4-EPSPS positive, but GUS
negative, showing segregation of the two T-DNAs of pMON67465, with
loss of the gus gene. 2/12 seed were CP4-EPSPS ELISA negative. Both
of these seed were CrtB negative and GUS positive, thus
demonstrating linkage between the identification sequence (crtB)
and the selectable marker CP4-EPSPS gene, and segregation of these
transgenic loci from the gus locus. The phenotypic ratio in
segregating seed was consistent with Mendelian segregation of two
dominant loci.
Example 4
R1 Seed Visual Analysis
[0114] Mature seed from A33908 were visually analyzed for color,
size, and shape (FIG. 5). A mixture of (i) marker-free normal (e.g.
yellow and smooth); (ii) orange and smooth; and (iii) orange and
shrunken seed was seen.
Example 5
PCR Analysis on GUS Positive Seed
[0115] INVADER PCR (e.g. Mein et al., 2000) was used to follow
segregation of the CP4-EPSPS, crtB, and gus genes delivered by
pMON67465 in seed of transgenic soybean plants. Event A33908 was
determined to contain a single copy of the CP4-EPSPS marker gene
and a single copy of the NPTII gene. Segregation of orange:normal
seed followed an expected 3:1 ratio in event A33908 (Table 1).
TABLE-US-00001 TABLE 1 Phenotype and genotype of progeny of
transgenic plants Expect. 3:1 Orange/ orange/ Invader Invader
Normal normal Chi Pedigree CP4 NPT II seed count seed count square
GM 1 1 86/26 84/28 0.19 GM 2 1 98/56 115.5/38.5 10.61 GM 1 1 130/72
151.5/50.5 12.20 GM 1 2 134/56 142.5/47.5 2.03 GM 2 0 62/30 69/23
2.84 GM 1 2 77/25 76.5/25.5 GM_A339 2 2 79/40 89.25/29.75 4.71
GM_A339 2 4 57/18 56.25/18.75 0.04
Example 6
Use of ATP PFK as an Identification Sequence
[0116] For starch-rich cereal grains including corn, manipulation
of sugar/starch metabolism resulting in a phenotype of shrunken or
abolished seed development may be utilized. Seed-specific
expression of sacB (Caimi et al. 1996), or seed-specific expression
of yeast ATP dependent phosphofructokinase (ATP PFK; e.g. GenBank
Accession NC_003423, bases 2297466..2300294) in corn ears results
in abolished kernel development (FIG. 12). The construct pMON99575
containing the CP4 selectable marker and ATP-PFK may be directly
used for co-transformation with a one T-DNA construct containing a
gene of interest by mixing cells of two Agrobacterium strains each
including one of these constructs and transforming a plant cell
with the mixed bacterial culture. Alternatively, the seed-specific
expressing ATP-PFK cassette may be subcloned into a 2T-DNA
construct as an identification sequence, for efficient
identification of marker free seeds. Kernels containing this gene
are extremely shrunken and do not germinate. Only the
identification sequence-free and marker gene free kernels show
normal appearance.
Example 7
Use of Genes Involved in Porphyrin Synthesis as Identification
Sequences
[0117] S-adenosyl-L-methionine-dependent uroporphyrinogen III
(uro'gen) methyl transferases (SUMT) produce bright red fluorescent
porphyrinoid compounds when overexpressed in E. coli, yeast, and
CHO cells. This property has enabled visual selection of
transformed E. coli colonies (Rossner & Scott 1995) and
automated sorting of transformed yeast and CHO cells (Wildt &
Deuschle 1999). This fluorescence is the result of intracellular
accumulation of di- and tri-methylated uro'gen
(dihydrosirohydrochlorin and trimethylpyrorocorphin), both of which
are compounds found in porphyrin synthesis pathways (i.e.,
chlorophyll and cobalamin).
[0118] Cells transformed with cobA encoding SUMT from
Propionibacterium freudenreichii (GenBank accession U13043;
incorporated herein by reference) yield a fluorescent signal with
absorbance peaks at 384 nm and 500 nm along with an emission band
at 605 nm. The fluorescent porphyrinoids generated by the cobA
uro'gen methyl transferase have a good spectral signature for
marking plant material. Excitation at either 384 or 500 nm avoids
strong chlorophyll absorbance and the resulting red emission is
readily detected as it has a substantial Stokes shift (from the 500
nm absorbance origin), but does not overlap with chlorophyll
autofluorescence in the far red (Haseloff, 1999).
[0119] The carboxy terminus of the maize SUMT (GenBank D83391),
Arabidopsis Upm1 (GenBank L47479), and E. coli CysG (GenBank
X14202) proteins are significantly similar to proteins encoded by
genes of P. freudenreichii (cobA), Pseudomonas denitrificans (cobA;
GenBank M59236), and of Synechocystis sp. (formerly Anacystis
nidulans; GenBank X70966), each incorporated herein by reference
(Sakakibara et al. 1996), and may be used similarly.
[0120] A construct including a promoter with kernel expression and
a gene encoding CobA, or a similar protein with SUMT activity,
allows the use of such a gene as an identification sequence by
screening for (lack of) visible red fluorescence in corn seed, for
instance. Plant siroheme synthases have been reported to be
localized in the chloroplast (Leustek et al., 1997). Thus use of a
porphyrin biosynthesis gene as an identification sequence may
include use of a chloroplast transit peptide to direct the gene
product to the chloroplast. The construct can be directly used as
an identification sequence, and a T-DNA comprising such an
identification sequence and a selectable marker may, for instance,
be co-transformed with a second construct comprising a T-DNA
containing a gene of interest by mixing two Agrobacterium strains
each containing one of these constructs, and transforming a plant
cell with the mixed bacterial culture. An SUMT expression cassette
can also be readily subcloned into other 2 T-DNA vectors, or into a
vector designed for use in microprojectile-mediated transformation,
and used as an identification sequence by a person of skill in the
art.
Example 8
Use of Gene Silencing to Produce a Detectable Seed Phenotype
[0121] An inverted repeat positioned within an intron of the marker
gene cassette can lead to efficient gene silencing in plant cells.
This is disclosed in detail in U.S. Application Publication No.
2006/0200878 (e.g., FIGS. 7, 8, 9, incorporated herein by
reference). To test if a dsRNA encoded by inverted repeats placed
within an intron was capable of eliciting gene silencing, inverted
repeats of a .about.400 bp segment of the luciferase gene (SEQ ID
NO:1) were placed into the intron of the rice Actin1 promoter in a
EPSPS-CP4 gene cassette (pMON73874) and the ability of the
construct to suppress the luciferase gene in a transient
transformation of corn leaf protoplasts was tested. As a control, a
similar plasmid was tested, except that the control plasmid had
inverted repeats of a segment of the GUS gene instead of the
luciferase gene (pMON73875). Finally, as an additional control,
pMON25492, which was identical except that it has no inverted
repeats, was also employed (FIG. 13).
[0122] When these three plasmids were tested in a corn leaf
protoplast transient gene silencing system testing for the
suppression of firefly luciferase and normalizing to the expression
of a RENILLA luciferase (Promega Corp., Madison, Wis.) internal
control, it was observed that plasmid with dsRNA encoding inverted
repeats within the intron (pMON73874) was able to suppress
luciferase relative to the controls pMON73875 and pMON25492 (FIG.
14). The experiment was repeated a second time with similar
results.
[0123] To test if a corn kernel phenotype may be generated via a
gene silencing approach, constructs designed to suppress the Waxy
gene were made. pMON81990 contains inverted repeats of part of the
Waxy gene. Transgenic corn plants containing pMON81990 displayed
silencing of the Waxy gene in at least 65% of the independent RO
plants, as determined by staining pollen and kernels with iodine
for starch production. In comparison, plants containing pMON81993,
which expresses a sense fragment of Waxy, do not display efficient
silencing of the Waxy gene.
[0124] Silencing of genes that encode zeins (seed storage
proteins), leading to a visible phenotype was also demonstrated.
pMON73567 contains inverted repeats of sequences of genes that
encode a-zeins in corn kernels. Transcription of the inverted
repeats results in silencing of these genes, reducing the levels of
the 19 kD and 22 kD .alpha.-zeins in 26 out of 29 R0 plants tested.
FIG. 15 demonstrates that kernels resulting from cells transformed
with this dsRNA-encoding sequence have an obvious visual phenotype,
wherein kernels with reduced zeins are less translucent than
wild-type kernels.
[0125] Thus, a dsRNA-encoding sequence embedded in the intron of a
marker gene may be used as an identification sequence according to
the present invention. Constructs containing, for instance, a
glyphosate resistance gene such as CP4 EPSPS as a selectable marker
and such a dsRNA-encoding sequence in an intron of the selectable
marker gene may be directly used for co-transformation with a one
T-DNA construct containing a gene of interest by mixing cells of
two Agrobacterium strains each comprising one of these constructs
and transforming a plant cell with the mixed bacterial culture.
Alternatively, the dsRNA-encoding cassette may be subcloned into a
2T-DNA construct as an identification sequence, for efficient
identification of marker free seeds. One of skill in the art could
also design analogous constructs for use in microprojectile
bombardment-mediated plant cell transformation.
Example 9
Use of KAS4 as an Identification Sequence
[0126] Binary vector pMON83530 (FIG. 16) contains a KAS4
(a-keto-acetyl-ACT synthase; GenBank accession AF060518) driven by
a soybean USP88 promoter (e.g. U.S. Pat. No. 7,078,588) with a CP4
plant expressible cassette as a selectable marker on the same
T-DNA. The seed-specific expression of the KAS4 gene results in
shrunken seeds which are easily distinguishable from the normal
seeds which do not contain the gene (FIG. 17). The construct can be
directly used as an identification sequence, and a T-DNA comprising
such an identification sequence and a selectable marker may be
co-transformed with a second construct comprising a T-DNA
containing a gene of interest by mixing two Agrobacterium strains
each containing one of these constructs and transforming a plant
cell with the mixed bacterial culture.
[0127] The KAS4 expression cassette is also present in a 2 T-DNA
plasmid as shown in pMON107314 (FIG. 18) wherein one T-DNA
comprises a splA gene (Sucrose phosphorylase from Agrobacterium
tumefaciens; GenBank Accession AE009432) as an identification
sequence and a marker gene and the other T-DNA may comprise a gene
of interest as constructed by routine cloning methods known to
those skilled in the art. The 2 T-DNA plasmid can then be used, for
instance, for soybean transformation. The identification gene is
used for selecting seeds without marker gene based on phenotype
provided by the identification gene.
Example 10
Use of an splA Gene as an Identification Sequence
[0128] Binary vector pMON68581 (FIG. 19) contains the splA (Sucrose
phosphorylase from Agrobacterium tumefaciens; GenBank Accession
AE009432) driven by a soybean 7S alpha promoter (e.g. GenBank
M13759; Doyle et al., 1986) with a CP4 plant expressible cassette
as a selectable marker on the same T-DNA. The seed-specific
expression of the splA gene results in shrunken seeds which are
easily distinguishable from the normal seeds which do not contain
the gene (FIG. 20). The construct can be directly used as an
identification sequence, and a T-DNA comprising such an
identification sequence and a selectable marker may be
co-transformed with a second construct comprising a T-DNA
containing a gene of interest by mixing two Agrobacterium strains
each containing one of these constructs and transforming a plant
cell with the mixed bacterial culture. The splA expression cassette
can also be readily sub-cloned into 2 T-DNA vectors wherein one
T-DNA comprises splA gene as an identification sequence and a
marker gene and the other T-DNA comprises a gene of interest by
routine cloning methods known to those skilled in the art. The 2
T-DNA plasmid can then be used, for instance, for soybean
transformation. The identification sequence is used for selecting
seeds without a selectable or screenable marker gene based on the
phenotype phenotype provided by the identification sequence.
Example 11
Use of Several Identification Sequences in Producing Marker-Free
Corn Seed
[0129] Multiple 2 T-DNA plant expression vectors were constructed.
In each construct, the first T-DNA segment comprised a plant
expressible uidA transgene as an example of a nucleic acid of
interest and the second T-DNA segment comprised of a plant
expressible CP4 EPSPS transgene as a selectable marker and an
identification sequence as shown in the table below. Sequences of
crtB designed for expression in monocots were prepared by methods
known in the art (e.g. by codon-optimization as found in SEQ ID
NO:2; SEQ ID NO:3). pMON68412 comprises SEQ ID NO:3. The first
T-DNA is flanked by right and left borders while the second T-DNA
is a located in the vector backbone, a 2 T-DNA format commonly
known as tandem format (Huang et al., 2005). Corn tissues were
transformed separately with each of the constructs by methods known
in the art. The expected phenotype with each identification gene is
indicated in Table 2 below. Alternative promoters for expression of
the identification sequences in endosperm include glutelin1
promoter from rice, the waxy promoter from corn, and the brittle2
promoter from corn.
TABLE-US-00002 TABLE 2 Exemplary phenotypes expected with given
identification sequences. Phenotype of seeds carrying the
identifica- Identification Sequence Cassette tion gene and
Identification Termina- the selectable Promoter sequence tor
Construct marker gene Maize 27 kD crtB Rice pMON68412 carotenoid
zein Glutelin1 pigment in endosperm; defective kernel development
Maize 27 kD 19 & 22 kD Rice pMON68413 opaque zein zein inverted
Glutelin1 endosperm repeats (US 20060200878) Maize 27 kD Phospho-
Rice pMON68414 shrunken zein fructokinase Glutelin1 endosperm (pfk)
Maize 27 kD B peru Rice pMON68415 anthocyanin zein Glutelin1
pigment in endosperm None None None pMON97371 none
[0130] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of the foregoing
embodiments and illustrative examples, it will be apparent to those
of skill in the art that variations, changes, modifications, and
alterations can be applied to the composition, methods, and in the
steps or in the sequence of steps of the methods described herein,
without departing from the concept, spirit, and scope of the
invention. More specifically, it will be apparent that certain
agents that are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope, and concept of the invention as defined
by the appended claims.
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Sequence CWU 1
1
311048DNAArtificial SequenceDescription of Artificial Sequence
Artificial Primer 1ccacaccctt aggtaaccca gtagatccag aggaattcat
tatcagtgca attgttttgt 60cacgatcaaa ggactctggt acaaaatcgt attcattaaa
accgggaggt agatgagatg 120tgacgaacgt gtacatcgac tgaaatccct
ggtaatccgt tttagaatcc atgataataa 180ttttctggat tattggtaat
tttttttgca cgttcaaaat tttttgcaac ccctttttgg 240aaacaaacac
tacggtaggc tgcgaaatgt tcatactgtt gagcaattca cgttcattat
300aaatgtcgtt cgcgggcgca actgcaactc cgataaataa cgcgcccaac
accggcataa 360agaattgaag agagttttca ctgcatacga cgattctgtg
atttgtattc agcccatatc 420gacgcgtgaa gacgccaaaa acataaagaa
aggcccggcg ccattctatc ctctagagga 480tggaaccgct ggagagcaac
tgcataaggc tatgaagaga tacgccctgg ttcctggaac 540aattgctttt
acagatgcac atatcgaggt gaacatcacg tacgcggaat acttcgaaat
600gtccgttcgg ttggcagaag ctatgaaacg atatgggctg aatacaaatc
acagaatcgt 660cgtatgcagt gaaaactctc ttcaattctt tatgccggtg
ttgggcgcgt tatttatcgg 720agttgcagtt gcgcccgcga acgacattta
taatgaacgt gaattgctca acagtatgaa 780catttcgcag cctaccgtag
tgtttgtttc caaaaagggg ttgcaaaaaa ttttgaacgt 840gcaaaaaaaa
ttaccaataa tccagaaaat tattatcatg gattctaaaa cggattacca
900gggatttcag tcgatgtaca cgttcgtcac atctcatcta cctcccggtt
ttaatgaata 960cgattttgta ccagagtcct ttgatcgtga caaaacaatt
gcactgataa tgaattcctc 1020tggatctact gggttaccta agggtgtg
10482930DNAArtificial SequenceDescription of Artificial Sequence
Artificial Primer 2atgtcccagc ctcctctcct ggatcatgct acccaaacta
tggctaacgg tagcaagtcc 60ttcgctaccg ccgctaagct cttcgatcct gctactaggc
gttccgtcct catgctgtac 120acctggtgta ggcattgcga cgatgtgatc
gacgatcaaa cccacggttt cgcgtccgag 180gctgcggccg aggaagaggc
gacccaaagg ctcgctcgcc tccgtaccct caccctcgcc 240gctttcgagg
gcgctgagat gcaagatcct gcgttcgccg ctttccaaga ggtcgccctc
300acccacggca tcactccaag gatggccctg gaccacctgg acggtttcgc
tatggacgtc 360gcccaaactc gctacgtgac tttcgaggac actctccggt
actgctacca tgtcgccggt 420gttgtcggcc tcatgatggc gcgcgtcatg
ggtgtccgcg acgagcgcgt cctcgacagg 480gcttgcgacc tcggcctcgc
gtttcagctt accaacattg ctagggacat catcgacgac 540gctgcgatag
ataggtgtta cctgcctgct gagtggcttc aggacgctgg tcttacgccc
600gagaactacg ctgccaggga gaacagggct gcccttgcta gagtcgctga
gcgtttgatc 660gacgcggctg aaccctatta cattagctcg caagcgggtt
tgcatgactt gcctccacgt 720tgtgcgtggg ccattgcgac ggcgcgtagt
gtttatcgtg aaattgggat caaggttaag 780gccgctggag gatcagcatg
ggaccggaga cagcatacgt ctaagggaga gaagatcgca 840atgctaatgg
cagctcccgg ccaagtcatc cgcgcaaaga caacgcgggt tacacccaga
900ccagcgggct tatggcagcg accagtatga 9303930DNAArtificial
SequenceDescription of Artificial Sequence Artificial Primer
3atgtcccagc ctcctctcct ggatcatgct acccaaacta tggctaacgg tagcaagtcc
60ttcgctaccg ccgctaagct cttcgatcct gctactaggc gttccgtcct catgctgtac
120acctggtgta ggcattgcga cgatgtgatc gacgatcaaa cccacggttt
cgcgtccgag 180gctgcggccg aggaagaggc gacccaaagg ctcgctcgcc
tccgtaccct caccctcgcc 240gctttcgagg gcgctgagat gcaagatcct
gcgttcgccg ctttccaaga ggtcgccctc 300acccacggca tcactccaag
gatggccctg gaccacctgg acggtttcgc tatggacgtc 360gcccaaactc
gctacgtgac tttcgaggac actctccggt actgctacca tgtcgccggt
420gttgtcggcc tcatgatggc gcgcgtcatg ggtgtccgcg acgagcgcgt
cctcgacagg 480gcttgcgacc tcggcctcgc gtttcagctt accaacattg
ctagggacat catcgacgac 540gctgcgatag ataggtgtta cctgcctgct
gagtggcttc aggacgctgg tcttacgccc 600gagaactacg ctgccaggga
gaacagggcc gcccttgcta gagtcgctga gcgtttgatc 660gacgcggctg
aaccctatta cattagctcg caagcgggtt tgcatgactt gcctccacgt
720tgtgcgtggg ccattgcgac ggcgcgtagt gtttatcgtg aaattgggat
caaggttaag 780gccgctggag gatcagcatg ggaccggaga cagcatacgt
ctaagggaga gaagatcgca 840atgctaatgg cagctcccgg ccaagtcatc
cgcgcaaaga caacgcgggt tacacccaga 900ccagcgggct tatggcagcg
accagtatga 930
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