U.S. patent application number 11/202401 was filed with the patent office on 2006-04-06 for enhanced zein reduction in transgenic corn seed.
Invention is credited to Shihshien Huang, Michael H. Luethy, Thomas M. Malvar.
Application Number | 20060075515 11/202401 |
Document ID | / |
Family ID | 39096043 |
Filed Date | 2006-04-06 |
United States Patent
Application |
20060075515 |
Kind Code |
A1 |
Luethy; Michael H. ; et
al. |
April 6, 2006 |
Enhanced zein reduction in transgenic corn seed
Abstract
Anti-sense-oriented RNA gene suppression agents in the form of a
loop of anti-sense-oriented RNA is produced in cells of transgenic
organisms, e.g. plants, by transcription from a recombinant DNA
construct that comprises in 5' to 3' order a promoter element
operably linked to more than one anti-sense-oriented DNA element
and one or more complementary DNA elements.
Inventors: |
Luethy; Michael H.; (Webster
Groves, MO) ; Malvar; Thomas M.; (N.Stonington,
CT) ; Huang; Shihshien; (Stonington, CT) |
Correspondence
Address: |
MONSANTO COMPANY
800 N. LINDBERGH BLVD.
ATTENTION: GAIL P. WUELLNER, IP PARALEGAL, (E2NA)
ST. LOUIS
MO
63167
US
|
Family ID: |
39096043 |
Appl. No.: |
11/202401 |
Filed: |
August 11, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60600859 |
Aug 11, 2004 |
|
|
|
Current U.S.
Class: |
800/278 ;
435/412; 435/468; 536/23.2; 800/320.1 |
Current CPC
Class: |
C12N 15/8254 20130101;
C12N 15/8247 20130101; A23L 7/198 20160801; A01H 5/10 20130101;
C12N 15/8251 20130101; C12N 9/0028 20130101; A23K 10/30 20160501;
C07K 14/415 20130101; C12N 15/8218 20130101 |
Class at
Publication: |
800/278 ;
800/320.1; 435/412; 435/468; 536/023.2 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C07H 21/04 20060101 C07H021/04; C12N 15/82 20060101
C12N015/82; C12N 5/04 20060101 C12N005/04; A01H 1/00 20060101
A01H001/00 |
Claims
1. A recombinant DNA construct for suppression of a plurality of
maize zein target genes, comprising in 5' to 3' order a promoter
element operably linked to a plurality of anti-sense-oriented DNA
elements from a plurality of maize zein target genes, the
anti-sense-oriented DNA elements designated in 5' to 3' order
element 1, element 2, and a plurality of sense-oriented DNA
elements from a plurality of maize zein target genes, designed in
5' to 3' order element 3, element 4, wherein the
anti-sense-oriented DNA element 2 has at least a portion of its
sequence at its 3' end that is not complementary either to itself
or to the sense-oriented DNA element 3, and wherein sense-oriented
RNA transcribed by the sense-oriented DNA elements is complementary
to the 5'-most end of anti-sense-oriented RNA transcribed by the
anti-sense-oriented DNA elements.
2. A recombinant DNA construct of claim 1 wherein the
anti-sense-oriented DNA elements 1 and 2 are from two different
genes targeted for suppression and the sense-oriented DNA elements
3 and 4 are from two different genes targeted for suppression.
3. A recombinant DNA construct of claim 1 wherein the
anti-sense-oriented DNA element 1 is from a 19-kD .alpha.-zein gene
targeted for suppression, the anti-sense-oriented DNA element 2 is
from a 22-kD .alpha.-zein gene targeted for suppression, the
sense-oriented DNA element 3 is from a 22-kD .alpha.-zein gene
targeted for suppression and is shorted than the
anti-sense-oriented DNA element 2 and is complementary to only the
5' end of element 2 and the sense-oriented DNA element 4 is from a
19-kD .alpha.-zein gene targeted for suppression and is
complementary to at least a portion of the 5' end of element 1.
4. A recombinant DNA construct of claim 1 wherein the more than one
maize zein target gene is selected from the group consisting of 19-
and 22-kD .alpha.-zeins, 16- and 27-kD .gamma.-zeins, 10-kD
.delta.-zeins, and 15-kD .beta.-zeins.
5. A recombinant DNA construct for suppression of more than one
maize zein target gene that comprises in 5' to 3' order a promoter
element operably linked to more than one anti-sense-oriented DNA
element from more than one maize zein target gene, the
anti-sense-oriented DNA elements designed in 5' to 3' order element
1, element 2, and at least one sense-oriented DNA element from at
least one maize zein target gene, designated element 3, wherein the
anti-sense-oriented DNA element 2 has at least a portion of its
sequence at its 3' end that is not complementary either to itself
or to the sense-oriented DNA element 3, and sense-oriented RNA
transcribed by the sense-oriented DNA element is complementary to
the 5'-most end of anti-sense-oriented RNA transcribed by the
anti-sense-oriented DNA element(s).
6. A recombinant DNA construct of claim 5 wherein the
anti-sense-oriented DNA elements 1 and 2 are from two genes
targeted for suppression and the sense-oriented DNA element 3 is
from a gene targeted for suppression.
7. A recombinant DNA construct of claim 5 wherein the
anti-sense-oriented DNA element 1 is from a 19-kD .alpha.-zein gene
targeted for suppression, the anti-sense-oriented DNA element 2 is
from a 22-kD .alpha.-zein gene targeted for suppression, and the
sense-oriented DNA element 3 is from a 19-kD .alpha.-zein gene
targeted for suppression.
8. A recombinant DNA construct of claim 5 wherein the more than one
maize zein target gene is selected from the group consisting of 19-
and 22-kD .alpha.-zeins, 16- and 27-kD .gamma.-zeins, 10-kD
.delta.-zeins, and 15-kD .beta.-zeins and wherein the at least one
maize zein target gene is selected from the group consisting of 19-
and 22-kD .alpha.-zeins, 16- and 27-kD .gamma.-zeins, 10-kD
.delta.-zeins, and 15-kD .beta.zeins.
9. A method for generating corn seeds having at least one of
enhanced lysine, enhanced oil and enhanced tryptophan comprising
the steps of: a) transforming a plant cell with the recombinant DNA
construct of claim 1 or claim 4 for suppression of more than one
maize zein target gene; and b) regenerating the plant cell into a
fertile transgenic plant, wherein the plant contains the construct
in its genome; and c) harvesting seed from the plant, wherein the
seed has at least one of enhanced lysine, enhanced oil and enhanced
tryptophan relative to seed of the same variety not transformed
with the construct.
10. A method for generating corn seeds having at least one of
enhanced lysine, enhanced oil and enhanced tryptophan comprising
the steps of: d) transforming a plant cell with the recombinant DNA
construct of claim 5 or claim 8 for suppression of more than one
maize zein target gene; and e) regenerating the plant cell into a
fertile transgenic plant, wherein the plant contains the construct
in its genome; and f) harvesting seed from the plant, wherein the
seed has at least one of enhanced lysine, enhanced oil and enhanced
tryptophan relative to seed of the same variety not transformed
with the construct.
11. A transgenic plant comprising within its genome a DNA construct
of claim 1 or claim 4 for suppression of more than one maize zein
target gene.
12. A harvested seed from a plant of claim 11.
13. A harvested seed from a plant of claim 11, wherein the
sense-oriented DNA elements are from two genes targeted for
suppression and the anti-sense-oriented DNA elements are from two
genes targeted for suppression.
14. A processed product of the seed of claim 12, wherein the
product is a feed, a meal, or a partially purified protein
composition.
15. A transgenic plant comprising within its genome a DNA construct
of claim 5 or claim 8 for suppression of more than one maize zein
target gene.
16. A harvested seed from a plant of claim 15.
17. A harvested seed from a plant of claim 15, wherein the
sense-oriented DNA element is from one gene targeted for
suppression and the anti-sense-oriented DNA elements are from two
genes targeted for suppression.
18. A processed product of the seed of claim 16, wherein the
product is a feed, a meal, or a partially purified protein
composition.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. 119(e) to
provisional applications Ser. No. 60/543,157, filed Feb. 10, 2004,
No. 60/543,187, filed Feb. 10, 2004 and No. 60/600,859, filed Aug.
11, 2004; and to utility application Ser. No. 11/057062, filed Feb.
10, 2005, the disclosures of all of which are incorporated herein
by reference in their entireties.
INCORPORATION OF SEQUENCE LISTING
[0002] A computer readable form of the sequence listing is
contained in the file named "53428B.ST25.txt" which is 21 kb
(measured in MS-Windows) and was created on Feb. 9, 2005 and is
located on a CDROM, which is filed herewith and herein incorporated
by reference.
FIELD OF THE INVENTION
[0003] Disclosed herein are seeds for transgenic corn having
elevated amino acid levels, recombinant DNA constructs for
producing gene-suppressing loops of anti-sense RNA and methods of
making and using such constructs and transgenic plants expressing
gene-suppressing loops of anti-sense RNA.
BACKGROUND
[0004] Certain plants have low levels of specific amino acids
compared to other plants, or compared to hypothetical nutritionally
"perfect" protein models based on milk or egg. By these standards,
corn has low levels of lysine, methionine and tryptophan. Efforts
to increase amino acid levels in transgenic plants include
expressing recombinant DNA which encodes proteins in an amino acid
synthesis pathway at higher levels than native genes. One such gene
for producing enhanced levels of lysine in corn is a bacterial
dihydropicolinic [is this correct?]acid synthase. A strategy for
achieving even higher levels of amino acids includes suppression of
genes encoding proteins in amino acid catabolic pathways.
[0005] Gene suppression includes any of the well-known methods for
suppressing transcription of a gene or the accumulation of the mRNA
corresponding to that gene, thereby preventing translation of the
transcript into protein. More particularly, gene suppression by
inserting a recombinant DNA construct with anti-sense oriented DNA
to regulate gene expression in plant cells is disclosed in U.S.
Pat. No. 5,107,065 (Shewmaker et al.) and U.S. Pat. No. 5,759,829
(Shewmaker et al.). Plants transformed using such anti-sense
oriented DNA constructs for gene suppression can comprise
integrated DNA arranged as an inverted repeat from co-insertion of
several copies of the transfer DNA (T-DNA) into plants by
Agrobacterium-mediated transformation, as disclosed by Redenbaugh
et al. in "Safety Assessment of Genetically Engineered Flavr
Savr.TM. Tomato, CRC Press, Inc. (1992). Inverted repeat insertions
can make up a part or all of the T-DNA, e.g. the T-DNA can contain
an inverted repeat of a complete or partial anti-sense construct.
Screening for inserted DNA comprising inverted repeat elements can
improve the efficiency of identifying transformation events
effective for gene silencing when the transformation construct is a
simple anti-sense DNA construct.
[0006] Gene suppression caused by an inserted recombinant DNA
construct with sense-oriented DNA is disclosed in U.S. Pat. No.
5,283,184 (Jorgensen et al.) and U.S. Pat. No. 5,231,020 (Jorgensen
et al.). Inserted T-DNA providing gene suppression in plants
transformed with such sense constructs by Agrobacterium is
organized predominantly in inverted repeat structures, as disclosed
by Jorgensen et al., Mol. Gen. Genet., 207: 471-477 (1987). See
also Stam et al., The Plant Journal, 12: 63-82 (1997) and De Buck
et al., Plant Mol. Biol. 46 433-445 (2001), who used segregation
studies to support Jorgensen's finding that in many events gene
silencing is mediated by multimeric transgene T-DNA where the
T-DNAs are arranged in inverted repeats. Screening for inserted DNA
comprising inverted repeat elements can improve the gene silencing
efficiency when transforming with simple sense-orientated DNA
constructs.
[0007] Gene silencing can also be effected by transcribing RNA from
both a sense and an anti-sense oriented DNA using two separate
transcription units, e.g. as disclosed by Shewmaker et al. in U.S.
Pat. No. 5,107,065 where in Example 1 a binary vector was prepared
with both sense and anti-sense aroA genes. Similar constructs are
disclosed in International Publication No. WO 99/53050 (Waterhouse
et al.). See also U.S. Pat. No. 6,326,193 where gene targeted DNA
is operably linked to opposing promoters.
[0008] Gene suppression can be achieved in plants by using
transformation constructs that are capable of generating an RNA
that can form double-stranded RNA along at least part of its
length. Gene suppression in plants is disclosed in EP 0426195 A1
(Goldbach et al.) where recombinant DNA constructs for
transcription into hairpin RNA provided transgenic plants with
resistance to tobacco spotted wilt virus. See also Sijen et al.,
The Plant Cell, Vol. 8, 2277-2294 (1996) which discloses the use of
constructs carrying inverted repeats (sense followed by anti-sense)
of a cowpea mosaic virus gene in transgenic plants to mediate virus
resistance. See also International Publication No. 98/53083
(Grierson et al.) and related U.S. Patent Application Publication
No. 2003/0175965 A1 (Lowe et al.) which disclose gene suppression
using a double stranded RNA construct comprising a gene coding
sequence preceded by an inverted repeat of the 5' UTR. Constructs
for posttranscriptional gene suppression in plants by
double-stranded RNA of the target gene are also disclosed in
International Publication No. WO 99/53050 (Waterhouse et al.) and
International Publication No. WO 99/49029 (Graham et al.). See also
U.S. Patent Application Publication No. 2002/0048814 A1 (Oeller)
where DNA constructs are transcribed to sense or anti-sense RNA
with a hairpin-forming poly(T)-poly(A) tail. See also U.S. Patent
Application Publication No. 2003/0018993 A1 (Gutterson et al.)
where sense or anti-sense DNA is followed by an inverted repeat of
the 3' untranslated region of the NOS gene. See also U.S. Patent
Application Publication No. 2003/0036197 A1 (Glassman et al.) where
RNA for reducing the expression of target mRNA comprises a segment
with homology to target mRNA and a segment with complementary RNA
regions that are unrelated to endogenous RNA.
[0009] The production of dsRNA in plants to inhibit gene
expression, e.g. in a nematode feeding on the plant, is disclosed
U.S. Pat. No. 6,506,559 (Fire et al.). Multi-gene suppression
vectors for use in plants are disclosed in U.S. patent application
Ser. No. 10/465,800 (Fillatti).
[0010] Transcriptional suppression such as promoter trans
suppression can be effected by a expressing a DNA construct
comprising a promoter operably linked to inverted repeats of
promoter DNA from a target gene. Constructs useful for such gene
suppression mediated by promoter trans suppression are disclosed by
Mette et al., The EMBO Journal, Vol. 18, pp. 241-148, (1999) and by
Mette et al., The EMBO Journal, Vol. 19, pp. 5194-5201-148, (2000),
both of which are incorporated herein by reference.
[0011] All of the above-described patents, applications and
international publications disclosing materials and methods for
gene suppression in plants are incorporated herein by
reference.
SUMMARY OF THE INVENTION
[0012] This invention provides methods and recombinant DNA
constructs useful for producing anti-sense-oriented RNA for gene
suppression in transgenic organisms. In one aspect of the invention
a recombinant DNA construct for suppressing a plurality of target
genes comprises in 5' to 3' order a promoter element operably
linked to an anti-sense-oriented DNA element and a sense-oriented
DNA element, in which the sense-oriented DNA element is shorter
than the anti-sense-oriented DNA element and sense-oriented RNA
transcribed from the sense-oriented DNA is complementary to the
5'-most end of anti-sense-oriented RNA transcribed from the
anti-sense-oriented DNA element, whereby the transcribed RNA forms
a into a loop of anti-sense-oriented RNA for suppressing the
plurality of target genes.
[0013] The sense-oriented DNA can be cloned as an inverted repeat
of a 5'-most segment of the anti-sense-oriented DNA element.
Constructs with such sense-oriented DNA are transcribed to RNA that
forms a loop of anti-sense-oriented RNA closed at its ends with a
double-stranded RNA (dsRNA) segment, e.g. as illustrated in FIG. 1.
To form an anti-sense-oriented RNA loop the complementary DNA
element is conveniently not more than about one-half the length of
the anti-sense-oriented DNA element, and preferably not more than
one-third the length of the anti-sense-oriented DNA element, e.g.
not more than one-quarter the length of the anti-sense-oriented DNA
element. The overall lengths of the combined DNA elements can vary.
For instance, the anti-sense-oriented DNA element can consist of
from 500 to 5000 nucleotides and the complementary DNA element can
consist of from 50 to 500 nucleotides. In many cases it will be
useful for the anti-sense-oriented DNA segment to be more than
twice the length of the sense-oriented DNA segment to allow for
formation of an anti-sense-oriented RNA loop.
[0014] The anti-sense transcription unit can be designed to
suppress multiple genes where the DNA is arranged with two or more
anti-sense-oriented elements from different genes targeted for
suppression followed by a complementary sense-oriented element,
e.g. complementary to at least a part of the 5'most anti-sense
element.
[0015] This invention also provides methods of suppressing the
expression of a gene by providing in the cells of a plant a
recombinant DNA construct of this invention that transcribes to an
anti-sense loop of RNA. In other aspects of the invention, e.g. for
providing traits other than plants with enhanced amino acid, the
gene targeted for suppression can be a plant gene, a plant pest
gene, a plant pathogen gene or a combination thereof. In the
constructs, methods and plants of this invention the gene targeted
for silencing can be a native gene or an exogenous gene or a gene
in an organism that ingests or contacts the tissues of the plant
that have cells comprising anti-sense RNA in a loop according to
this invention. Plant pathogens include viruses such as cucumber
mosaic virus, bacteria such as Erwinia stewartii (Stewart's wilt of
corn) and fungi such as Phakopsora pachyrhizi (soybean rust
fungus); plant pests include nematodes such as soybean cyst
nematode and root knot nematode, and insects of various orders
including Lepidoptera (e.g., European corn borer), Coleoptera
(e.g., spotted cucumber beetle) and Homoptera (e.g., aphids).
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a schematic illustration of a recombinant DNA
construct useful in this invention to produce an
anti-sense-oriented loop of RNA.
[0017] FIG. 2 is a Western analysis indicating gene suppression
using a construct of this invention.
[0018] FIG. 3 shows mass spectroscopy spectra indicating zein
content in seeds.
[0019] FIG. 4 is a graphic illustration of the design of the vector
constructs used in an aspect of this invention.
[0020] FIG. 5 shows mass spectroscopy spectra indicating zein
content in wild type and transgenic seeds.
[0021] FIG. 6 illustrates the correlation between the protein
content of the kernels and their levels of free amino acids.
DETAILED DESCRIPTION
[0022] SEQ ID NO:1 and SEQ ID NO:2 are nucleotide sequences of
recombinant DNA constructs useful for transcribing RNA that can
form an anti-sense-oriented RNA loop for suppressing one or
multiple genes in transgenic plants. See Tables 1 and 2 for a
description of elements of those constructs.
[0023] As used herein, "complementary" refers to polynucleotides
that are capable of hybridizing, e.g. sense and anti-sense strands
of DNA or self-complementary strands of RNA, due to complementarity
of aligned nucleotides permitting C-G and A-T or A-U bonding.
[0024] As used herein "vector" means a DNA molecule capable of
replication in a host cell and/or to which another DNA segment can
be operatively linked so as to bring about replication of the
attached segment. A plasmid is an exemplary vector.
[0025] As used herein a "transgenic" organism, e.g. plant or seed,
is one whose genome has been altered by the incorporation of
recombinant DNA comprising exogenous genetic material or additional
copies of native genetic material, e.g. by transformation or
recombination of the organism or an ancestral organism. Transgenic
plants include progeny plants of an original plant derived from a
transformation process including progeny of breeding transgenic
plants with wild type plants or other transgenic plants. Crop
plants of particular interest in the present invention include, but
are not limited to maize, soybean, cotton, canola (rape), wheat,
rice, sunflower, safflower and flax. Other crops of interest
include plants producing vegetables, fruit, grass and wood.
Recombinant DNA Constructs for Plant Transformation
[0026] Recombinant DNA constructs for producing looped, anti-sense
RNA, gene suppression agents in transgenic plants can be readily
prepared by those skilled in the art. Typically, such a DNA
construct comprises as a minimum a promoter active in the tissue
targeted for suppression, a transcribable DNA element having a
sequence that is complementary to nucleotide sequence of a gene
targeted for suppression and a transcription terminator element.
The targeted gene element copied for use in transcribable DNA in
the gene suppression construct can be a promoter element, an intron
element, an exon element, a 5' UTR element, or a 3'UTR element.
Although the minimum size of DNA copied from sequence of a gene
targeted for suppression is believed to be about 21 or 23
nucleotides; larger nucleotide segments are preferred, e.g. up the
full length of a targeted gene. Useful lengths of either DNA
segment are in the range of 50 to 5000 nucleotides, say
anti-sense-oriented DNA of 500 to 5000 nucleotides in length and
complementary DNA elements can be 50 to 500 or more nucleotides in
length. The DNA element can comprise multiple parts of a gene, e.g.
nucleotides that are complementary to contiguous or separated gene
elements of UTR, exon and intron. Such constructs may also comprise
other regulatory elements, DNA encoding transit peptides, signal
peptides, selective markers and screenable markers as desired.
[0027] With reference to FIG. 1 there is schematically shown a
recombinant DNA construct comprising a promoter element, an
anti-sense-oriented DNA element (denoted "a/s DNA"), a
complementary sense-oriented DNA element (denoted "s DNA") and DNA
providing polyadenylation signals and site (denoted "polyA site").
The DNA construct is transcribed to RNA comprising an
anti-sense-oriented RNA segment and a complementary RNA segment
that is complementary to the 5'-most end of the anti-sense-oriented
RNA segment. The 5' and 3' ends of the anti-sense RNA can self
hybridize to form a double-stranded RNA segment that closes a loop
of anti-sense-oriented RNA. For example, if the nucleotide sequence
of the 5'-most end of the strand of transcribed anti-sense-oriented
DNA is 5'-CGGCATA---, the sequence of the 3'-most end of the
transcribed strand of the inverted repeat DNA will be ---TATGCCG-3'
which is readily cloned from the source DNA providing the
anti-sense element. With such sequences the loop of
anti-sense-oriented RNA will extend from one side of a dsRNA
segment, e.g. TABLE-US-00001 5'-GCCGUAU--------
3'-CGGCAUA--------
[0028] The anti-sense-oriented DNA and its self-complementary DNA
can be contiguous or separated by vector DNA, e.g. up to about 100
nucleotides or so of vector DNA separating restriction sites used
for vector assembly.
[0029] Recombinant DNA constructs can be assembled using
commercially available materials and methods known to those of
ordinary skill in the art. A useful technology for building DNA
constructs and vectors for transformation is the GATEWAY.TM.
cloning technology (available from Invitrogen Life Technologies,
Carlsbad, Calif.) uses the site specific recombinase LR cloning
reaction of the Integrase att system from bacterophage lambda
vector construction, instead of restriction endonucleases and
ligases. The LR cloning reaction is disclosed in U.S. Pat. Nos.
5,888,732 and 6,277,608, U.S. Patent Application Publications
2001283529, 2001282319 and 20020007051, all of which are
incorporated herein by reference. The GATEWAY.TM. Cloning
Technology Instruction Manual that is also supplied by Invitrogen
also provides concise directions for routine cloning of any desired
DNA into a vector comprising operable plant expression
elements.
[0030] An alternative vector fabrication method employs
ligation-independent cloning as disclosed by Aslanidis, C. et al.,
Nucleic Acids Res., 18, 6069-6074, 1990 and Rashtchian, A. et al.,
Biochem., 206, 91-97, 1992 where a DNA fragment with
single-stranded 5' and 3' ends are ligated into a desired vector
that can then be amplified in vivo.
[0031] Numerous promoters that are active in plant cells have been
described in the literature. These include promoters present in
plant genomes as well as promoters from other sources, including
nopaline synthase (nos) promoter and octopine synthase (ocs)
promoters carried on tumor-inducing plasmids of Agrobacterium
tumefaciens, caulimovirus promoters such as the cauliflower mosaic
virus or figwort mosaic virus promoters. For instance, see U.S.
Pat. Nos. 5,322,938 and 5,858,742 which disclose versions of the
constitutive promoter derived from cauliflower mosaic virus
(CaMV35S), U.S. Pat. No. 5,378,619 which discloses a Figwort Mosaic
Virus (FMV) 35S promoter, U.S. Pat. No. 5,420,034 which discloses a
napin promoter, U.S. Pat. No. 6,437,217 which discloses a maize
RS81 promoter, U.S. Pat. No. 5,641,876 which discloses a rice actin
promoter, U.S. Pat. No. 6,426,446 which discloses a maize RS324
promoter, U.S. Pat. No. 6,429,362 which discloses a maize PR-1
promoter, U.S. Pat. No. 6,232,526 which discloses a maize A3
promoter, U.S. Pat. No. 6,177,611 which discloses constitutive
maize promoters, U.S. Pat. No. 6,433,252 which discloses a maize L3
oleosin promoter, U.S. Pat. No. 6,429,357 which discloses a rice
actin 2 promoter and intron, U.S. Pat. No. 5,837,848 which
discloses a root specific promoter, U.S. Pat. No. 6,084,089 which
discloses cold inducible promoters, U.S. Pat. No. 6,294,714 which
discloses light inducible promoters, U.S. Pat. No. 6,140,078 which
discloses salt inducible promoters, U.S. Pat. No. 6,252,138 which
discloses pathogen inducible promoters, U.S. Pat. No. 6,175,060
which discloses phosphorus deficiency inducible promoters, U.S.
Pat. No. 6,635,806 which discloses a coixin promoter, U.S.
2002/0192813A1 which discloses 5', 3' and intron elements useful in
the design of effective plant expression vectors, U.S. 2004/0216189
A1 which discloses a maize chloroplast aldolase promoter, and U.S.
2004/0123347A1 which discloses water-deficit inducible promoters,
all of which are incorporated herein by reference. These and
numerous other promoters that function in plant cells are known to
those skilled in the art and available for use in recombinant
polynucleotides of the present invention to provide for expression
of desired genes in transgenic plant cells.
[0032] Furthermore, the promoters may be altered to contain
multiple "enhancer sequences" to assist in elevating gene
expression. Such enhancers are known in the art. By including an
enhancer sequence with such constructs, the expression of the
selected protein may be enhanced. These enhancers often are found
5' to the start of transcription in a promoter that functions in
eukaryotic cells, but can often be inserted upstream (5') or
downstream (3') to the coding sequence. In some instances, these 5'
enhancing elements are introns. Particularly useful as enhancers
are the 5' introns of the rice actin 1 (see U.S. Pat. No.
5,641,876) and rice actin 2 genes, the maize alcohol dehydrogenase
gene intron, the maize heat shock protein 70 gene intron (U.S. Pat.
No. 5,593,874) and the maize shrunken 1 gene.
[0033] In other aspects of the invention, sufficient expression in
plant seed tissues is desired to effect improvements in seed
composition. Exemplary promoters for use for seed composition
modification include promoters from seed genes such as napin (U.S.
Pat. No. 5,420,034), maize L3 oleosin (U.S. Pat. No. 6,433,252),
zein Z27 (Russell et al. (1997) Transgenic Res. 6(2):157-166),
globulin 1 (Belanger et al (1991) Genetics 129:863-872), glutelin 1
(Russell (1997) supra), and peroxiredoxin antioxidant (Per1) (Stacy
et al. (1996) Plant Mol Biol. 31(6):1205-1216).
[0034] Recombinant DNA constructs prepared in accordance with the
invention will often include a 3' element that typically contains a
polyadenylation signal and site, especially if the recombinant DNA
is intended for protein expression as well as gene suppression.
Well-known 3' elements include those from Agrobacterium tumefaciens
genes such as nos 3', tml 3', tmr 3', tms 3', ocs 3', tr7 3', e.g.
disclosed in U.S. Pat. No. 6,090,627, incorporated herein by
reference; 3' elements from plant genes such as wheat (Triticum
aesevitum) heat shock protein 17 (Hsp17 3'), a wheat ubiquitin
gene, a wheat fructose-1,6-biphosphatase gene, a rice glutelin gene
a rice lactate dehydrogenase gene and a rice beta-tubulin gene, all
of which are disclosed in U.S. published patent application
2002/0192813 A1, incorporated herein by reference; and the pea
(Pisum sativum) ribulose biphosphate carboxylase gene (rbs 3'), and
3' elements from the genes within the host plant.
[0035] The gene-suppressing recombinant DNA constructs can also be
stacked with DNA imparting other traits of agronomic interest
including DNA providing herbicide resistance or insect resistance
such as using a gene from Bacillus thuringensis to provide
resistance against lepidopteran, coliopteran, homopteran,
hemiopteran, and other insects. Herbicides for which resistance is
useful in a plant include glyphosate herbicides, phosphinothricin
herbicides, oxynil herbicides, imidazolinone herbicides,
dinitroaniline herbicides, pyridine herbicides, sulfonylurea
herbicides, bialaphos herbicides, sulfonamide herbicides and
glufosinate herbicides. Persons of ordinary skill in the art are
enabled in providing stacked traits by reference to U.S. patent
application publications 2003/0106096A1 and 2002/0112260A1 and U.S.
Pat. Nos. 5,034,322; 5,776,760; 6,107,549 and 6,376,754 and to
insect/nematode/virus resistance by reference to U.S. Pat. Nos.
5,250,515; 5,880,275; 6,506,599; 5,986,175 and U.S. Patent
Application Publication 2003/0150017 A1, all of which are
incorporated herein by reference.
[0036] Transformation Methods--Numerous methods for transforming
plant cells with recombinant DNA are known in the art and may be
used in the present invention. Two commonly used methods for plant
transformation are Agrobacterium-mediated transformation and
microprojectile bombardment. Microprojectile bombardment methods
are illustrated in U.S. Pat. Nos. 5,015,580 (soybean); 5,550,318
(corn); 5,538,880 (corn); 5,914,451 (soybean); 6,160,208 (corn);
6,399,861 (corn) and 6,153,812 (wheat) and Agrobacterium-mediated
transformation is described in U.S. Pat. Nos. 5,159,135 (cotton);
5,824,877 (soybean); 5,591,616 (corn); and 6,384,301 (soybean), all
of which are incorporated herein by reference. For Agrobacterium
tumefaciens based plant transformation system, additional elements
present on transformation constructs will include T-DNA left and
right border sequences to facilitate incorporation of the
recombinant polynucleotide into the plant genome.
[0037] In general it is useful to introduce recombinant DNA
randomly, i.e. at a non-specific location, in the genome of a
target plant line. In special cases it may be useful to target
recombinant DNA insertion in order to achieve site-specific
integration, e.g. to replace an existing gene in the genome, to use
an existing promoter in the plant genome, or to insert a
recombinant polynucleotide at a predetermined site known to be
active for gene expression. Several site specific recombination
systems exist that are known to function implants include cre-lox
as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in
U.S. Pat. No. 5,527,695, both incorporated herein by reference.
[0038] Transformation methods of this invention are preferably
practiced in tissue culture on media and in a controlled
environment. "Media" refers to the numerous nutrient mixtures that
are used to grow cells in vitro, that is, outside of the intact
living organism. Recipient cell targets include, but are not
limited to, meristem cells, callus, immature embryos and gametic
cells such as microspores, pollen, sperm and egg cells. It is
contemplated that any cell from which a fertile plant may be
regenerated is useful as a recipient cell. Callus may be initiated
from tissue sources including, but not limited to, immature
embryos, seedling apical meristems, microspores and the like. Cells
capable of proliferating as callus are also recipient cells for
genetic transformation. Practical transformation methods and
materials for making transgenic plants of this invention, e.g.
various media and recipient target cells, transformation of
immature embryos and subsequent regeneration of fertile transgenic
plants are disclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526,
which are incorporated herein by reference.
[0039] The seeds of transgenic plants can be harvested from fertile
transgenic plants and be used to grow progeny generations of
transformed plants of this invention including hybrid plants line
for screening of plants having an enhanced agronomic trait. In
addition to direct transformation of a plant with a recombinant
DNA, transgenic plants can be prepared by crossing a first plant
having a recombinant DNA with a second plant lacking the DNA. For
example, recombinant DNA can be introduced into first plant line
that is amenable to transformation to produce a transgenic plant
that can be crossed with a second plant line to introgress the
recombinant DNA into the second plant line. A transgenic plant with
recombinant DNA providing an enhanced agronomic trait, e.g.
enhanced yield, can be crossed with transgenic plant line having
other recombinant DNA that confers another trait, e.g. herbicide
resistance or pest resistance, to produce progeny plants having
recombinant DNA that confers both traits. Typically, in such
breeding for combining traits the transgenic plant donating the
additional trait is a male line and the transgenic plant carrying
the base traits is the female line. The progeny of this cross will
segregate such that some of the plants will carry the DNA for both
parental traits and some will carry DNA for one parental trait;
such plants can be identified by markers associated with parental
recombinant DNA Progeny plants carrying DNA for both parental
traits can be crossed back into the female parent line multiple
times, e.g. usually 6 to 8 generations, to produce a progeny plant
with substantially the same genotype as one original transgenic
parental line but for the recombinant DNA of the other transgenic
parental line
[0040] In the practice of transformation DNA is typically
introduced into only a small percentage of target cells in any one
transformation experiment. Marker genes are used to provide an
efficient system for identification of those cells that are stably
transformed by receiving and integrating a transgenic DNA construct
into their genomes. Preferred marker genes provide selective
markers that confer resistance to a selective agent, such as an
antibiotic or herbicide. Any of the herbicides to which plants of
this invention may be resistant are useful agents for selective
markers. Potentially transformed cells are exposed to the selective
agent. In the population of surviving cells will be those cells
where, generally, the resistance-conferring gene is integrated and
expressed at sufficient levels to permit cell survival. Cells may
be tested further to confirm stable integration of the exogenous
DNA. Commonly used selective marker genes include those conferring
resistance to antibiotics such as kanamycin and paromomycin
(nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or
resistance to herbicides such as glufosinate (bar or pat) and
glyphosate (aroA or EPSPS). Examples of such selectable are
illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and
6,118,047, all of which are incorporated herein by reference.
Screenable markers that provide an ability to visually identify
transformants can also be employed, e.g., a gene expressing a
colored or fluorescent protein such as a luciferase or green
fluorescent protein (GFP) or a gene expressing a beta-glucuronidase
or uidA gene (GUS) for which various chromogenic substrates are
known.
[0041] Cells that survive exposure to the selective agent, or cells
that have been scored positive in a screening assay, may be
cultured in regeneration media and allowed to mature into plants.
Developing plantlets can be transferred to plant growth mix, and
hardened off, e.g., in an environmentally controlled chamber at
about 85% relative humidity, 600 ppm CO.sub.2, and 25-250
microeinsteins m.sup.-2 s.sup.-1 of light, prior to transfer to a
greenhouse or growth chamber for maturation. Plants are regenerated
from about 6 weeks to 10 months after a transformant is identified,
depending on the initial tissue. Plants may be pollinated using
conventional plant breeding methods known to those of skill in the
art and seed produced, e.g. self-pollination is commonly used with
transgenic corn. The regenerated transformed plant or its progeny
seed or plants can be tested for expression of the recombinant DNA
and screened for the presence of enhanced agronomic trait.
Transgenic Plants and Seeds
[0042] Transgenic plant seed provided by this invention are grown
to generate transgenic plants having an enhanced trait as compared
to a control plant. Such seed for plants with enhanced agronomic
trait is identified by screening transformed plants or progeny seed
for enhanced trait. For efficiency a screening program is designed
to evaluate multiple transgenic plants (events) comprising the
recombinant DNA, e.g. multiple plants from 2 to 20 or more
transgenic events.
[0043] Transgenic plants grown from transgenic seed provided herein
demonstrate improved agronomic traits that contribute to increased
yield or other trait that provides increased plant value,
including, for example, improved seed quality. Of particular
interest are plants having enhanced yield resulting from improved
plant growth and development, stress tolerance, improved seed
development, higher light response, improved flower development, or
improved carbon and/or nitrogen metabolism
[0044] Many transgenic events that survive to fertile transgenic
plants that produce seeds and progeny plants will not exhibit an
enhanced agronomic trait. Screening is necessary to identify the
transgenic plant having enhanced agronomic traits from populations
of plants transformed as described herein by evaluating the trait
in a variety of assays to detect an enhanced agronomic trait. These
assays also may take many forms, including but not limited to,
analyses to detect changes in the chemical composition, biomass,
physiological properties, morphology of the plant.
[0045] The following examples illustrate aspects of the
invention.
EXAMPLE 1
[0046] This example illustrates preparation of a transformation
vector useful for inserting a recombinant DNA construct of this
invention into a transgenic plant to practice a method of this
invention.
[0047] The LKR/SDH gene encodes a pre-protein for lysine
ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH)
which are enzymes in a lysine catabolic pathway. Suppression of LKR
is manifest in modification, e.g. increase, of lysine content.
Suppression of LKR is effected by expressing in a plant a
recombinant DNA construct that produces a stabilized anti-sense RNA
transcribed from anti-sense-oriented LKR DNA and sense-oriented LKR
DNA that forms a loop of anti-sense-oriented RNA.
[0048] A transformation vector is prepared comprising two
transcription units between right and left borders from
Agrobacterium tumefaciens. One transcription unit for a marker
comprised: [0049] (a) DNA of a rice actin promoter and rice actin
intron, [0050] (b) DNA of a chloroplast transit peptide from
Arabidopsis EPSPS [0051] (c) DNA of A. tumefaciens aroA (a
glyphosate-resistant marker), and [0052] (d) DNA of A. tumefaciens
NOS terminator, The other transcription unit for LKR gene
suppression comprised: [0053] (a) DNA of Zea mays GLB1 promoter,
[0054] (b) DNA of a Zea mays ADH1 intron, [0055] (c)
Anti-sense-oriented DNA fragment of Zea mays LKR, [0056] (d)
Sense-oriented DNA fragment of Zea mays LKR, and [0057] (e) DNA of
Zea mays GLB1 terminator.
[0058] SEQ ID NO: 1 is DNA sequence of a transformation vector
comprising the above-described marker and gene suppression
elements. See Table 1 below for a description of the elements of
the transformation vector contained within SEQ ID NO:1.
TABLE-US-00002 TABLE 1 Bases of SEQ ID NO: 1 Description of DNA
segment 1-357 A. tumefaciens right border 376-1774 DNA of a rice
actin promoter and rice actin intron 1784-2011 DNA of A.
tumefaciens EPSPS chloroplast transit peptide 2012-3379 DNA of A.
tumefaciens aroA (glyphosate-resistant marker) 3395-3647 DNA of A.
tumefaciens NOS terminator 3691-4686 DNA of Zea mays Glb1
terminator 4692-5145 Sense-oriented DNA element from Zea mays LKR
5152-6118 Anti-sense-oriented DNA element from Zea mays LKR
6123-6680 DNA of a Zea mays ADH1 intron 6687-8082 DNA of Zea mays
GLB1 promoter 8149-8590 A. tumefaciens left border
[0059] A vector prepared with the elements listed in Table 1 was
used to transform corn plant tissue. Transgenic corn plants were
obtained by Agrobacterium-mediated transformation. Transgenic
plants from two separate transgenic insertion events were grown to
produce F1 seed. Six mature seeds from each event were analyzed to
determine success of transformation and suppression of LKR. The
mature transgenic seeds were dissected to extract protein that was
analyzed by Western analysis. With reference to FIG. 2, seed from
one of the events showed no reduction in LKR as compared to wild
type; and seed from the other event was shown to be segregating
(1:1 hemizygous:wild type) as three of the six seeds showed
substantial reduction in LKR as compared to wild type.
EXAMPLE 2
[0060] This example illustrates a wide scope of embodiments of
transformation vectors useful for inserting a recombinant DNA
construct of this invention into a transgenic plant to practice a
method of this invention. Transformation vectors were prepared
using the following DNA elements where: [0061] (a) "pGcx" refers to
DNA for a promoter derived from a gamma coixin gene from Coix
lacryma-jobi; [0062] (b) "pZ27" refers to DNA for a promoter
derived from a gamma zein gene from Zea mays; [0063] (c) "pZ27t"
refers to DNA for a truncated promoter having 59 nucleotides leader
sequence deleted from the 3' region of pZ27; [0064] (d) "Z19as"
refers to DNA for an antisense-oriented segment of 351 nucleotides
from the coding sequence of a 19 kilo dalton alpha zein gene from
Zea mays; [0065] (e) "Z19s" refers to DNA for a sense-oriented
segment of 351 nucleotides from the coding sequence of a 19 kilo
dalton alpha zein gene from Zea mays, which is an inverted repeat
of Z19as; [0066] (f) "Z22as" refers to DNA for an
antisense-oriented segment of 789 nucleotides from the coding
sequence of a 22 kilo dalton alpha zein gene from Zea mays; [0067]
(g) "Z22asL" refers to DNA for an antisense-oriented segment of 785
nucleotides from the coding sequence of a 22 kilo dalton alpha zein
gene from Zea mays; [0068] (h) "Z22asSI" refers to DNA for an
antisense-oriented segment of 789 nucleotides from the coding
sequence of a 22 kilo dalton alpha zein gene from Zea mays having a
520 nucleotide long spliceable intron from a GB1 gene intron 3 from
Zea mays inserted in the unpaired region; [0069] (i) "Z22s" refers
to DNA for a sense-oriented segment of 289 nucleotides from the
coding sequence of a 22 kilo dalton alpha zein gene from Zea mays,
which is an inverted repeat of the 5' end of Z22as; and
[0070] (j) "TE9" refers to DNA for a sense oriented polyadenylation
signal and site element from an RbcS2 gene from Pisum sativum.
[0071] With reference to Table 2 and SEQ ID NO:2 a transformation
vector comprising "construct 2a" was made in the manner of Example
1 except that the transcription unit for LKR gene suppression was
replaced by a transcription unit comprising the elements
illustrated in the following schematic:
[0072] "Construct 2a" pZ27 - Z19as - Z22asL - Z22s - Z19s - TE9
TABLE-US-00003 TABLE 2 Bases of SEQ ID NO: 2 description of DNA
segment 1-357 A. tumefaciens right border 376-1774 DNA of a rice
actin promoter and rice actin intron 1784-2011 DNA of A.
tumefaciens EPSPS chloroplast transit peptide 2012-3379 DNA of A.
tumefaciens aroA (glyphosate-resistant marker) 3395-3647 DNA of A.
tumefaciens NOS terminator 3479-4391 DNA of Pisum sativum RbcS2
terminator 4398-4748 DNA for Z19s 4755-5043 DNA for Z22s 5050-5835
DNA of Z22asL 5842-6192 DNA of Z19as 6204-7305 DNA of Zea mays Z27
promoter 7353-7794 A. tumefaciens left border
[0073] Corn callus was transformed and events with a single copy of
the transformation vector were selected for growth into plants.
Seed from plants grown from 26 of 29 single copy events showed
substantial reduction of the 19 kilo dalton alpha zeins and the 22
kilo Dalton alpha zeins.
[0074] Other transformation vectors were made in a similar manner
using the elements illustrated in the following Table 3.
TABLE-US-00004 TABLE 3 Construct 2b1
pGcx-Z19as-Z22asSI-Z22s-Z19s-TE9 Construct 2b2*
pGcx-Z19as-Z22asSI-Z22s-Z19s-TE9 Construct 2c
pZ27-Z19as-Z22asSI-Z22s-Z19s-TE9 Construct 2d
PZ27t-Z19as-Z22asSI-Z22s-Z19s-TE9 Construct 2e
PZ27-Z19as-Z22asL-Z19s-TE9 *construct 2b2 was inserted into a
transformation vector that also included a transcription unit for
expressing another gene having a promoter contiguous to pGcx.
[0075] The efficiency of suppressing the alpha zeins in seeds
produced by plants grown from single copy events is reported in
Table 4 which reports the number of transgenic events with
reduction of zeins as compared to the total number of transgenic
events generated in each construct tested. The zein reduction
phenotype is observed by MALDI-TOF MS
(Matrix-Assisted-Laser-Desorption Ionization Time-Of-Flight Mass
Spectrometry) analysis. FIG. 3 is illustrates typical spectra
evidencing zein reduction. TABLE-US-00005 TABLE 4 Construct 19 kD
zein 19 and 22 kD zein 2a 26/29 26/29 2b1 0/21 0/21 2b2 5/7 0/7 2c
20/21 18/21 2d 7/8 1/8 2e 12/14 2/14
EXAMPLE 3
[0076] This example illustrates zein reduction in transgenic plants
harboring construct 2a (pMON73566; Table 2 and FIG. 4A) and
construct 2e (pMON73566; Table 3, Table 5, and FIG. 4B) as well as
the oil, protein, and amino acid profiles of bulked kernels derived
from plants grown from single copy events as reported in Table
4.
[0077] With reference to Table 5 and SEQ ID NO:2 a transformation
vector comprising "construct 2e" was made in the manner of Example
1 except that the transcription unit for LKR gene suppression was
replaced by a transcription unit comprising the elements
illustrated in the following schematic:
[0078] "Construct 2e" PZ27 - Z19as - Z22asL - Z19s - TE9
TABLE-US-00006 TABLE 5 Bases of SEQ ID NO: 2 description of DNA
segment 1-357 A. tumefaciens right border 376-1774 DNA of a rice
actin promoter and rice actin intron 1784-2011 DNA of A.
tumefaciens EPSPS chloroplast transit peptide 2012-3379 DNA of A.
tumefaciens aroA (glyphosate-resistant marker) 3395-3647 DNA of A.
tumefaciens NOS terminator 3479-4391 DNA of Pisum sativum RbcS2
terminator 4398-4748 DNA for Z19s 4750-5535 DNA of Z22asL 5542-5892
DNA of Z19as 5904-7003 DNA of Zea mays Z27 promoter 7353-7794 A.
tumefaciens left border
[0079] Corn immature embryo was transformed and events with a
single copy of the transformation vector were selected for growth
into plants. Seed from plants grown from 2 of 14 single copy events
showed substantial reduction of both the 19 kilo Dalton alpha zeins
and the 22 kilo Dalton alpha zeins (Table 4).
Zein Reduction
[0080] In transgenic plants harboring pMON73567, which contains
dsRNA against both 19- and 22-kD .alpha.-zein sequences, 26 of 29
plants display reduction in both 19- and 22-kD .alpha.-zein
accumulation (Table 4, construct 2a; FIG. 4A; FIG. 5B).
Additionally, in transgenic plants harboring pMON73566, which
contains dsRNA against only a 19-kD .alpha.-zein sequence and which
uses the 22-kD .alpha.-zein sequence as the loop, 2 of 14 plants
display reduction in both 19- and 22-kD .alpha.-zein accumulation
(Table 4, construct 2e; FIG. 4B; FIG. 5B). Ten other pMON73566
events exhibit mostly 19-kD .alpha.-zein reduction (Table 4,
construct 2e; FIG. 4B; FIG. 5C). Two representative events were
selected from each construct for advancement to the next generation
for collection of homozygous ears for compositional analyses.
Events M80442 and M82186 containing the pMON73567 construct, and
events M80780 and M80791 containing the pMON73566 construct were
selected. M80442, M82186 and M80791 exhibit both 19- and 22-kD
.alpha.-zein reduction. M80780, exhibits only 19-kD .alpha.-zein
reduction.
Oil Content
[0081] Four events (M80442, M82186, M80780 and M80791) from 2 zein
reduction constructs, pMON73566 and pMON73567, were grown in the
field and zygosity was determined by a molecular assay. Oil was
determined for kernels of homozygous transgene-positive ears and
control ears by a wet chemistry oil extraction method. About 100 mg
of ground sample was weighed in an 11-ml pressure cell half filled
with sand. Additional sand was added to fill the cell, a filter was
placed on the top, and the cell was capped with a screw cap. The
cell was placed on the carousel of the Dionex Accelerated Solvent
Extractor (Dionex, Sunnyvale, Calif.) following the manufacturer's
protocol. The oil was extracted with petroleum ether at 1000 psig
(pounds per square inch gauge) at 105.degree. C. in three
extraction steps. The final extraction product was added to a
pre-weighed vial. The solvent was evaporated at 37.degree. C. for
two hours under a stream of either nitrogen or air and the vial was
then weighed. Analysis of oil content of the samples was done in
triplicate. Oil concentration of control kernels averaged 3.8%
(Table 6). Average oil concentration of the transgene-positive
kernels ranged from 4.0% to 5.2%. The average oil content of seeds
from all 4 transgenic events represents an increase over the
average oil content of seeds from wild type plants; and the
increase in oil content from the M82186 event is considered to be
statistically significant. TABLE-US-00007 TABLE 6 Proximate assay
and size of bulked kernels Wild Type pMON73566 pMON73567 % +
SD.sup.a LH244 M80780 M80791 M80442 M82186 Oil.sup.b 3.8 .+-. 0.3
4.1 .+-. 0.2 4.4 .+-. 0.1 4.0 .+-. 0.5 5.2 .+-. 0.5 Protein 9.6
.+-. 1.1 8.7 .+-. 1.0 9.9 .+-. 1.2 9.3 .+-. 1.0 10.0 .+-. 0.7
Starch 70.4 .+-. 0.9 68.7 .+-. 0.4 67.3 .+-. 0.3 68.5 .+-. 0.6 67.8
.+-. 1.0 Moisture 9.0 .+-. 0.4 10.5 .+-. 0.2 10.4 .+-. 0.3 10.5
.+-. 0.2 10.5 .+-. 0.7 Density 1.31 .+-. 0.00 1.20 .+-. 0.01 1.20
.+-. 0.02 1.20 .+-. 0.01 1.21 .+-. 0.01 Size.sup.c 24.9 .+-. 1.4
24.3 .+-. 2.0 24.0 .+-. 0.9 23.9 .+-. 1.4 23.2 .+-. 2.2 Table 6.
The oil, protein and starch contents are calculated on dry matter
base. Except oil, the proximate contents of bulked kernels from
each ear were determined by near infrared transmission (NIT)
analysis. .sup.bThe oil content was obtained by a wet chemistry
method. The numbers in bold are statistically different from the
LH244 numbers by Dunnett's test (.alpha. = 0.05). .sup.aData are
means .+-. standard deviations. .sup.cThe sizes were measured in
100 kernel weights in grams.
Amino Acid Profile
[0082] Transgenic plants transformed with antisense constructs
targeting 19-kD .alpha.-zeins have exhibited an increased lysine
content of up to 35% in mature corn seeds (Huang et al., 2004;
Huang et al., 2005). In the transgenic plants haboring the
pMON73567 and pMON73566 constructs that target both the 19- and
22-kD .alpha.-zeins, the increases in lysine content are
significantly greater. As shown in Table 7, among the events
analyzed, the least increase in lysine content, 66% ((4035 ppm
transgenic-2438 ppm wild type)/2438 ppm wild type), is observed in
M80780 and the most, 105% ((5003 ppm transgenic-2438 ppm wild
type)/2438 ppm wild type), is found in M80791. Similarly, the
transgenic plants have much higher tryptophan contents as compared
to the wild type, LH244 (Table 7). TABLE-US-00008 TABLE 7 Total
amino acid analysis of ground kernels Wild Type pMON73566 pMON73567
Ave .+-. SD.sup.a LH244 M80780 M80791 M80442 M82186 Ala 6687 .+-.
594 5497 .+-. 631 6417 .+-. 855 5862 .+-. 999 6458 .+-. 322 Arg
4342 .+-. 293 6060 .+-. 708 7313 .+-. 1048 6665 .+-. 1203 7165 .+-.
655 Asx 5555 .+-. 377 8928 .+-. 1651 11977 .+-. 2034 10253 .+-.
2803 11143 .+-. 886 Glx 17788 .+-. 1623 15873 .+-. 2421 18610 .+-.
2762 16860 .+-. 3617 18603 .+-. 1322 Gly 3400 .+-. 166 4537 .+-.
463 5377 .+-. 770 4973 .+-. 783 5290 .+-. 410 His 1498 .+-. 126
2350 .+-. 234 2253 .+-. 441 2305 .+-. 518 2470 .+-. 160 Ile 3265
.+-. 255 3030 .+-. 368 3700 .+-. 678 3373 .+-. 587 3578 .+-. 261
Leu 11265 .+-. 1074 7318 .+-. 788 8327 .+-. 1106 7718 .+-. 1264
8270 .+-. 497 Lys 2438 .+-. 132 4035 .+-. 574 5003 .+-. 866 4533
.+-. 780 4800 .+-. 443 Phe 3760 .+-. 282 3032 .+-. 295 3637 .+-.
506 3288 .+-. 571 3455 .+-. 260 Ser 4067 .+-. 364 4235 .+-. 465
4620 .+-. 425 4355 .+-. 779 4785 .+-. 325 Thr 3062 .+-. 226 3572
.+-. 406 4047 .+-. 446 3783 .+-. 676 4130 .+-. 279 Trp 598 .+-. 48
877 .+-. 117 1087 .+-. 158 940 .+-. 201 1040 .+-. 96 Tyr 3720 .+-.
307 3430 .+-. 414 4093 .+-. 432 3652 .+-. 624 4045 .+-. 270 Val
4710 .+-. 325 5312 .+-. 598 6553 .+-. 1068 5977 .+-. 1030 6293 .+-.
483 Sum 76155 .+-. 5996 78087 .+-. 10057 93013 .+-. 12760 84535
.+-. 16220 91553 .+-. 6286 Lys % (P).sup.b 2.83 .+-. 0.23 5.23 .+-.
0.17 5.62 .+-. 0.29 5.40 .+-. 0.37 5.33 .+-. 0.28 Trp % (P).sup.b
0.69 .+-. 0.05 1.14 .+-. 0.04 1.22 .+-. 0.03 1.12 .+-. 0.11 1.15
.+-. 0.05 Leu % (P).sup.b 13.00 .+-. 0.34 9.51 .+-. 0.15 9.38 .+-.
0.12 9.21 .+-. 0.56 9.20 .+-. 0.34 Table 7. Samples were ground
mills of bulked mature kernels of individual ears. .sup.aData (ppm)
are averages of ears within an event .+-. standard deviations. Four
homologous ears from each event were measured. .sup.bThey are
expressed as the percentages of protein measured in Table 6 without
the subtraction of moisture. The numbers in bold are statistically
different from the LH244 numbers by Dunnett's test (.alpha. =
0.05). Asx, asparagine and Aspartate; Glx, glutamine and
glutamate.
[0083] Aspartate, asparagine and glutamate are among the free amino
acids that exhibit a significant increase in the seeds of these
transgenic plants (Table 8). The increased accumulation of these
free amino acids has been shown to enhance lysine biosynthesis in
the presence of CordapA (Huang et al., 2005; Monsanto patent
application Ser. No. 11/077089, filed Mar. 10, 2005).
TABLE-US-00009 TABLE 8 Free amino acid analysis of ground kernels
Wid Type pMON73566 pMON73567 Ave .+-. SD.sup.a LH244 M80780 M80791
M80442 M82186 Ala 95 .+-. 39 114 .+-. 73 194 .+-. 91 172 .+-. 103
313 .+-. 122 Arg 50 .+-. 24 92 .+-. 64 121 .+-. 40 110 .+-. 53 160
.+-. 38 Asn 232 .+-. 40 2341 .+-. 667 3726 .+-. 1102 2844 .+-. 1184
2995 .+-. 402 Asp 143 .+-. 30 692 .+-. 386 1306 .+-. 375 1040 .+-.
479 1323 .+-. 207 Glu 256 .+-. 33 582 .+-. 492 1064 .+-. 472 823
.+-. 670 1207 .+-. 119 Gln 85 .+-. 56 182 .+-. 186 270 .+-. 180 255
.+-. 264 319 .+-. 40 Gly 18 .+-. 6 25 .+-. 13 37 .+-. 13 31 .+-. 15
44 .+-. 7 His 24 .+-. 4 47 .+-. 21 79 .+-. 25 63 .+-. 27 76 .+-. 5
Ile 13 .+-. 5 18 .+-. 12 25 .+-. 8 21 .+-. 9 36 .+-. 9 Leu 10 .+-.
3 15 .+-. 14 25 .+-. 11 19 .+-. 14 34 .+-. 10 Lys 25 .+-. 12 40
.+-. 26 66 .+-. 39 57 .+-. 40 70 .+-. 11 Phe 37 .+-. 30 19 .+-. 12
34 .+-. 8 25 .+-. 11 40 .+-. 11 Ser 50 .+-. 21 69 .+-. 41 115 .+-.
50 88 .+-. 53 176 .+-. 76 Thr 16 .+-. 6 46 .+-. 41 84 .+-. 36 60
.+-. 46 110 .+-. 29 Trp 8 .+-. 1 17 .+-. 5 23 .+-. 4 21 .+-. 5 25
.+-. 1 Tyr 34 .+-. 6 94 .+-. 52 163 .+-. 18 120 .+-. 48 182 .+-. 52
Val 33 .+-. 10 46 .+-. 32 76 .+-. 29 62 .+-. 33 101 .+-. 26 Sum
1134 .+-. 259 4447 .+-. 1962 7417 .+-. 2334 5819 .+-. 3052 7225
.+-. 336 Table 8. Samples were ground mills of bulked mature
kernels of individual ears. .sup.aData (ppm) are averages of ears
within an event .+-. standard deviations. Four homologous ears from
each event were measured. The numbers in bold are statistically
different from the LH244 numbers by Dunnett's test (.alpha. =
0.05).
Protein Content
[0084] The constitutive expression of asparagine synthetase is
thought to increase the protein content of maize seeds by fueling
the flux of free amino acids from vegetative tissues into
developing ears. Not only do these zein reduction lines have
elevated levels of free amino acids (Table 8), but also the amount
of protein accumulation is proportional to the level of free amino
acids (FIG. 6). These results suggest a possible synergistic effect
of combining zein reduction and the expression of asparagine
synthetase that could result in improving both the quantity and the
quality of the maize proteins.
[0085] This invention contemplates the use of anti-sense-oriented
DNA elements and sense-oriented DNA elements from other maize zein
proteins, including but not limited to additional members of the
19- and 22-kD .alpha.-zeins, 16- and 27-kD .gamma.-zeins, 10-kD
.delta.-zeins, and 15-kD .beta.-zeins, as a method for suppressing
transcription of more than one gene or the accumulation of the mRNA
corresponding to those genes thereby preventing translation of the
transcript into protein. In particular, the contemplated
.alpha.-zeins include all variants in the 19-kD size range and all
variants in the 22-kD size range.
[0086] All of the materials and methods disclosed and claimed
herein can be made and used without undue experimentation as
instructed by the above disclosure. Although the materials and
methods of this invention have been described in terms of preferred
embodiments and illustrative examples, it will be apparent to those
of skill in the art that variations may be applied to the materials
and methods described herein without departing from the concept,
spirit and scope of the invention. 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.
Sequence CWU 1
1
2 1 8590 DNA Artificial recombinant DNA construct in plasmid
between Agrabacterium borders 1 aggatttttc ggcgctgcgc tacgtccgcg
accgcgttga gggatcaagc cacagcagcc 60 cactcgacct tctagccgac
ccagacgagc caagggatct ttttggaatg ctgctccgtc 120 gtcaggcttt
ccgacgtttg ggtggttgaa cagaagtcat tatcgcacgg aatgccaagc 180
actcccgagg ggaaccctgt ggttggcatg cacatacaaa tggacgaacg gataaacctt
240 ttcacgccct tttaaatatc cgattattct aataaacgct cttttctctt
aggtttaccc 300 gccaatatat cctgtcaaac actgatagtt taaactgaag
gcgggaaacg acaatctgat 360 ccccatcaag cttactcgag gtcattcata
tgcttgagaa gagagtcggg atagtccaaa 420 ataaaacaaa ggtaagatta
cctggtcaaa agtgaaaaca tcagttaaaa ggtggtataa 480 agtaaaatat
cggtaataaa aggtggccca aagtgaaatt tactcttttc tactattata 540
aaaattgagg atgtttttgt cggtactttg atacgtcatt tttgtatgaa ttggttttta
600 agtttattcg cttttggaaa tgcatatctg tatttgagtc gggttttaag
ttcgtttgct 660 tttgtaaata cagagggatt tgtataagaa atatctttag
aaaaacccat atgctaattt 720 gacataattt ttgagaaaaa tatatattca
ggcgaattct cacaatgaac aataataaga 780 ttaaaatagc tttcccccgt
tgcagcgcat gggtattttt tctagtaaaa ataaaagata 840 aacttagact
caaaacattt acaaaaacaa cccctaaagt tcctaaagcc caaagtgcta 900
tccacgatcc atagcaagcc cagcccaacc caacccaacc cagcccaccc cagtccagcc
960 aactggacaa tagtctccac acccccccac tatcaccgtg agttgtccgc
acgcaccgca 1020 cgtctcgcag ccaaaaaaaa aaagaaagaa aaaaaagaaa
aagaaaaaac agcaggtggg 1080 tccgggtcgt gggggccgga aacgcgagga
ggatcgcgag ccagcgacga ggccggccct 1140 ccctccgctt ccaaagaaac
gccccccatc gccactatat acataccccc ccctctcctc 1200 ccatcccccc
aaccctacca ccaccaccac caccacctcc acctcctccc ccctcgctgc 1260
cggacgacga gctcctcccc cctccccctc cgccgccgcc gcgccggtaa ccaccccgcc
1320 cctctcctct ttctttctcc gttttttttt ccgtctcggt ctcgatcttt
ggccttggta 1380 gtttgggtgg gcgagaggcg gcttcgtgcg cgcccagatc
ggtgcgcggg aggggcggga 1440 tctcgcggct ggggctctcg ccggcgtgga
tccggcccgg atctcgcggg gaatggggct 1500 ctcggatgta gatctgcgat
ccgccgttgt tgggggagat gatggggggt ttaaaatttc 1560 cgccgtgcta
aacaagatca ggaagagggg aaaagggcac tatggtttat atttttatat 1620
atttctgctg cttcgtcagg cttagatgtg ctagatcttt ctttcttctt tttgtgggta
1680 gaatttgaat ccctcagcat tgttcatcgg tagtttttct tttcatgatt
tgtgacaaat 1740 gcagcctcgt gcggagcttt tttgtaggta gaagtgatca
accatggcgc aagttagcag 1800 aatctgcaat ggtgtgcaga acccatctct
tatctccaat ctctcgaaat ccagtcaacg 1860 caaatctccc ttatcggttt
ctctgaagac gcagcagcat ccacgagctt atccgatttc 1920 gtcgtcgtgg
ggattgaaga agagtgggat gacgttaatt ggctctgagc ttcgtcctct 1980
taaggtcatg tcttctgttt ccacggcgtg catgcttcac ggtgcaagca gccggcccgc
2040 aaccgcccgc aaatcctctg gcctttccgg aaccgtccgc attcccggcg
acaagtcgat 2100 ctcccaccgg tccttcatgt tcggcggtct cgcgagcggt
gaaacgcgca tcaccggcct 2160 tctggaaggc gaggacgtca tcaatacggg
caaggccatg caggcgatgg gcgcccgcat 2220 ccgtaaggaa ggcgacacct
ggatcatcga tggcgtcggc aatggcggcc tcctggcgcc 2280 tgaggcgccg
ctcgatttcg gcaatgccgc cacgggctgc cgcctgacga tgggcctcgt 2340
cggggtctac gatttcgaca gcaccttcat cggcgacgcc tcgctcacaa agcgcccgat
2400 gggccgcgtg ttgaacccgc tgcgcgaaat gggcgtgcag gtgaaatcgg
aagacggtga 2460 ccgtcttccc gttaccttgc gcgggccgaa gacgccgacg
ccgatcacct accgcgtgcc 2520 gatggcctcc gcacaggtga agtccgccgt
gctgctcgcc ggcctcaaca cgcccggcat 2580 cacgacggtc atcgagccga
tcatgacgcg cgatcatacg gaaaagatgc tgcagggctt 2640 tggcgccaac
cttaccgtcg agacggatgc ggacggcgtg cgcaccatcc gcctggaagg 2700
ccgcggcaag ctcaccggcc aagtcatcga cgtgccgggc gacccgtcct cgacggcctt
2760 cccgctggtt gcggccctgc ttgttccggg ctccgacgtc accatcctca
acgtgctgat 2820 gaaccccacc cgcaccggcc tcatcctgac gctgcaggaa
atgggcgccg acatcgaagt 2880 catcaacccg cgccttgccg gcggcgaaga
cgtggcggac ctgcgcgttc gctcctccac 2940 gctgaagggc gtcacggtgc
cggaagaccg cgcgccttcg atgatcgacg aatatccgat 3000 tctcgctgtc
gccgccgcct tcgcggaagg ggcgaccgtg atgaacggtc tggaagaact 3060
ccgcgtcaag gaaagcgacc gcctctcggc cgtcgccaat ggcctcaagc tcaatggcgt
3120 ggattgcgat gagggcgaga cgtcgctcgt cgtgcgtggc cgccctgacg
gcaaggggct 3180 cggcaacgcc tcgggcgccg ccgtcgccac ccatctcgat
caccgcatcg ccatgagctt 3240 cctcgtcatg ggcctcgtgt cggaaaaccc
tgtcacggtg gacgatgcca cgatgatcgc 3300 cacgagcttc ccggagttca
tggacctgat ggccgggctg ggcgcgaaga tcgaactctc 3360 cgatacgaag
gctgcctgat gagctcgaat tcccgatcgt tcaaacattt ggcaataaag 3420
tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt atcatataat ttctgttgaa
3480 ttacgttaag catgtaataa ttaacatgta atgcatgacg ttatttatga
gatgggtttt 3540 tatgattaga gtcccgcaat tatacattta atacgcgata
gaaaacaaaa tatagcgcgc 3600 aaactaggat aaattatcgc gcgcggtgtc
atctatgtta ctagatcggg gatgggggat 3660 ccactagtga tatccgtcga
gtggcggccg cgttttatga ataataataa tgcatatctg 3720 tgcattacta
cctgggatac aagggcttct ccgccataac aaattgagtt gcgatgctga 3780
gaacgaacgg ggaagaaagt aagcgccgcc caaaaaaaac gaacatgtac gtcggctata
3840 gcaggtgaaa gttcgtgcgc caatgaaaag ggaacgatat gcgttgggta
gttgggatac 3900 ttaaatttgg agagtttgtt gcatacacta atccactaaa
gttgtctatc tttttaacag 3960 ctctaggcag gatataagat ttatatctaa
tctgttggag ttgcttttag agtaactttt 4020 ctctctgttt cgtttatagc
cgattagcac aaaattaaac taggtgacga gaaataaaga 4080 aaaacggagg
cagtaaaaaa tacccaaaaa aatacttgga gatttttgtc tcaaaattat 4140
cttctaattt taaaagctac atattaaaaa tactatatat taaaaatact tcgagatcat
4200 tgcttgggat gggcagggcc aatagctaat tgctaaggat gggctatatt
tatgtatcgt 4260 ctgaaacatg taggggctaa tagttagatg actaatttgc
tgtgttcgta cggggtgctg 4320 tttgagccta gcgatgaagg gtcatagttt
catacaagaa ctcacttttg gttcgtctgc 4380 tgtgtctgtt ctcagcgtaa
cggcatcaat ggatgccaaa ctccgcaagg ggacaaatga 4440 agaagcgaag
agattataga acacgcacgt gtcattattt atttatggac ttgcctcagt 4500
agcttacagc atcgtacccg cacgtacata ctacagagcc acacttattg cactgcctgc
4560 cgcttacgta catagttaac acgcagagag gtatatacat acacgtccaa
cgtctccact 4620 caggctcatg ctacgtacgc acgtcggtcg cgcgccaccc
tctcgttgct tcctgctcgt 4680 tttggcgaat tccgatttgg caagtgttcc
agagcaaaag ctggaagctc tcgtagtctg 4740 agcctctttg ctgattcata
caagttatga ccatctacat ggatcgtctc accaagaaat 4800 ttgtagactg
caggattttt ccctgaccgg agtgcaccag ctgggttcca actgaattta 4860
taggcaagcg gattgtttgc tgcagctgga gatggcaatc caccacagta agatgtaaat
4920 gcctttattt ttccctttcg tgcatgagct tcatcaatca tcttcattga
catcaagtga 4980 tctatgccag gatctaggcc catttcacaa agtatagtta
cacctgcatc tttggcagct 5040 tggctcaagt ttgacatgga ttcatcaaca
tagcttgccg ttaccatgtg cttcttcaac 5100 tctatgcata ctcctgcaat
ggcagcatga aaactagcag gcagcaccgg ttggacatca 5160 ttgagacagc
tggaggttca tttcacttgg ttagatgtga agttggacaa agcacggatg 5220
atatgtcgta ctcagagctt gaagtaggag cagatgatac tgccacattg gataaaatta
5280 ttgattcctt gacttcttta gctaatgaac atggtggaga tcacgatgcc
gggcaagaaa 5340 ttgaattagc tctgaagata ggaaaagtca atgagtatga
aactgacgtc acaattgata 5400 aaggagggcc aaagatttta attcttggag
ctggaagagt ctgtcggcca gctgctgagt 5460 ttctggcatc ttacccagac
atatgtacct atggtgttga tgaccatgat gcagatcaaa 5520 ttcatgttat
cgtggcatct ttgtatcaaa aagatgcaga agagacagtt gatggtattg 5580
aaaatacaac tgctacccag cttgatgttg ctgatattgg aagcctttca gatcttgttt
5640 ctcaggttga ggttgtaatt agcttgctgc ctgctagttt tcatgctgcc
attgcaggag 5700 tatgcataga gttgaagaag cacatggtaa cggcaagcta
tgttgatgaa tccatgtcaa 5760 acttgagcca agctgccaaa gatgcaggtg
taactatact ttgtgaaatg ggcctagatc 5820 ctggcataga tcacttgatg
tcaatgaaga tgattgatga agctcatgca cgaaagggaa 5880 aaataaaggc
atttacatct tactgtggtg gattgccatc tccagctgca gcaaacaatc 5940
cgcttgccta taaattcagt tggaacccag ctggtgcact ccggtcaggg aaaaatcctg
6000 cagtctacaa atttcttggt gagacgatcc atgtagatgg tcataacttg
tatgaatcag 6060 caaagaggct cagactacga gagcttccag cttttgctct
ggaacacttg ccaaatcggg 6120 atccgcagct gcacgggtcc aggaaagcaa
tcgcatagtc aagctaaatc atcaagatgc 6180 aaacttttcg cccttgctaa
acacggtaaa attcgaatgg acatgtgtgg agcagcaaag 6240 gccttacgtc
cgagaaacag ggccactcaa cgagttagtt aaattcaaag aaagaaacgc 6300
ctccttgcaa gttgcaacat tcttagatca tactgatgaa aatgacgtct ttcattaaag
6360 aacagggaag atagatcttt gctcaatatc gtatgatgtg ttcagccaga
ctgtcggatg 6420 gaccacacgg taatagcagt gctggacgat gttacatcga
gaaagattac tagccttttc 6480 atgggagtga aggatataaa agaaataagt
tcaccacgat tgcaggatag catacaagat 6540 cagcgccact gcggcactgt
tcatcgaaaa aaaaactgtg gacgaagcta gctttcccca 6600 aaattactca
acgaatcata aaccaagatt agtcagatca agagacagag gagaaacaag 6660
gcggaccttt gcacttgatc ggatccttgg gttggctgta tgcagaacta aagcggaggt
6720 ggcgcgcatt tataccagcg ccgggccctg gtacgtggcg cggccgcgcg
gctacgtgga 6780 ggaaggctgc gtggcagcag acacacgggt cgccacgtcc
cgccgtactc tccttaccgt 6840 gcttatccgg gctccggctc ggtgcacgcc
agggtgtggc cgcctctgag cagactttgt 6900 cgtgttccac agtggtgtcg
tgttccgggg actccgatcc gcggcgagcg accgagcgtg 6960 taaaagagtt
cctactaggt acgttcattg tatctggacg acgggcagcg gacaatttgc 7020
tgtaagagag gggcagtttt tttttagaaa aacagagaat tccgttgagc taattgtaat
7080 tcaacaaata agctattagt tggttttagc ttagattaaa gaagctaacg
actaatagct 7140 aataattagt tggtctatta gttgactcat tttaaggccc
tgtttcaatc tcgcgagata 7200 aactttagca gctatttttt agctactttt
agccatttgt aatctaaaca ggagagctaa 7260 tggtggtaat tgaaactaaa
ctttagcact tcaattcata tagctaaagt ttagcaggaa 7320 gctaaacttt
atcccgtgag attgaaacgg ggcctaaatc tctcagctat ttttgatgca 7380
aattactgtc actactggaa tcgagcgctt tgccgagtgt caaagcctga aaaacactcc
7440 gtaaagactt tgcctagtgt gacactcgac aaagagatct cgacgaacag
tacatcgaca 7500 acggcttctt tgtcgagtac tttttatcgg acacttgaca
aagtctttgt cgagtgaact 7560 acattgaaac tctatgattt tatgtgtagg
tcacttaggt ttctacacat agtacgtcac 7620 aactttaccg aaacattatc
aaatttttat cacaacctct atatatgata tcatgacatg 7680 tggacaagtt
tcattaattt ctgactttat ttgtgtttta tacaattttt aaacaactag 7740
ataacaagtt cacggtcatg tttagtgagc atggtgcttg aagattctgg tctgcttctg
7800 aaatcggtcg taacttgtgc tagataacat gcatatcatt tattttgcat
gcacggtttt 7860 ccatgtttcg agtgacttgc agtttaaatg tgaattttcc
gaagaaattc aaataaacga 7920 actaaatcta atatttatag aaaacatttt
tgtaaatatg taattgtgcc aaaatggtac 7980 atgtagatct acatagtgta
ggaacatacc acaaaaagtt tggttggcaa aataaaaaaa 8040 ataaaatata
ctttatccga gtgtccaagg tatggcactc ggcccgggtg gccaagctta 8100
ctagcccggg cgcgccttaa ttaagcggcc gcatcgatcg tgaagtttct catctaagcc
8160 cccatttgga cgtgaatgta gacacgtcga aataaagatt tccgaattag
aataatttgt 8220 ttattgcttt cgcctataaa tacgacggat cgtaatttgt
cgttttatca aaatgtactt 8280 tcattttata ataacgctgc ggacatctac
atttttgaat tgaaaaaaaa ttggtaatta 8340 ctctttcttt ttctccatat
tgaccatcat actcattgct gatccatgta gatttcccgg 8400 acatgaagcc
atttacaatt gaatatatcc tgccgccgct gccgctttgc acccggtgga 8460
gcttgcatgt tggtttctac gcagaactga gccggttagg cagataattt ccattgagaa
8520 ctgagccatg tgcaccttcc ccccaacacg gtgagcgacg gggcaacgga
gtgatccaca 8580 tgggactttt 8590 2 7794 DNA Artificial recombinant
DNA construct in plasmid between Agrobacterium borders 2 aggatttttc
ggcgctgcgc tacgtccgcg accgcgttga gggatcaagc cacagcagcc 60
cactcgacct tctagccgac ccagacgagc caagggatct ttttggaatg ctgctccgtc
120 gtcaggcttt ccgacgtttg ggtggttgaa cagaagtcat tatcgcacgg
aatgccaagc 180 actcccgagg ggaaccctgt ggttggcatg cacatacaaa
tggacgaacg gataaacctt 240 ttcacgccct tttaaatatc cgattattct
aataaacgct cttttctctt aggtttaccc 300 gccaatatat cctgtcaaac
actgatagtt taaactgaag gcgggaaacg acaatctgat 360 ccccatcaag
cttactcgag gtcattcata tgcttgagaa gagagtcggg atagtccaaa 420
ataaaacaaa ggtaagatta cctggtcaaa agtgaaaaca tcagttaaaa ggtggtataa
480 agtaaaatat cggtaataaa aggtggccca aagtgaaatt tactcttttc
tactattata 540 aaaattgagg atgtttttgt cggtactttg atacgtcatt
tttgtatgaa ttggttttta 600 agtttattcg cttttggaaa tgcatatctg
tatttgagtc gggttttaag ttcgtttgct 660 tttgtaaata cagagggatt
tgtataagaa atatctttag aaaaacccat atgctaattt 720 gacataattt
ttgagaaaaa tatatattca ggcgaattct cacaatgaac aataataaga 780
ttaaaatagc tttcccccgt tgcagcgcat gggtattttt tctagtaaaa ataaaagata
840 aacttagact caaaacattt acaaaaacaa cccctaaagt tcctaaagcc
caaagtgcta 900 tccacgatcc atagcaagcc cagcccaacc caacccaacc
caacccaccc cagtccagcc 960 aactggacaa tagtctccac acccccccac
tatcaccgtg agttgtccgc acgcaccgca 1020 cgtctcgcag ccaaaaaaaa
aaagaaagaa aaaaaagaaa aagaaaaaac agcaggtggg 1080 tccgggtcgt
gggggccgga aacgcgagga ggatcgcgag ccagcgacga ggccggccct 1140
ccctccgctt ccaaagaaac gccccccatc gccactatat acataccccc ccctctcctc
1200 ccatcccccc aaccctacca ccaccaccac caccacctcc acctcctccc
ccctcgctgc 1260 cggacgacga gctcctcccc cctccccctc cgccgccgcc
gcgccggtaa ccaccccgcc 1320 cctctcctct ttctttctcc gttttttttt
ccgtctcggt ctcgatcttt ggccttggta 1380 gtttgggtgg gcgagaggcg
gcttcgtgcg cgcccagatc ggtgcgcggg aggggcggga 1440 tctcgcggct
ggggctctcg ccggcgtgga tccggcccgg atctcgcggg gaatggggct 1500
ctcggatgta gatctgcgat ccgccgttgt tgggggagat gatggggggt ttaaaatttc
1560 cgccgtgcta aacaagatca ggaagagggg aaaagggcac tatggtttat
atttttatat 1620 atttctgctg cttcgtcagg cttagatgtg ctagatcttt
ctttcttctt tttgtgggta 1680 gaatttgaat ccctcagcat tgttcatcgg
tagtttttct tttcatgatt tgtgacaaat 1740 gcagcctcgt gcggagcttt
tttgtaggta gaagtgatca accatggcgc aagttagcag 1800 aatctgcaat
ggtgtgcaga acccatctct tatctccaat ctctcgaaat ccagtcaacg 1860
caaatctccc ttatcggttt ctctgaagac gcagcagcat ccacgagctt atccgatttc
1920 gtcgtcgtgg ggattgaaga agagtgggat gacgttaatt ggctctgagc
ttcgtcctct 1980 taaggtcatg tcttctgttt ccacggcgtg catgcttcac
ggtgcaagca gccggcccgc 2040 aaccgcccgc aaatcctctg gcctttccgg
aaccgtccgc attcccggcg acaagtcgat 2100 ctcccaccgg tccttcatgt
tcggcggtct cgcgagcggt gaaacgcgca tcaccggcct 2160 tctggaaggc
gaggacgtca tcaatacggg caaggccatg caggcgatgg gcgcccgcat 2220
ccgtaaggaa ggcgacacct ggatcatcga tggcgtcggc aatggcggcc tcctggcgcc
2280 tgaggcgccg ctcgatttcg gcaatgccgc cacgggctgc cgcctgacga
tgggcctcgt 2340 cggggtctac gatttcgaca gcaccttcat cggcgacgcc
tcgctcacaa agcgcccgat 2400 gggccgcgtg ttgaacccgc tgcgcgaaat
gggcgtgcag gtgaaatcgg aagacggtga 2460 ccgtcttccc gttaccttgc
gcgggccgaa gacgccgacg ccgatcacct accgcgtgcc 2520 gatggcctcc
gcacaggtga agtccgccgt gctgctcgcc ggcctcaaca cgcccggcat 2580
cacgacggtc atcgagccga tcatgacgcg cgatcatacg gaaaagatgc tgcagggctt
2640 tggcgccaac cttaccgtcg agacggatgc ggacggcgtg cgcaccatcc
gcctggaagg 2700 ccgcggcaag ctcaccggcc aagtcatcga cgtgccgggc
gacccgtcct cgacggcctt 2760 cccgctggtt gcggccctgc ttgttccggg
ctccgacgtc accatcctca acgtgctgat 2820 gaaccccacc cgcaccggcc
tcatcctgac gctgcaggaa atgggcgccg acatcgaagt 2880 catcaacccg
cgccttgccg gcggcgaaga cgtggcggac ctgcgcgttc gctcctccac 2940
gctgaagggc gtcacggtgc cggaagaccg cgcgccttcg atgatcgacg aatatccgat
3000 tctcgctgtc gccgccgcct tcgcggaagg ggcgaccgtg atgaacggtc
tggaagaact 3060 ccgcgtcaag gaaagcgacc gcctctcggc cgtcgccaat
ggcctcaagc tcaatggcgt 3120 ggattgcgat gagggcgaga cgtcgctcgt
cgtgcgtggc cgccctgacg gcaaggggct 3180 cggcaacgcc tcgggcgccg
ccgtcgccac ccatctcgat caccgcatcg ccatgagctt 3240 cctcgtcatg
ggcctcgtgt cggaaaaccc tgtcacggtg gacgatgcca cgatgatcgc 3300
cacgagcttc ccggagttca tggacctgat ggccgggctg ggcgcgaaga tcgaactctc
3360 cgatacgaag gctgcctgat gagctcgaat tcccgatcgt tcaaacattt
ggcaataaag 3420 tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt
atcatataat ttctgttgaa 3480 ttacgttaag catgtaataa ttaacatgta
atgcatgacg ttatttatga gatgggtttt 3540 tatgattaga gtcccgcaat
tatacattta atacgcgata gaaaacaaaa tatagcgcgc 3600 aaactaggat
aaattatcgc gcgcggtgtc atctatgtta ctagatcggg gatgggggat 3660
ccactagtga tatccgtcga ctggtaccta cgcgtagcta gcccgggcgc gccttaatta
3720 agcggccgct tcgagtggct gcaggtcgat tgatgcatgt tgtcaatcaa
ttggcaagtc 3780 ataaaatgca ttaaaaaata ttttcatact caactacaaa
tccatgagta taactataat 3840 tataaagcaa tgattagaat ctgacaagga
ttctggaaaa ttacataaag gaaagttcat 3900 aaatgtctaa aacacaagag
gacatacttg tattcagtaa catttgcagc ttttctaggt 3960 ctgaaaatat
atttgttgcc tagtgaataa gcataatggt acaactacaa gtgttttact 4020
cctcatatta acttcggtca ttagaggcca cgatttgaca catttttact caaaacaaaa
4080 tgtttgcata tctcttataa tttcaaattc aacacacaac aaataagaga
aaaaacaaat 4140 aatattaatt tgagaatgaa caaaaggacc atatcattca
ttaactcttc tccatccatt 4200 tccatttcac agttcgatag cgaaaaccga
ataaaaaaca cagtaaatta caagcacaac 4260 aaatggtaca agaaaaacag
ttttcccaat gccataatac tcaaactcag taggattctg 4320 gtgtgtgcgc
aatgaaactg atgcattgaa cttgacgaac gttgtcgaaa ccgatgatac 4380
gaacgaaagc tgaattccta gctggctgaa tggtagtagt tgttgctgct gtaaataagc
4440 aggagagttc aatgctgtca gttggttgaa tggaagaaat tgctgggggt
aggcagcaga 4500 tagctggctg aatggtagtt gttgttgttg caaataagaa
gcagagttca atgcagctag 4560 ttggttgaat ggaagaaact gctgttgctg
agagtaggca gcaaggtttg ctagcacaag 4620 ttgttgtagt tgttgtgccc
tgatgttttg tgccaataaa tgcaccaaag gtaactgctg 4680 taatagggct
gatgattgtt ggaggaacaa gggtgataaa ggtaagatgc cagctgcgat 4740
tgcctgttat gcataaagat ggcacctcca acgatgggtt gctgcaaggc agggttcatc
4800 aaagagaact ggttgtatgg cagcaattgt tgttgctgct gcaggaaggt
agcgaccaat 4860 gggttagcca ctgccaatgg attaagtaac tgttgtcgct
gttgtaggta cgcagcagag 4920 tttgacacag ccagttggtt gaatggaagc
aactgttgta agtaggcagc agggtttgcc 4980 acagctagct gagtcagagc
tggtacaatt tgttgcagca actgttgttg taggtacgta 5040 ggtgggcccg
ctaccaagat attagccctc cttgcgcttc ttgccctttt agtgagcgca 5100
acaaatgcgt tcattattcc acagtgctca cttgctccta gtgccagtat tccacagttc
5160 ctcccaccag ttacttcaat gggcttcgaa catccagccg tgcaagccta
caggctacaa 5220 ctagcgcttg cggcgagcgc cttacaacaa ccaattgccc
aattgcaaca acaatccttg 5280 gcacatctaa ccctacaaac cattgcaacg
caacaacaac aacaacagtt tctgccatca 5340 ctgagccacc tagccgtggt
gaaccctgtc acctacttgc aacagcagct gcttgcatcc 5400 aacccacttg
ctctggcgaa cgtagctgca taccagcaac aacaacagct gcaacagttt 5460
atgccagtgc tcagtcaact agccatggtg aaccctgccg tctacctaca actactttca
5520 tctagcccgc tcgcggtggg caatgcacct acgtacctac aacaacagtt
gctgcaacaa 5580 attgtaccag ctctgactca gctagctgtg gcaaaccctg
ctgcctactt acaacagttg 5640 cttccattca accaactggc tgtgtcaaac
tctgctgcgt acctacaaca gcgacaacag 5700 ttacttaatc cattggcagt
ggctaaccca ttggtcgcta ccttcctgca gcagcaacaa 5760 caattgctgc
catacaacca gttctctttg atgaaccctg ccttgcagca acccatcgtt 5820
ggaggtgcca tctttaccgg taacaggcaa tcgcagctgg catcttacct ttatcaccct
5880 tgttcctcca acaatcatca gccctattac agcagttacc tttggtgcat
ttattggcac 5940 aaaacatcag ggcacaacaa ctacaacaac ttgtgctagc
aaaccttgct gcctactctc 6000 agcaacaaca gtttcttcca ttcaaccaac
tagctgcatt gaactctgct tcttatttgc 6060 aacaacaaca actaccattc
agccagctat ctgctgccta cccccagcaa tttcttccat 6120 tcaaccaact
gacagctttg aactctcctg cttatttaca gcagcaacaa ctactaccat 6180
tcagccagct agggatccgg taccgggttc ttctgcgctc tggagtagat aaagctaatg
6240 gtctgaagac
ccagtggtgg tgatggagaa gtgcacaggc atgcgagcgt tatttatagc 6300
tttgattaat taacacaatt tcttgtgttc ttatgccacc gagacggctg taggcagctt
6360 catggtttct tgccaaatgt atatgactcg tcactctctt tacgtagcac
gtcgatggtt 6420 catctggaat cattctgtac ttctgcgtgg ctcagttttg
ttgccttcta caggttgttg 6480 atctacgtaa aacgaattag atttagcttg
acatatggct ttttttttgt tgtaaattta 6540 ctttacacgt caaggatttt
tgtcctgtcc ggcctatttt atttttcatg aaacgatctt 6600 tgtaatgcaa
tatgagttgt ttgtaatgtc ttgtgagctg taagcatgta tatcagatga 6660
gtatgatctc ggcatgactc accgtgtttc tttgcacaca gagaggattt gtttgattgt
6720 ttcttaccca atacccttga cgtgcaattt tggttgatgt tctgtgagtt
gttaaggata 6780 caacaaattc ttggagcttt acatgccaat gcatggttgt
ttcgtgttcc tcaccacttt 6840 aggacttata cggttgcacc tggatgatcg
aaggggattg ggagagatta aatctccttc 6900 tattcaattt tgactaggaa
gagatttaat cgtttccaac ccctttcgat ccagacgtaa 6960 gcgaacaagt
tttttatttg gataccctct tattcatctt aatacacaca tgtattaagt 7020
tgcactagtt atatgcccgt gcattgctac ggtttatata tatatatata tatatgtata
7080 tatatatata tgatatatga taaattttgt tttaataaaa catatgtttt
ctattgatta 7140 ggttgtgtga atatggagcc aacaaccaat atccagaaca
cttatacata atttcacctt 7200 attttgtaca taaactctct tattatagta
gtagagaaga gattataaga gtgcgggttg 7260 attataaaga aatgtaggag
ttttttaata atattgacgc gggacaagct tactagtagc 7320 ttgttaacgc
ggccgcatcg atcgtgaagt ttctcatcta agcccccatt tggacgtgaa 7380
tgtagacacg tcgaaataaa gatttccgaa ttagaataat ttgtttattg ctttcgccta
7440 taaatacgac ggatcgtaat ttgtcgtttt atcaaaatgt actttcattt
tataataacg 7500 ctgcggacat ctacattttt gaattgaaaa aaaattggta
attactcttt ctttttctcc 7560 atattgacca tcatactcat tgctgatcca
tgtagatttc ccggacatga agccatttac 7620 aattgaatat atcctgccgc
cgctgccgct ttgcacccgg tggagcttgc atgttggttt 7680 ctacgcagaa
ctgagccggt taggcagata atttccattg agaactgagc catgtgcacc 7740
ttccccccaa cacggtgagc gacggggcaa cggagtgatc cacatgggac tttt
7794
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