U.S. patent application number 14/101865 was filed with the patent office on 2016-08-04 for regulatory sequences for expressing gene products in plant reproductive tissue.
This patent application is currently assigned to SYNGENTA PARTICIPATIONS AG. The applicant listed for this patent is Syngenta Participations AG. Invention is credited to Lawrence Mark LAGRIMINI, Moez Rajabali MEGHJI, Michael L. NUCCIO.
Application Number | 20160222402 14/101865 |
Document ID | / |
Family ID | 35197459 |
Filed Date | 2016-08-04 |
United States Patent
Application |
20160222402 |
Kind Code |
A1 |
NUCCIO; Michael L. ; et
al. |
August 4, 2016 |
REGULATORY SEQUENCES FOR EXPRESSING GENE PRODUCTS IN PLANT
REPRODUCTIVE TISSUE
Abstract
Expression cassettes causing specific regulatory control of
transgene expression in plants, wherein the expression cassettes
include regulatory sequences from the MADS gene family for
expression of recombinant gene products in the reproductive tissue
of plants for the purpose of generating abiotic stress tolerant
plants.
Inventors: |
NUCCIO; Michael L.;
(Research Triangle Park, NC) ; LAGRIMINI; Lawrence
Mark; (Lincoln, NE) ; MEGHJI; Moez Rajabali;
(St. Louis, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Syngenta Participations AG |
Basel |
|
CH |
|
|
Assignee: |
SYNGENTA PARTICIPATIONS AG
Basel
CH
|
Family ID: |
35197459 |
Appl. No.: |
14/101865 |
Filed: |
December 10, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12764211 |
Apr 21, 2010 |
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14101865 |
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11109594 |
Apr 19, 2005 |
8129588 |
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12764211 |
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60563687 |
Apr 20, 2004 |
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60563678 |
Apr 20, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/827 20130101;
C12N 15/8271 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Claims
1. A method of increasing abiotic stress tolerance in a plant, the
method comprising: a) inserting an expression cassette into a plant
cell, wherein the expression cassette comprises a
trehalose-6-phosphate phosphatase polynucleotide operably linked to
a 5'-regulatory polynucleotide of a MADS6 gene from Oryza sativa
wherein the 5'-regulatory polynucleotide confers expression of the
trehalose-6-phosphate phosphatase polynucleotide in maternal plant
reproductive tissue; b) regenerating transgenic plants from the
plant cell of a); and c) evaluating the plants of b) for increased
abiotic stress tolerance.
2. The method of claim 1, wherein the trehalose-6-phosphate
phosphatase polynucleotide is obtained from Oryza sativa or
maize.
3. The method of claim 2, wherein the trehalose-6-phosphate
phosphatase polynucleotide is obtained from Oryza sativa.
4. The method of claim 1, wherein the trehalose-6-phosphate
phosphatase polynucleotide encodes a polypeptide having 90%, 95%,
98%, 99% or 100% sequence identity to SEQ ID NO: 532.
5. The method of claim 1, wherein the trehalose-6-phospate
phosphatase polynucleotide comprises a polynucleotide having 90%,
95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 531.
6. The method of claim 5, wherein the trehalose-6-phospate
phosphatase polynucleotide comprises SEQ ID NO: 531.
7. The method of claim 1, wherein the 5'-regulatory polynucleotide
comprises a polynucleotide having 90%, 95%, 98%, 99% or 100%
sequence identity to base pairs 13 to 1478 of SEQ ID NO: 12.
8. The method of claim 7, wherein the 5'-regulatory polynucleotide
comprises base pairs 13 to 1478 of SEQ ID NO: 12.
9. The method of claim 1, wherein the 5'-regulatory polynucleotide
comprises SEQ ID NO: 12.
10. The method of claim 1, wherein the expression cassette
comprises SEQ ID NO: 531 and SEQ ID NO: 12.
11. The method of claim 1, wherein the plant is a monocot.
12. The method of claim 1, wherein the plant is a dicot.
13. The method of claim 1, wherein the plant is selected from the
group consisting of sugarcane, sugarbeet, sorghum, wheat, rice,
oat, barley and maize.
14. The method of claim 1, wherein increased abiotic stress has a
positive effect on grain yield relative to control plants not
comprising said expression cassette.
15. An expression cassette comprising a trehalose-6-phosphate
phosphatase polynucleotide operably linked to a 5'-regulatory
polynucleotide of a MADS6 gene from Oryza sativa resulting in the
expression of the trehalose-6-phosphate phosphatase polynucleotide
in maternal plant reproductive tissue.
16. The expression cassette of claim 15, wherein the
trehalose-6-phosphate phosphatase polynucleotide is obtained from
Oryza sativa or maize.
17. The expression cassette of claim 16, wherein the
trehalose-6-phosphate phosphatase polynucleotide is obtained from
Oryza sativa.
18. The expression cassette of claim 15, wherein the
trehalose-6-phosphate phosphatase polynucleotide encodes a
polypeptide having 90%, 95%, 98%, 99% or 100% sequence identity to
SEQ ID NO: 532.
19. The expression cassette of claim 15, wherein the
trehalose-6-phospate phosphatase polynucleotide comprises a
polynucleotide having 90%, 95%, 98%, 99% or 100% sequence identity
to SEQ ID NO: 531.
20. The expression cassette of claim 19, wherein the
trehalose-6-phospate phosphatase polynucleotide comprises SEQ ID
NO: 531.
21. The expression cassette of claim 15, wherein the 5'-regulatory
polynucleotide comprises a polynucleotide having 90%, 95%, 98%, 99%
or 100% sequence identity to base pairs 13 to 1478 of SEQ ID NO:
12.
22. The expression cassette of claim 21, wherein the 5'-regulatory
polynucleotide comprises base pairs 13 to 1478 of SEQ ID NO:
12.
23. The expression cassette of claim 15, wherein the 5'-regulatory
polynucleotide comprises SEQ ID NO: 12.
24. The expression cassette of claim 15, wherein the expression
cassette comprises SEQ ID NO: 531 and SEQ ID NO: 12.
25. A host cell comprising the expression cassette of claim 15.
26. The host cell of claim 25, wherein the host cell is a plant
cell.
27. A transgenic plant comprising the expression cassette of claim
15.
28. A transgenic plant comprising the expression cassette of claim
24.
29. A transgenic plant generated by the method of claim 1.
30. An expression cassette comprising a 5'-regulatory
polynucleotide from an Oryza sativa OsMADS6 gene; wherein the
5'-regulatory sequence confers expression of a polynucleotide in
maternal plant reproductive tissue.
31. The expression cassette of claim 30, wherein the 5'-regulatory
polynucleotide comprises a polynucleotide having 90%, 95%, 98%, 99%
or 100% sequence identity to base pairs 13 to 1478 of SEQ ID NO:
12.
32. The expression cassette of claim 31, wherein the 5'-regulatory
sequence comprises base pairs 13 to 1478 of SEQ ID NO: 12.
33. The expression cassette of claim 30, wherein the 5'-regulatory
polynucleotide comprises SEQ ID NO: 12.
34. A host cell comprising the expression cassette of claim 30.
35. The host cell of claim 34, wherein the host cell is a plant
cell.
36. A transgenic plant comprising the expression cassette of claim
30.
37. The plant of claim 36, wherein the plant is maize.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/764,211, filed Apr. 21, 2010 (pending),
which is a continuation of U.S. patent application Ser. No.
11/109,594, filed Apr. 19, 2005 (now U.S. Pat. No. 8,129,588), and
which claims the benefit of U.S. patent application Ser. No.
60/563,687, filed Apr. 20, 2004 (expired) and U.S. patent
application Ser. No. 60/563,678, filed Apr. 20, 2004 (expired). All
of the foregoing applications are hereby incorporated by reference
in their entirety, including their respective sequence
listings.
STATEMENT REGARDING ELECTRONIC SUBMISSION OF A SEQUENCE LISTING
[0002] A Sequence Listing in ASCII text format, submitted under 37
C.F.R. .sctn.1.821, entitled
"70369-US-REG-C-NAT-4_Sequence_Listing_ST24", 1,103,000 bytes in
size, generated on Dec. 9, 2013, and filed via EFS-Web is provided
in lieu of a paper copy. This Sequence Listing is hereby
incorporated by reference into the specification for its
disclosures.
FIELD OF THE INVENTION
[0003] The present invention includes expression cassettes that
contain regulatory sequences derived from a target gene, for
example, regulatory sequences from the MADS gene family, for tissue
specific expression of recombinant gene products in plants.
BACKGROUND OF THE INVENTION
[0004] In agricultural biotechnology, plants can be modified
according to one's needs. One way to accomplish this is by using
modern genetic engineering techniques. For example, by introducing
a gene of interest into a plant, the plant can be specifically
modified to express a desirable phenotypic trait. For this, plants
are transformed most commonly with a heterologous gene comprising a
promoter region, a coding region and a termination region. When
genetically engineering a heterologous gene for expression in
plants, the selection of a promoter is often a critical factor.
While it may be desirable to express certain genes constitutively,
i.e. throughout the plant at all times and in most tissues and
organs, other genes are more desirably expressed only in response
to particular stimuli or confined to specific cells or tissues.
[0005] Promoters consist of several regions that are necessary for
full function of the promoter. Some of these regions are modular,
in other words they can be used in isolation to confer promoter
activity or they may be assembled with other elements to construct
new promoters. The first of these promoter regions lies immediately
upstream of the coding sequence and forms the "core promoter
region" containing consensus sequences, normally 20-70 base pairs
immediately upstream of the coding sequence. The core promoter
region contains a TATA box and often an initiator element as well
as the initiation site. The precise length of the core promoter
region is not fixed but is usually well recognizable. Such a region
is normally present, with some variation, in most promoters. The
base sequences lying between the various well-characterized
elements appear to be of lesser importance. The core promoter
region is often referred to as a minimal promoter region because it
is functional on its own to promote a basal level of
transcription.
[0006] The presence of the core promoter region defines a sequence
as being a promoter: if the region is absent, the promoter is
non-functional. The core region acts to attract the general
transcription machinery to the promoter for transcription
initiation. However, the core promoter region is insufficient to
provide full promoter activity. A series of regulatory sequences,
often upstream of the core, constitute the remainder of the
promoter. The regulatory sequences determine expression level, the
spatial and temporal pattern of expression and, for a subset of
promoters, expression under inductive conditions (regulation by
external factors such as light, temperature, chemicals and
hormones). Regulatory sequences may be short regions of DNA
sequence 6-100 base pairs that define the binding sites for
trans-acting factors, such as transcription factors. Regulatory
sequences may also be enhancers, longer regions of DNA sequence
that can act from a distance from the core promoter region,
sometimes over several kilobases from the core region. Regulatory
sequence activity may be influenced by trans-acting factors
including general transcription machinery, transcription factors
and chromatin assembly factors.
[0007] Frequently, it is desirable to have tissue-specific
expression of a gene of interest in a plant. Tissue-specific
promoters promote expression exclusively in one set of tissues
without expression throughout the plant; tissue-preferred promoters
promote expression at a higher level in a subset of tissues with
significantly less expression in the other tissues of the plant.
For example, one may desire to express a value-added product only
in corn seed but not in the remainder of the plant. Another example
is the production of male sterility by tissue-specific
ablation.
[0008] Tissue specific promoters may be expressed in specific
tissue at a specific time or times during the plant growth cycle.
However, sufficient expression levels of gene products, especially
those gene products directed to expression in specific tissues, is
difficult to obtain. Iyer M., et al. (2001). It is known that the
5' untranslated leader sequence of mRNA, introns, and the 3'
untranslated region of mRNA effect expression for particular genes.
For example, Sieburth, L. E. and Meyerowitz, E. M. (1997) show that
intragenic sequences appear to be necessary for the expression of
the AGAMOUS (AG) gene, an Arabidopsis MADS box gene, in the
distinct expression patterns of normal early and later flower
development. Larkin J. C., et al. (1993) show that deletion of the
3' noncoding region of the Arabidopsis GLABROUS1 (GL1) gene
negatively affects GL1 function. However, to date, identifying and
specific regulatory regions and incorporating them into a robust
trait delivery platform has not been accomplished. Important
aspects of the present invention are based on the discovery that
DNA sequences from the MADS gene family are exceptionally useful in
the development of robust expression cassettes that express
recombinant genes in the reproductive tissues of plants.
SUMMARY OF THE INVENTION
[0009] The present invention includes a number of different
aspects, including specific regulatory control of transgene
expression in plants by identifying regulatory sequences from the
MADS gene family and incorporating such sequences into expression
cassettes for expression of recombinant gene products in the
reproductive tissue of plants.
[0010] The present invention relates to a method of constructing
expression cassettes by identifying the target gene, using the
relevant cDNA sequence to annotate the gDNA sequence for the
purpose of identifying regulatory sequences of the target gene, and
incorporating one or more of the regulatory sequences into an
expression cassette with a nucleic acid molecule. A plant
transformed with an expression cassette of the invention expresses
the product of the nucleic acid molecule in a manner that mimics
the expression of the target gene.
[0011] The present invention relates to the specific regulatory
control of transgene expression in plants, and includes targeting
transgene expression to developing reproductive tissue in maize,
rice and other monocots. Use of the expression cassettes of the
present invention includes expressing a glucose or sucrose
transporter to increase reproductive sink strength. Sink strength
can also be increased by flower-specific expression of an invertase
gene or one or more of the trehalose metabolism genes. The
invention further encompasses enhancing the capacity for small
molecule uptake via increased expression of specific
transporters.
DEFINITIONS
[0012] The terms "open reading frame" and "ORF" refer to the amino
acid sequence encoded between translation initiation and
termination codons of a coding sequence. The terms "initiation
codon" and "termination codon" refer to a unit of three adjacent
nucleotides (`codon`) in a coding sequence that specifies
initiation and chain termination, respectively, of protein
synthesis (mRNA translation).
[0013] The term "abiotic stress" refers to nonliving environmental
factors such as frost, drought, excessive heat, high winds, etc.,
that can have harmful effects on plants.
[0014] The term "nucleic acid" refers to a polynucleotide of high
molecular weight which can be single-stranded or double-stranded,
composed of monomers (nucleotides) containing a sugar, phosphate
and a base which is either a purine or pyrimidine. A "nucleic acid
fragment" is a fraction of a given nucleic acid molecule. In higher
plants, deoxyribonucleic acid (DNA) is the genetic material while
ribonucleic acid (RNA) is involved in the transfer of information
contained within DNA into proteins. A "genome" is the entire body
of genetic material contained in each cell of an organism. The term
"nucleotide sequence" refers to a polymer of DNA or RNA which can
be single- or double-stranded, optionally containing synthetic,
non-natural or altered nucleotide bases capable of incorporation
into DNA or RNA polymers. Unless otherwise indicated, a particular
nucleic acid sequence of this invention also implicitly encompasses
conservatively modified variants thereof (e.g. degenerate codon
substitutions) and complementary sequences and as well as the
sequence explicitly indicated. Specifically, degenerate codon
substitutions may be achieved by generating sequences in which the
third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer,
et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka, et al., J. Biol.
Chem. 260:2605-2608 (1985); and Rossolini, et al., Mol. Cell.
Probes 8:91-98 (1994)). The term nucleic acid is used
interchangeably with gene, cDNA, and mRNA encoded by a gene.
[0015] "Operably-linked" refers to the association of nucleic acid
sequences on a single nucleic acid fragment so that the function of
one is affected by the other. For example, a promoter is
operably-linked with a coding sequence or functional RNA when it is
capable of affecting the expression of that coding sequence or
functional RNA (i.e., that the coding sequence or functional RNA is
under the transcriptional control of the promoter). Coding
sequences in sense or antisense orientation can be operably-linked
to regulatory sequences.
[0016] "Promoter" refers to a nucleotide sequence, usually upstream
(5') to its coding sequence, which controls the expression of the
coding sequence by providing the recognition for RNA polymerase and
other factors required for proper transcription. "Promoter
regulatory sequences" consist of proximal and more distal upstream
elements. Promoter regulatory sequences influence the
transcription, RNA processing or stability, or translation of the
associated coding sequence. Regulatory sequences include enhancers,
promoters, untranslated leader sequences, introns, and
polyadenylation signal sequences. They include natural and
synthetic sequences as well as sequences that may be a combination
of synthetic and natural sequences. An "enhancer" is a DNA sequence
that can stimulate promoter activity and may be an innate element
of the promoter or a heterologous element inserted to enhance the
level or tissue specificity of a promoter. It is capable of
operating in both orientations (normal or flipped), and is capable
of functioning even when moved either upstream or downstream from
the promoter. The meaning of the term "promoter" includes "promoter
regulatory sequences."
[0017] "Primary transformant" and "T0 generation" refer to
transgenic plants that are of the same genetic generation as the
tissue that was initially transformed (i.e., not having gone
through meiosis and fertilization since transformation). "Secondary
transformants" and the "T1, T2, T3, etc. generations" refer to
transgenic plants derived from primary transformants through one or
more meiotic and fertilization cycles. They may be derived by
self-fertilization of primary or secondary transformants or crosses
of primary or secondary transformants with other transformed or
untransformed plants.
[0018] "Gene" refers to a nucleic acid fragment that expresses
mRNA, functional RNA, or specific protein, including regulatory
sequences. The term "Native gene" refers to a gene as found in
nature. The term "chimeric gene" refers to any gene that contains
1) DNA sequences, including regulatory and coding sequences, that
are not found together in nature, or 2) sequences encoding parts of
proteins not naturally adjoined, or 3) parts of promoters that are
not naturally adjoined. Accordingly, a chimeric gene may comprise
regulatory sequences and coding sequences that are derived from
different sources, or comprise regulatory sequences and coding
sequences derived from the same source, but arranged in a manner
different from that found in nature. A "transgene" refers to a gene
that has been introduced into the genome by transformation and is
stably maintained. Transgenes may include, for example, genes that
are either heterologous or homologous to the genes of a particular
plant to be transformed. Additionally, transgenes may comprise
native genes inserted into a non-native organism, or chimeric
genes. The term "endogenous gene" refers to a native gene in its
natural location in the genome of an organism. A "foreign" gene
refers to a gene not normally found in the host organism but one
that is introduced into the organism by gene transfer.
[0019] "Expression cassette" as used herein means a DNA sequence
capable of directing expression of a particular nucleotide sequence
in an appropriate host cell, comprising a promoter operably linked
to the nucleotide sequence of interest which is operably linked to
termination signals. It also typically comprises sequences required
for proper translation of the nucleotide sequence. The coding
region usually codes for a protein of interest but may also code
for a functional RNA of interest, for example antisense RNA or a
nontranslated RNA, in the sense or antisense direction. The
expression cassette comprising the nucleotide sequence of interest
may be chimeric, meaning that at least one of its components is
heterologous with respect to at least one of its other
components.
[0020] "Intron" refers to an intervening section of DNA which
occurs almost exclusively within a eukaryotic gene, but which is
not translated to amino acid sequences in the gene product. The
introns are removed from the pre-mature mRNA through a process
called splicing, which leaves the exons untouched, to form an mRNA.
For purposes of the present invention, the definition of the term
"intron" includes modifications to the nucleotide sequence of an
intron derived from a target gene, provided the modified intron
does not significantly reduce the activity of its associated 5'
regulatory sequence.
[0021] "Exon" refers to a section of DNA which carries the coding
sequence for a protein or part of it. Exons are separated by
intervening, non-coding sequences (introns). For purposes of the
present invention, the definition of the term "exon" includes
modifications to the nucleotide sequence of an exon derived from a
target gene, provided the modified exon does not significantly
reduce the activity of its associated 5' regulatory sequence.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIGS. 1A and 1B are schematic representations of the OsMADS5
cDNA and of the annotation of the OsMADS5 gDNA with OsMADS5 cDNA
sequence.
[0023] FIG. 2A is a schematic representation of the OsMADS5
assembly vector.
[0024] FIG. 2B is a schematic representation of the OsMADS5 binary
vector.
[0025] FIGS. 3A and 3B are schematic representations of the OsMADS
6 cDNA and of the annotation of the OsMADS6 gDNA with OsMADS6 cDNA
sequence.
[0026] FIG. 4A is a schematic representation of the OsMADS6
assembly vector.
[0027] FIG. 4B is a schematic representation of the OsMADS6 binary
vector.
[0028] FIG. 5 is a schematic representation of the annotation of
the OsMADS8 gDNA with OsMADS8 cDNA sequence
[0029] FIG. 6A is a schematic representation of the OsMADS8
assembly vector.
[0030] FIG. 6B is a schematic representation of the OsMADS8 binary
vector.
[0031] FIG. 7 is a schematic representation of the annotation of
the OsMADS 13 gDNA with OsMADS 13 cDNA sequence.
[0032] FIG. 8 is a schematic representation of the OsMADS13
Assembly Vector.
[0033] FIG. 9 is a schematic representation of the OsMADS13 Binary
Vector.
[0034] FIG. 10 shows an alignment of T6PP protein sequences from
Zea mays (SEQ ID NO:540), Oryza sativa (SEQ ID NO:541), Zea mays
(SEQ ID NO:542), Oryza sativa (SEQ ID NO:531), Arabidopsis thaliana
(SEQ ID NO:543), Zea mays (SEQ ID NO:544), Oryza sativa (SEQ ID
NO:545), and Arabidopsis thaliana (SEQ ID NO:546).
[0035] FIG. 11A shows the OsMADS6-OsT6PP-Assembly vector.
[0036] FIG. 11B shows the OsMADS6-OsT6PP-Binary vector.
[0037] FIG. 12 shows the effect of the OsMADS6-T6PP-3 transgene on
yield in a drought stress study.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention includes a method for constructing
expression cassettes based on identifying a target gene and
incorporating into the expression cassettes modified regulatory
elements of the selected target gene. For example, regulatory
elements from genes that are expressed in roots, stalks, leaves, or
reproductive tissues that provide insect resistance, herbicide
tolerance, or abiotic stress tolerance are incorporated into
expression cassettes for the purpose of producing a transgenic
event in a plant that closely mimics the expression profile of the
original target gene. Thus, the target gene may be identified from
gene expression data.
[0039] The present invention is also directed to expression
cassettes that incorporate the regulatory mechanisms of target
genes of interest to express in plants the products of nucleic acid
molecules of interest in a manner that mimics the expression
profile of the original target genes.
[0040] The present invention further includes expression cassettes
that incorporate 5'-MADS gene regulatory sequences to express the
products of nucleic acid molecules in plant reproductive tissues,
and further includes expression cassettes incorporating both MADS
5'- and 3'-regulatory sequences.
[0041] The present invention also includes expression cassettes
that incorporate 5'-MADS gene regulatory sequences, and further
incorporate a 5'-MADS gene exon.
[0042] The present invention also includes expression cassettes
that incorporate 5'-MADS gene regulatory sequences, and further
incorporates a 5'-MADS gene exon, and a 5'-MADS gene intron.
[0043] The present invention further includes expression cassettes
that incorporate 5'-MADS gene regulatory sequences, and further
incorporates a 5'-MADS gene exon, a 5'-MADS gene intron, and a
second exon.
[0044] The present invention also includes and further includes
expression cassettes incorporating both MADS 5'- and 3'-regulatory
sequences, wherein said 3'-regulatory sequence include the
3'-non-translated sequence, and the 3'-nontranscribed sequence.
[0045] For purposes of this invention, the definition of the term
"3'-non-translated sequence" includes includes modifications to the
nucleotide sequence of a 3'-non-translated sequence derived from a
target gene, provided the modified 3'-non-translated sequence does
not significantly reduce the activity of its associated 3'
regulatory sequence.
[0046] For purposes of this invention, the definition of the term
"3'-nontranscribed sequence" includes modifications to the
nucleotide sequence of a 3'-nontranscribed sequence derived from a
target gene, provided the modified 3'-nontranscribed sequence does
not significantly reduce the activity of its associated 3'
regulatory sequence. The 3'-nontranscribed sequence extends
approximately 0.5 to 1.5 kb downstream of the transcription
termination site.
[0047] The present invention also includes expression cassettes
incorporating both MADS 5'- and 3'-regulatory sequences, wherein
said 3'-regulatory sequence includes the 3'-non-translated
sequence, and the 3'-nontranscribed sequence, and may further
include an intron of said MADS gene.
[0048] In general MADS genes contribute to the development of plant
reproductive structures (De Bodt et al., 2003). For example, the
DoMADS3 gene is expressed specifically in pedicel tissue (Yu and
Goh, 2000). The genes of the OsMADS gene family were selected for
expression cassette development because they encode
MADS-transcription factors that are expressed in young rice flowers
(Kang and An, 1997). The proteins encoded by genes of the OsMADS
gene family are similar to the orchid DoMADS3 gene (GenBank
accession AF198176). The present invention recognizes that one
method of stabilizing or increasing yield in monocots such as maize
is to increase sink strength in reproductive tissue. Thus,
transgenic methods for production of plants having increased sink
strength in reproductive tissue would benefit from the use of
promoters that result in specific expression in a plant's
reproductive tissues. The present invention therefore includes the
use of OsMADS gene 5'- and 3'-regulatory sequences in expression
cassettes to target transgene expression to developing reproductive
tissues. The MADS genes from which gene regulatory sequences were
identified and utilized according to the present invention encode
the following list of MADS proteins (TABLE 1). The MADS proteins
are compared by percent identity and similarity to the protein
encoded by the DoMADS3 gene.
TABLE-US-00001 TABLE 1 Whole Protein MADS Domain Only MADS gene
identity similarity gaps identity similarity AB003322 42% 58% 0%
68% 78% AB003324 59% 74% 3% 80% 95% AB003328 48% 64% 0% 77% 91%
AF077760 40% 59% 2% 64% 80% AF095645 40% 61% 5% 64% 90% AF139664
50% 66% 1% 78% 95% AF139665 48% 66% 0% 80% 95% AF141964 40% 60% 11%
66% 86% AF141965 51% 67% 0% 84% 91% AY174093 42% 63% 0% 63% 87%
AF204063 60% 72% 3% 91% 98% AF345911 50% 68% 5% 80% 95% AF424549
39% 59% 2% 63% 87% AJ293816 35% 52% 8% 65% 79% AY115556 39% 61% 1%
60% 85% AY177695 39% 58% 0% 66% 87% AY177696 38% 62% 4% 61% 87%
AY177698 41% 61% 3% 68% 87% AY177699 37% 59% 3% 63% 78% AY177700
41% 61% 0% 66% 87% AY177702 38% 59% 5% 70% 89% AY224482 38% 59% 5%
70% 89% AY250075 42% 67% 5% 64% 88% L37527 37% 60% 5% 63% 85%
L37528 45% 68% 1% 84% 94% U78891 62% 75% 4% 94% 99% U78782 58% 69%
4% 91% 99% (OsMADS6) U78892 60% 73% 6% 94% 99% (OsMADS8) U78890 57%
72% 2% 92% 97% (OsMADS5) AF151693 46% 67% 1% 84% 94% (OsMADS13)
AF095646 55% 67% 6% 94% 99%
[0049] The present invention therefore includes an expression
cassette for expression of a nucleic acid molecule product
primarily in the reproductive tissue of a plant comprising a
promoter, a first exon; a first intron, and a second exon of a MADS
gene, wherein said promoter, first exon, intron, and second exon
are the 5'-regulatory sequence of said expression cassette; wherein
said 5'-regulatory sequence is engineered to include a
translational initiation codon at approximately the 3'-end of said
5'-regulatory sequence, and not to contain restriction endonuclease
sites that hinder manipulation by recombinant DNA methods or
additional translation initiation codons upstream of said
translation initiation codon; a 3'-regulatory sequence of a MADS
gene that does not to contain restriction endonuclease sites that
hinder manipulation by recombinant DNA methods; and a nucleic acid
molecule operably linked to said 5'-regulatory sequence and said
3'-regulatory sequence.
[0050] Recombinant DNA methods require the presence of specific
restriction endonuclease sites at the termini of the DNA molecules
to be joined. The most efficient practice requires the sites in one
molecule complement the sites in the other molecule. For example, a
plasmid with SacI and NotI restriction endonuclease sites is
required to clone a gene of interest with SacI and Not I
restriction endonuclease sites at its termini Ideally, these sites
are unique, that is they should not occur at any other place in
either molecule. If these sites occur internally, they hinder
manipulation by recombinant DNA methods and should be eliminated.
Site-directed mutagenesis is one method of eliminating such sites.
Techniques such as partial digestion followed by gel-purification
of the appropriately sized fragment will also accomplish this
without eliminating the internal restriction endonuclease sites,
but are far less efficient and therefore less desirable.
[0051] The present invention recognizes that chemical synthesis,
that is use of synthetic chemical technology as opposed
enzyme-mediated technology, of a polynucleotide molecule can
replace or substitute for recombinant DNA methods in the
construction of a polynucleotide molecule comprising a specific
nucleotide sequence.
[0052] The present invention further includes a method for
constructing an expression cassette comprising the steps of
selecting a target gene based on its expression data or its encoded
protein's similarity to a protein encoded by another gene of
interest; identifying the open reading frame on said target gene
cDNA; identifying the positions of the translational start codon,
translational stop codon, the first intron, first exon, second
exon, the 3'-untranslated sequence and the 3'-nontranscribed
sequence of said target gene gDNA by using the cDNA to annotate the
target gene gDNA; incorporating into an expression cassette a
5'-regulatory sequence comprising said promoter, first exon, first
intron, and second exon and a 3'-regulatory sequence comprising the
3'-untranslated sequence and the 3'-nontranscribed sequence; and
operably linking a nucleic acid molecule to said 5'-regulatory
sequence and said 3'-regulatory sequence of said expression
cassette, wherein said nucleic acid molecule is expressed in a
manner that mimics the expression profile of said target gene of
interest.
EXAMPLE 1
Method of Constructing Expression Cassettes Comprising Regulatory
Sequences from the MADS Gene Family
[0053] 1. Identifying target MADS genes. [0054] 2. Identifying high
quality sequence for both the target's genomic DNA (gDNA) and cDNA.
[0055] 3. Identifying the target gene's open reading frame on the
cDNA. In general this is the longest open reading frame. [0056] 4.
Using a candidate gene's cDNA sequence to annotate gDNA sequence
and marking positions of the translation start codon, translation
stop codon, introns, exons, the 5'-untranslated leader and the
3'-untranslated sequence. As is known in the art, marking the
translation start codon and the translation stop codon identifies
the 5'-regulatory sequence and the 3'-regulatory sequence of the
gene. According to the present invention, the promoter, which
includes the promoter regulatory sequence, is the sequence that
extends approximately 1.5 to 2.5 kb upstream from the translation
start codon, wherein the 3'-regulatory sequence of the present
invention includes the 3'-untranslated sequence located immediately
downstream of the translation stop codon and all or a part of the
3'-nontranscribed sequence, which extends 0.5 to 1.5 kb downstream
of the transcription termination site. In one embodiment of the
invention, the 5'-regulatory sequence includes the promoter, the
first exon, the first intron and the second exon.
[0057] By way of example only, FIGS. 1A and 1B illustrates
annotating the OsMADS5 gDNA with the OsMADS5 cDNA sequence. The
gDNA contigs (AB026295 from GenBank and CL000624.108 (SEQ ID NO.
500) plus CL019873.131 (SEQ ID NO. 521) were aligned with OsMADS5
cDNA sequence (GenBank accession U78890). The cDNA sequence is
broken into corresponding exons. The exons are labeled according to
cDNA base numbers. Both sequences align precisely and the
intervening sequences (introns) are flanked by GT..AG borders. Gaps
in between exons represent introns. The AB026295 fragment is a
portion of the entire bacterial artificial chromosome (BAC)
sequence. The AB026295 (promoter) is an additional fragment from
that BAC which defines sequence used for promoter development.
[0058] 5. Designing expression cassettes that incorporate the
following components from a MADS gene(s): [0059] a. The promoter, a
sequence that begins at the translation start codon and extends
approximately 1.5 to 2.5 kb upstream of the translation start
codon. [0060] b. The first exon [0061] c. The first intron [0062]
d. The 5'-most portion of the second exon [0063] e. The terminus,
including the 3'-untranslated sequence and the 3'-nontranscribed
sequence, which extends 0.5 to 1.5 kb downstream of the
transcription termination site. The terminus can further include an
intron.
[0064] For simplicity the "5'-regulatory sequence" of the present
invention includes components a-d and the "3'-regulatory sequence"
of the present invention refers to component e. [0065] 6.
Amplifying the 5'-regulatory sequence from the appropriate gDNA
template by high-fidelity PCR and cloning into a suitable bacterial
vector.
[0066] The 5'-regulatory sequence from rice genomic DNA (gDNA) is
amplified using high-fidelity PCR. A 50 .mu.L reaction mixture
contains 100 ng rice gDNA, 200 .mu.M dNTPs (dATP, dCTP, dGTP, TTP),
1 .mu.L 20 .mu.M each of oligonucleotide primers designed to
amplify the 5'-regulatory gDNA (Table 2), 1 .mu.L 10.times. Expand
High Fidelity buffer and 1 .mu.L Expand High Fidelity polymerase
(Roche Diagnostics, Cat. No. 1 759 078). The thermocycling program
is 95.degree. C. for 2 minutes followed by 40 cycles of (94.degree.
C. for 15 seconds, 68.degree. C. for 7.5 minutes) followed by
68.degree. C. for 10 minutes. The 5'-regulatory gDNA product is
cloned with the TOPO XL PCR cloning kit (Invitrogen, Cat. No.
K4750-20). pCR-XL-TOPO-5'-regulatory-gDNA is identified by
digesting 5 .mu.L pCR-XL-TOPO-5'-regulatory-gDNA miniprep DNA
(prepared using the QIAprep Spin Miniprep procedure from Qiagen,
Cat. No. 27106) with EcoRI (New England Biolabs) in a 20 .mu.L
reaction containing 2 .mu.g BSA and 2 .mu.L 10.times. EcoRI
restriction endonuclease buffer (New England Biolabs). The reaction
is incubated at 37.degree. C. for 2 hours and the
pCR-XL-TOPO-5'-regulatory-gDNA (EcoRI) products are resolved on 1%
TAE agarose. The pCR-XL-TOPO-5'-regulatory-gDNA clone is sequenced
using the ABI PRISM dye terminator cycle sequencing kit (Perkin
Elmer). [0067] 7. Amplifying the "3'-regulatory sequence" from the
appropriate gDNA template by high-fidelity PCR and clone into a
suitable bacterial vector.
[0068] The 3'-regulatory sequence from rice gDNA is amplified using
high-fidelity PCR. The 50 .mu.L reaction mixture consists of 100 ng
rice gDNA, 200 .mu.M dNTPs, 1 .mu.L 20 .mu.M each of the
oligonucleotide primers designed to amplify the 3'-regulatory gDNA
(Table 2), 1 .mu.L 10.times. Expand High Fidelity buffer and 1
.mu.L Expand High Fidelity polymerase. The thermocycling program is
95.degree. C. for 2 minutes followed by 40 cycles of (94.degree. C.
for 15 seconds, 68.degree. C. for 7.5 minutes) followed by
68.degree. C. for 10 minutes. The 3'-regulatory gDNA product is
cloned with the TOPO XL PCR cloning kit (Invitrogen, Cat. No.
K4750-20) following manufactures' instructions. The
pCR-XL-TOPO-3'-regulatory-gDNA is identified by digesting 5 .mu.L
pCR-XL-TOPO-3'-regulatory-gDNA miniprep DNA with EcoRI in a 20
.mu.L reaction containing 2 .mu.g BSA and 2 .mu.L 10.times. EcoRI
restriction endonuclease buffer. The reaction is incubated at
37.degree. C. for 2 hours and the pCR-XL-TOPO-3'-regulatory-gDNA
(EcoRI) products are resolved on 1% TAE agarose. The
pCR-XL-TOPO-3'-regulatory-gDNA clone is then sequenced. [0069] 8.
Assembling the 5'-regulatory sequence and 3'-regulatory sequence in
any bacterial plasmid.
[0070] The expression cassettes of the present invention were
assembled in the vector pNOV6901, also known as the "Assembly
Vector". This vector contains the coding sequence for GUS reporter
gene (which is disrupted by an intron to prevent bacterial
expression) flanked at its 5'- and 3'-termini by unique restriction
sites (polylinkers) to facilitate recombinant DNA procedures. Any
number of other vectors may be used as is known to those persons
skilled in the art. [0071] 9. Incorporating restriction sites in
the expression cassettes, as necessary, to facilitate recombinant
DNA procedures.
[0072] The "engineered" translation initiation codon, below, is the
ATG in the NcoI restriction site (CCATGG). If there are any NcoI
restriction sites in the "expression cassette 5'-regulatory
sequence" they must be eliminated by mutagenesis. Likewise,
restriction sites that are used to assemble the expression cassette
must be eliminated by mutagenesis. Incorporation of the first
intron in the "expression cassette 5'-regulatory sequence" requires
the sequence be modified to avoid creating fusions between native
coding sequence, which is normally translated into protein encoded
by target gene, and the "gene of interest" (nucleic acid molecule)
to be driven by the expression cassette. This is accomplished by
any of a number of mutagenic procedures, including the procedure
performed by the Stratagene QuikChange Multi Site-Directed
Mutagenesis Kit (Cat. No. 200513). Modifications to the expression
cassette 5'-regulatory sequence include: [0073] a. Modifying the
target gene's natural translation initiation codon so that the
target gene's protein coding sequence is silent. [0074] b.
Modifying any other translation initiation codons that exist in the
sequence between the "silenced" translation initiation codon and
the "engineered" translation initiation codon to insure such codons
are not operable. [0075] c. Modifying any NcoI sites in the
5'-regulatory sequence. [0076] d. Modifying restriction
endonuclease sites, as necessary, to facilitate expression cassette
assembly.
[0077] In this embodiment of the invention, the procedure does not
eliminate nucleotides. Rather, it modifies them to preserve the
length of the 5'-regulatory sequence in the expression cassette,
yet still silencing the candidate gene's protein coding sequence.
However, it is contemplated that one or more of the nucleotides
could be eliminated to silence undesired protein expression,
provided that 5'- and 3'-regulatory sequences of the cassette
continue to enhance expression of the candidate gene in plant
reproductive tissue. Furthermore, those skilled in the art do not
consider it unreasonable to alter the sequence of nucleotides in a
polynucleotide molecule comprising a regulatory sequence so long as
the modified regulatory sequence retains a majority of the activity
associated with the original regulatory sequence.
[0078] Table 2 lists the primers designed to accomplish this task
for each 5'-regulatory sequence derived from the MADs gene family.
The Stratagene QuikChange Multi Site-Directed Mutagenesis Kit uses
each gene's pCR-XL-TOPO-5'-regulatory-gDNA clone as a template and
the primers listed to mutagenize that clone according to the
present invention. The primers must contain a 5'-phosphate to work.
Furthermore, alterations may require more than one round of
mutagenesis. The modified pCR-XL-TOPO-5'-regulatory clone is
sequenced using the ABI PRISM dye terminator cycle sequencing kit
(Perkin Elmer). [0079] 10. Modifying, in some cases, the
3'-regulatory sequence to eliminate restriction endonuclease sites
to facilitate recombinant DNA procedures. The "engineered"
translation initiation codon is the ATG in the NcoI restriction
site (CCATGG). If there are any NcoI restriction sites in the
"expression cassette 3'-regulatory sequence" they must be
eliminated by mutagenesis. Again, this is accomplished any of a
number of mutagenic procedures. The present invention therefore
includes: [0080] a. Modifying any NcoI sites in the 3'-regulatory
sequence. [0081] b. Modifying restriction endonuclease sites, as
necessary, to facilitate expression cassette assembly.
[0082] In this embodiment of the present invention, the procedure
does not eliminate nucleotides. Rather, it modifies them to
preserve the length of the 3'-regulatory sequence in the expression
cassette. However, it is contemplated that one or more of the
nucleotides could be eliminated to silence undesired protein
expression, provided that 5'- and 3'-regulatory sequences of the
cassette continue to enhance expression of the candidate gene in
plant reproductive tissue. Furthermore, those skilled in the art do
not consider it unreasonable to alter the sequence of nucleotides
in a polynucleotide molecule comprising a regulatory sequence so
long as the modified regulatory sequence retains a majority of the
activity associated with the original regulatory sequence. Table 2
lists the primers designed to accomplish this task for each
3'-regulatory sequence derived from the MADS gene family. Each
gene's pCR-XL-TOPO-3'-regulatory-gDNA clone is used as a template
and the primers listed are used to mutagenize these clones. The
modified pCR-XL-TOPO-3'-regulatory clones are sequenced. [0083] 11.
Cloning the 3'-regulatory sequence into pNOV6901, using PCR with
the modified pCR-XL-TOPO-3'-regulatory clone as template and the
appropriate primer set disclosed in Table 2.
[0084] These primers are a 5'-oligonucleotide primer that
introduces unique restriction site from the GUS 3'-terminal
polylinker in pNOV6901 and 3'-oligonucleotide primer that
introduces a rare cutting restriction site (either AscI, PacI, SgfI
or RsrII) followed by a restriction endonuclease site unique to the
3'-terminal polylinker. In Table 2, these primers are listed as
"primers to clone 3'-regulatory sequence in pNOV6901".
[0085] High-fidelity PCR is used to amplify the 3'-regulatory
sequence from the modified pCR-XL-TOPO-3'-regulatory clone. A 50
.mu.L reaction mixture consists of 1 .mu.L miniprep DNA, 200 .mu.M
dNTPs, 1 .mu.L each of 20 .mu.M oligonucleotide primers (Table 2),
5 .mu.L 10.times. Cloned PFU buffer and 2.5 Units of Pfuturbo DNA
polymerase (Stratagene, Cat. No. 600252). The thermocycling program
was 95.degree. C. for 30 seconds then 40 cycles of (95.degree. C.
for 10 seconds, 50.degree. C. for 60 seconds, 72.degree. C. for 6
minutes) then 72.degree. C. for 10 minutes. The amplified
3'-regulatory sequence DNA fragment is recovered using the QIAquick
PCR purification kit (Qiagen, Cat. No. 28106). The recovered
3'-regulatory sequence DNA fragment is precipitated with 20 .mu.g
glycogen, 0.3 M CH.sub.2COONa (pH 5.2) and 2.5 volumes ethanol at
-20.degree. C. for more than 2 hours. The 3'-regulatory sequence
DNA fragment is recovered by micro centrifugation, washed with 70%
ethanol, dried under vacuum and resuspended in 14 .mu.L ddH.sub.2O.
The 3'-regulatory sequence DNA fragment is digested in a 20 .mu.L
reaction containing 2 .mu.g BSA, 2 .mu.L of the appropriate
10.times. restriction endonuclease buffer and 2 .mu.L of the
appropriate restriction endonuclease(s). The reaction is incubated
at 37.degree. C. for more than 6 hours. The digested 3'-regulatory
sequence DNA products are resolved on 1.0% TAE agarose and the
appropriate 3'-regulatory sequence (digested) band is excised. The
3'-regulatory sequence (digested) DNA is extracted and recovered
using the QIAquick Gel extraction kit (Qiagen, Cat. No. 28704). The
recovered 3'-regulatory sequence (digested) DNA is ethanol
precipitated with glycogen carrier. The 3'-regulatory sequence
(digested) DNA fragment is recovered by micro centrifugation,
washed with 70% ethanol, dried under vacuum and resuspended in 5
.mu.L ddH.sub.2O.
[0086] 2 .mu.g of pNOV6901 miniprep DNA is digested in a 20 .mu.L
reaction mixture containing 2 .mu.g BSA, 2 .mu.L of the appropriate
10.times. restriction endonuclease buffer (used to generate the
3'-gene regulatory sequence,) and 2 .mu.L of the appropriate
restriction endonuclease (used to generate the 3'-gene regulatory
sequence). The reaction mixture is incubated at 37.degree. C. for
more than 6 hours, then at 70.degree. C. for 20 minutes. Then 1
.mu.L of the appropriate 10.times. restriction endonuclease buffer,
1 .mu.L 1 Unit/.mu.L calf-intestinal alkaline phosphatase (CIP-New
England Biolabs) and 8 .mu.L ddH.sub.2O is added to the reaction
mixture and incubated at 37.degree. C. for 30 minutes. The pNOV6901
(digested/CIP) DNA is resolved on 1.0% TAE agarose and the 4.7 kb
pNOV6901 (digested/CIP) band is excised. The pNOV6901
(digested/CIP) DNA is extracted and recovered using the QIAquick
Gel extraction kit (Qiagen, Cat. No. 28704). The recovered pNOV6901
(digested/CIP) DNA is ethanol precipitated with glycogen carrier.
The pNOV6901 (digested/CIP) DNA is recovered by micro
centrifugation, washed with 70% ethanol, and dried under vacuum and
resuspend in 5 .mu.L ddH.sub.2O. 4.0 .mu.L 3'-regulatory sequence
(digested) is ligated to 4.0 .mu.L pNOV6901 (digested/CIP) in a 10
.mu.L ligation mixture containing 1 .mu.L 10.times. T4 DNA ligase
buffer and 1 .mu.L T4 DNA ligase (400 Units/.mu.L-New England
Biolabs). The ligation mixture is incubated for more than 8 hours
at 16.degree. C. 5.0 .mu.L of ligation mixture is transformed into
50 .mu.L Top10 competent cells (Invitrogen, Cat. No. C4040-03). The
pNOV6901-3'-regulatory-sequence recombinants are verified by
digesting 2 .mu.L pNOV6901-3'-regulatory-sequence miniprep DNA with
1 .mu.L of the appropriate restriction endonuclease in 10 .mu.L
reactions containing 1 .mu.g BSA and 1 .mu.L of the appropriate
10.times. restriction endonuclease buffer. Digests are incubated at
37.degree. C. for 2 hours then resolved on 1% TAE agarose. The
positive pNOV6901-3'-regulatory-sequence recombinants are
sequenced. [0087] 12. Cloning the 5'-regulatory sequence into
pNOV6901-3'-regulatory-sequence, using PCR with the modified
pCR-XL-TOPO-5'-regulatory clone as template and the appropriate
primer set disclosed in Table 2.
[0088] These primers are a 5'-oligonucleotide primer that
introduces the same rare cutting restriction site used for the
3'-regulatory sequence preceded by a unique restriction
endonuclease site in the 5'-terminal polylinker of pNOV6901 and a
3'-oligonucleotide primer that introduces an NcoI site preceded by
a Kozak sequence (CCACCATGG) at the "engineered" translation
initiation codon. Table 2 lists these primers as "primers to clone
5'-regulatory sequence in pNOV6901".
[0089] High-fidelity PCR is used to amplify the 5'-regulatory
sequence from the modified pCR-XL-TOPO-5'-regulatory clone. A 50
.mu.L reaction mixture consists of 1 .mu.L miniprep DNA, 200 .mu.M
dNTPs, 1 .mu.L each of 20 .mu.M oligonucleotide primers (Table 2),
5 .mu.L 10.times. Cloned PFU buffer and 2.5 Units of Pfuturbo DNA
polymerase (Stratagene, Cat. No. 600252). The thermocycling program
was 95.degree. C. for 30 seconds then 40 cycles of (95.degree. C.
for 10 seconds, 50.degree. C. for 60 seconds, 72.degree. C. for 6
minutes) then 72.degree. C. for 10 minutes. The amplified
5'-regulatory sequence DNA fragment is recovered using the QIAquick
PCR purification kit. The recovered 5'-regulatory sequence DNA
fragment is ethanol precipitated with glycogen carrier. The
5'-regulatory sequence DNA fragment is recovered by micro
centrifugation, washed with 70% ethanol, dried under vacuum and
resuspended in 14 .mu.L ddH.sub.2O. The 5'-regulatory sequence DNA
fragment is digested in a 20 .mu.L reaction containing 2 .mu.g BSA,
2 .mu.L of the appropriate 10.times. restriction endonuclease
buffer and 2 .mu.L of the appropriate restriction endonuclease(s).
The reaction is incubated at 37.degree. C. for more than 6 hours.
The digested 5'-regulatory sequence DNA products are resolved on
1.0% TAE agarose and the appropriate 5'-regulatory sequence
(digested) band is excised. The 5'-regulatory sequence (digested)
DNA is extracted and recovered using the QIAquick Gel extraction
kit (Qiagen, Cat. No. 28704). The recovered 5'-regulatory sequence
(digested) DNA is ethanol precipitated with glycogen carrier. The
5'-regulatory sequence (digested) DNA fragment is recovered by
micro centrifugation, washed with 70% ethanol, dried under vacuum
and resuspended in 5 .mu.L ddH.sub.2O.
[0090] 2 .mu.g of the pNOV6901-3'-regulatory-sequence miniprep DNA
is digested in a 20 .mu.L reaction containing 2 .mu.g BSA, 2 .mu.L
of the appropriate 10.times. restriction endonuclease buffer and 2
.mu.L of the appropriate restriction endonuclease. The reaction is
incubated at 37.degree. C. for more than 6 hours, then at
70.degree. C. for 20 minutes. Then 1 .mu.L of the appropriate
10.times. restriction endonuclease buffer, 1 .mu.L 1 Unit/.mu.L CIP
and 8 .mu.L ddH.sub.2O are added to the reaction and it is further
incubated at 37.degree. C. for 30 minutes. The
pNOV6901-3'-regulatory-sequence (digested/CIP) DNA is resolved on
1.0% TAE agarose and the pNOV6901-3'-regulatory-sequence
(digested/CIP) band is excised. The pNOV6901-3'-regulatory-sequence
(digested/CIP) DNA is extracted and recovered. The recovered
pNOV6901-3'-regulatory-sequence (digested/CIP) DNA is ethanol
precipitated with glycogen carrier. The
pNOV6901-3'-regulatory-sequence (digested/CIP) DNA is recovered by
micro centrifugation, washed with 70% ethanol, dried under vacuum
and resuspended in 5 .mu.L ddH.sub.2O.
[0091] 4.0 .mu.L of the 5'-regulatory sequence (digested) is
ligated to 4.0 .mu.L pNOV6901-3'-regulatory-sequence (digested/CIP)
in a 10 .mu.L ligation mixture containing 1 .mu.L 10.times. T4 DNA
ligase buffer and 1 .mu.L T4 DNA ligase (400 Units/.mu.L). The
ligation mixture is incubated for more than 8 hours at 16.degree.
C. 5.0 .mu.L of ligation mixture is transformed into 50 .mu.L Top10
competent cells. The pNOV6901-3'/5'-regulatory-sequence
recombinants are verified by digesting 2 .mu.L
pNOV6901-3'/5'-regulatory-sequence miniprep DNA with 1 .mu.L of the
appropriate restriction endonuclease in 10 .mu.L reaction mixtures
containing 1 .mu.g BSA and 1 .mu.L of the appropriate 10.times.
restriction endonuclease buffer. Digests are incubated at
37.degree. C. for 2 hours then pNOV6901-3'/5'-regulatory-sequence
(digested) DNA is resolved on 1% TAE agarose. The positive
pNOV6901-3'/5'-regulatory-sequence recombinants are sequenced.
[0092] The expression cassette of the present invention includes a
GUS reporter construct in the Assembly Vector. It is flanked by the
engineered, rare-cutting restriction site. In this embodiment of
the present invention the GUS reporter gene can be replaced with
any gene of interest using methods known to those individuals
skilled in the art. [0093] 13. The expression cassette can now be
mobilized into the agrobacterium binary vector pNOV6900, by
digesting the assembly vector with the rare-cutting enzyme and
purifying the cassette DNA.
[0094] 2 .mu.g pNOV6900 is digested in a 20 .mu.L reaction mixture
containing 2 .mu.g BSA, 2 .mu.L of the appropriate 10.times.
restriction endonuclease buffer and 2 .mu.L of the appropriate
restriction endonuclease. The reaction mixture is incubated at
37.degree. C. for more than 6 hours, then at 70.degree. C. for 20
minutes. Then 1 .mu.L of the appropriate 10.times. restriction
endonuclease buffer, 1 .mu.L 1 Unit/.mu.L CIP and 8 .mu.L
ddH.sub.2O are added to the reaction and it is further incubated at
37.degree. C. for 30 minutes. 2 .mu.g of the
pNOV6901-3'/5'-regulatory-sequence miniprep DNA is digested in a 20
.mu.L reaction containing 2 .mu.g BSA, 2 .mu.L of the same
10.times. restriction endonuclease buffer used for pNOV6900 and 2
.mu.L of the same restriction endonuclease used for pNOV6900. The
reaction is incubated at 37.degree. C. for more than 6 hours.
[0095] The digested plasmid DNA, pNOV6900 (digested/CIP) and
pNOV6901-3'/5'-regulatory-sequence (digested) are resolved on 1.0%
TAE agarose and the 9.2 kb pNOV6900 (digested/CIP) and the
appropriate pNOV6901-3'/5'-regulatory-sequence (digested) bands are
excised. The pNOV6900 (digested/CIP) and the
pNOV6901-3'/5'-regulatory-sequence (digested) DNAs are extracted
and recovered. The recovered pNOV6900 (digested/CIP) and the
pNOV6901-3'/5'-regulatory-sequence (digested) DNAs are ethanol
precipitated with glycogen. The pNOV6900 (digested/CIP) and the
pNOV6901-3'/5'-regulatory-sequence (digested) DNA fragments are
recovered by micro centrifugation, washed with 70% ethanol, dried
under vacuum and resuspended each in Silt ddH.sub.2O.
[0096] 4.0 .mu.L of the pNOV6900 (digested/CIP) is ligated to 4.0
.mu.L pNOV6901-3'/5'-regulatory-sequence (digested) in a 10 .mu.L
ligation mixture containing 1 .mu.L 10.times. T4 DNA ligase buffer
and 1 .mu.L T4 DNA ligase (400 U/.mu.L). The ligation mixture is
incubated for more than 8 hours at 16.degree. C. 5.0 .mu.L of
ligation mixture is transformed into 50 .mu.L Top 10 competent
cells. The pNOV6900-pNOV6901-3'/5'-regulatory-sequence recombinants
are verified by digesting 7.5 .mu.L
pNOV6900-pNOV6901-3'/5'-regulatory-sequence miniprep DNA with 1.0
.mu.L NcoI in 10 .mu.L reaction mixtures containing 1 .mu.g BSA and
1 .mu.L 10.times. restriction endonuclease buffer 4 (New England
Biolabs). Digests are incubated at 37.degree. C. for 2 hours then
resolved on 1% TAE agarose. The junction sequence of positive
pNOV6900-pNOV6901-3'/5'-regulatory-sequence recombinants is
verified. [0097] 14. The expression cassette can now be transformed
into agrobacterium and then transformed into plants in accordance
with methods known to those persons skilled in the art.
TABLE-US-00002 [0097] TABLE 2 Primers for constructing expression
cassettes comprising regulatory sequences from the MADS gene family
primers to clone 5'-regulatory sequence target gene name sequence
AB003322 AB003322-1 5'-CGTTGCCCAGCGAGGAGCATGCGTAAAATC-3' (SEQ. ID
No. 133) AB003322-2 5'-GATTTCATTCATACTGTCCAACAGAAGGCA-3' (SEQ. ID
No. 134) AB003324 AB003324-1 5'-TCTAAATAGGGCCCAACATACTCA-3' (SEQ.
ID No. 145) AB003324-2 5'-TCAAGCGTCTTAAGCATGCTGAAATATGA-3' (SEQ ID
No. 146) AB003328 AB003328-1 5'-AACAAAACCAATAATCTCCAATGCCC-3' (SEQ.
ID No. 158) AB003328-2 5'-CATCTTCGATCGCCGATGAACCAACC-3' (SEQ. ID
No. 158) AB077760 AB077760-1 5'-ATTCGTTGGGGGTGACATGACTAGTC-3' (SEQ.
ID No. 167) AB077760-2 5'-CAAAGATCCCCTTGATGCTGCAGCAG-3' (SEQ. ID
No. 168) AB095645 AB095645-1 5'-CTCGTCGCCGTCGTTGGCTCGGCGT-3' (SEQ.
ID No. 179) AB095645-2 5'-CAAGATTCTTGATAGCCTTATACATG-3' (SEQ. ID
No. 180) AF139664 AF139664-1 5'-TGCATCCTTACAAAGAGACAGATAGATC-3'
(SEQ. ID No. 193) AF139664-2 5'-CAAGGATTTTGTCCATACTGTGAAAAATG-3'
(SEQ. ID No. 194) AF139665 AF139665-1
5'-ACCTGGGGTTTGGAAATGGGGAGCG-3' (SEQ. ID No. 207) AF139665-2
5'-CAAGGATCCCTTCCATACTACAGAAGAAG-3' (SEQ. ID No. 208) AF141964
AF141964-1 5'-AAAACACTGATTAAAGGGTTGTTGAAAGGAAAACACC-3' (SEQ. ID No.
218) AF141964-2 5'-CATAGGTACCTTCAATGCTGAAAAAGAAAACAAGGCA-3' (SEQ.
ID No. 219) AF141965 AF141965-1 5'-TGTTTCTCGATTAGGCTACAAGTTAAC-3'
(SEQ. ID No. 232) AF141965-2 5'-GATGGTTTTCTGTAGGCTGCAGAACAG-3'
(SEQ. ID No. 233) AF174093 AF174093-1
5'-AAAATTCACAAGTATAATTCGTCAC-3' (SEQ. ID No. 246) AF174093-2
5'-CAGCTCCATCAAGCTTAACAAAGCA-3' (SEQ. ID No. 247) AF204063
AF204063-1 5'-ACAAATTTGTTGTACCACCTACCTAGGGGT-3' (SEQ. ID No. 258)
AF204063-2 5'-CAAGGTTTTGTACATGCTGGAAGTGAA-3' (SEQ. ID No. 259)
AF345911 AF345911-1 5'-GAAAATCTCAAGGTTTCGAAAACGAC-3' (SEQ. ID No.
271) AF345911-2 5'-CAAGGATTTTGTCCATCCTGCAGGGAAAAC-3' (SEQ. ID No.
272) AF424549 AF424549-1 5'-TACAAAGTGCTGGAAGTGATAGTATGT-3' (SEQ. ID
No. 283) AF424549-2 5'-GATCCCCTTGATGCTGCAGCAGGATGCA-3' (SEQ. ID No.
284) AJ293816 AJ293816-1 5'-CAAACATTTTAAACTTTAACCATTAATAG-3' (SEQ.
ID No. 295) AJ293816-2 5'-CAATAATCTGTTCCATGCTTCATCAATG-3' (SEQ. ID
No. 296) AY115556 AY115556-1 5'-GATATAAAAACCTACTTTATGTTCATG-3'
(SEQ. ID No. 305) AY115556-2 5'-CAGCTCCTCCATGGTTCTGTTCAAAGAAATC-3'
(SEQ. ID No. 306) AY177695 AY177695-1
5'-TCAGTCCATCATTTCGTCTACAACTAA-3' (SEQ. ID No. 316) AY177695-2
5'-TATTATTGATTTCATGCTAACAAAAAG-3' (SEQ. ID No. 317) AY177696
AY177696-1 5'-CAAGCAAAGAAACAAATTTCGCAATTAG-3' (SEQ. ID No. 330)
AY177696-2 5'-ATAACTGACTTCATGCTGCATATTTGCA-3' (SEQ. ID No. 331)
AY177698 AY177698-1 5'-AAGATTTGAACTACTGCCTTGTCTTC-3' (SEQ. ID No.
342) AY177698-2 5'-CAAAGATATTTGCAATCCTGCCAAAAG-3' (SEQ. ID No. 343)
AY177699 AY177699-1 5'-TCCGGTCGGCCCTCGTCCTCCCCGT-3' (SEQ. ID No.
356) AY177699-2 5'-CAAACTCCCTCACGCTGCGCCAAGAAAG-3' (SEQ. ID No.
357) AY177700 AY177700-1 5'-ATAAGTATTTCAGAAAGCTGAAGTTGA-3' (SEQ. ID
No. 366) AY177700-2 5'-CAAGTTATGCATATTTCTTGCATTTTG-3' (SEQ. ID No.
367) AY177702 AY177702-1 5'-TCTTTTCGCAAACTAAACAAGGCCT-3' (SEQ. ID
No. 379) AY177702-2 5'-CACAGTTTTCATGCTAGCATGCAAGTAAAG-3' (SEQ. ID
No. 380) AY224482 AY224482-1 5'-TCTTTTCGCAAACTAAACAAGGCCTTAACA-3'
(SEQ. ID No. 392) AY224482-2 5'-TATCACAGTTTTCATGCTAGCATGCAAGT-3'
(SEQ. ID No. 393) AY250075 AY250075-1
5'-GAAATGGTTTCATTTTGGGACAAGTTATTG-3' (SEQ. ID No. 405) AY250075-2
5'-CAAGGATTCGCTCCATACTATCAATAAAATATG-3' (SEQ. ID No. 406) L37527
L37527-1 5'-GCCAAGAGAGCCCCCTTGCTGCTGGT-3' (SEQ. ID No. 419)
L37527-2 5'-CAAGATCCTTGACAGCCTAAAACGCCA-3' (SEQ. ID No. 420) L37528
L37528-1 5'-ACCACCTCAATCTCCACTGTTTGATTG-3' (SEQ. ID No. 432)
L37528-2 5'-CAGCTCAGATCGGTCTGGACACAAAC-3' (SEQ. ID No. 433) U78891
U78891-1 5'-TCTTCTTCTTGCCTTCATTTGAGTTAATTACA-3' (SEQ. ID No. 443)
U78891-2 5'-CAAGCGTTTTAGTCATGCTACAAAGTTC-3' (SEQ. ID No. 444)
AF095646-1 5'-TGTTGCATGCATGCGTACTGGTGATGGCCGCA-3' (SEQ ID No. 508)
AF095646-2 5'-AATGTCTTGTTTATTCTGCAAACAAAAATAGG-3' (SEQ ID No. 509)
name sequence primers to clone 3'-regulatory sequence AB003322-3
5'-TAACTGAAAGGAAAAAAAAAGCATCCTTGC-3' (SEQ. ID No. 135) AB003322-4
5'-GAATAAATCTGGCAACTGAAATCCAACCAT-3' (SEQ. ID No. 136) AB003324-3
5'-CTAGCTAGCTAGCTACCGTTTCAGCTTC-3' (SEQ. ID No. 147) AB003324-4
5'-TGGAGATCGACCACAATTAAATTCGT-3' (SEQ. ID No. 148) AB003328-3
5'-TAAGTAACAGGCCAGGAATAAGCTGG-3' (SEQ. ID No. 160) AB003328-4
5'-ATTGCCGGGCTATAGCCTTTCTCCCT-3' (SEQ. ID No. 161) AB077760-3
5'-TGATATATCATCGCCGCCGCCGCCG-3' (SEQ. ID No. 169) AB077760-4
5'-CCGTGGTACTGAAATCGAAAAAAGAAATG-3' (SEQ. ID No. 170) AB095645-3
5'-TAAGCTGCTAGGTTGCCCCGCCACT-3' (SEQ. ID No. 181) AB095645-4
5'-GCACGGCTACCTCTCGCCGGAGTACG-3' (SEQ. ID No. 182) AF139664-3
5'-TAAGGAGGCTTCAGATCCATACCAG-3' (SEQ. ID No. 195) AF139664-4
5'-GGATAAACATTGTGAAGCAACATTTC-3' (SEQ. ID No. 196) AF139665-3
5'-TGAAGGCATCTGTTGATCTCAAACGTC-3' (SEQ. ID No. 209) AF139665-4
5'-ATGAACTCCACCTCGGGAACTCAGCCT-3' (SEQ. ID No. 210) AF141964-3
5'-TGAGCAGGAAGCACAGGTGTCCTGT-3' (SEQ. ID No. 220) AF141964-4
5'-ATGAGATACAATCTAGTACAACGAAT-3' (SEQ. ID No. 221) AF141965-3
5'-TGAAGAAGGCCAGCCACAGCAACAGCTG-3' (SEQ. ID No. 234) AF141965-4
5'-CCTTATCGAATATTCAAATCTCGATG-3' (SEQ. ID No. 235) AF174093-3
5'-TGACTTCCTGGAAGCAGTAGGAAC-3' (SEQ. ID No. 248) AF174093-4
5'-CCCCTTCTCCTCCTCCGGGAAGAAG-3' (SEQ. ID No. 249) AF204063-3
5'-TGATGTGTGTGTTCAGTTCAGGCTT-3' (SEQ. ID No. 260) AF204063-4
5'-CTGCATATACTTGCAACATTGCAATTTTA-3' (SEQ. ID No. 261) AF345911-3
5'-TAAGATGATCATCGTCGTCGTCGTCG-3' (SEQ. ID No. 273) AF345911-4
5'-TTGACTTAAATGGCAAGATAAATTAGATATAG-3' (SEQ. ID No. 274) AF424549-3
5'-TGAGATATCATCGCCGCCGCCGCCGCCG-3' (SEQ. ID No. 285) AF424549-4
5'-TTTTATTATCTTGGCGAGGCCCAAGCACCTTTG-3' (SEQ. ID No. 286)
AJ293816-3 5'-TGATGGCTGGAAACTAAAACTGAGAGGA-3' (SEQ. ID No. 297)
AJ293816-4 5'-TTTGAAACATTTCGGCCCTGGCTCTCCT-3' (SEQ ID No. 298)
AY115556-3 5'-TAACACTAATAATGGCCTGGGGGATAC-3' (SEQ. ID No. 307)
AY115556-4 5'-AACTTTGCACGAATGATTAAATTGCAT-3' (SEQ. ID No. 308)
AY177695-3 5'-TAACACATGATCAAGTGTTAAAAACAG-3' (SEQ. ID No. 318)
AY177695-4 5'-GGCTCGGTCAAACCAGCGTGGTGAGCT-3' (SEQ. ID No. 319)
AY177696-3 5'-TGACCAGAAAAACATTGTTTTGCTAC-3' (SEQ. ID No. 332)
AY177696-4 5'-TTTGGGATTGGACTGCATCCCAGGAA-3' (SEQ. ID No. 333)
AY177698-3 5'-TGATTGCCCTCTTTCCATCCCAATAGG-3' (SEQ. ID No. 344)
AY177698-4 5'-GTTAACCTAAAAGAAATATTGTTCAG-3' (SEQ. ID No. 345)
AY177699-3 5'-TAGAAACAGATGGACGCTTGACGTTCA-3' (SEQ. ID No. 358)
AY177699-4 5'-AGGCTTGGTGCAAGGATACGAAATCTTG-3' (SEQ. ID No. 359)
AY177700-3 5'-TAGCCGTCAAAGGACCTTGGTCAATTC-3' (SEQ. ID No. 368)
AY177700-4 5'-TAATTTTACTAGTACTTTTACTTTGAC-3' (SEQ. ID No. 369)
AY177702-3 5'-TAAGACTATGCCGTACAAGCTGGACGA-3' (SEQ. ID No. 381)
AY177702-4 5'-CCGTGGTTGTCACAGAAGACCAATCTATAC-3' (SEQ. ID No. 382)
AY224482-3 5'-TAAGACTATGCCGTACAAGCTGGACGA-3' (SEQ. ID No. 394)
AY224482-4 5'-CCGTGGTTGTCACAGAAGACCAATCTATAC-3' (SEQ. ID No. 395)
AY250075-3 5'-TAGAAGATGTTCAGATGAAATGGTCCCT-3' (SEQ. ID No. 407)
AY250075-4 5'-AAAAAAATCACCGGCGTCAAATATTTAGGGA-3' (SEQ. ID No. 408)
L37527-3 5'-TAGGCTGCTGAGCACTGCCAATTTG-3' (SEQ. ID No. 421) L37527-4
5'-TGCTGGTGCTCGAGGTGCTGAGCGCG-3' (SEQ. ID No. 422) L37528-3
5'-TAGTTTTGGTGTAGACACCGTACGTAC-3' (SEQ. ID No. 434) L37528-4
5'-TGAAAGCTACATTTTAGCCTTGTATTTG-3' (SEQ. ID No. 435) U78891-3
5'-TGAACAGTGCGTGCATGAACACCTACATG-3' (SEQ. ID No. 445) U78891-4
5'-GATGTTTTTTTTTGTCTTTTGATGCAAGGCC-3' (SEQ. ID No. 446) AF095646-3
5'-TGAAGTCCAAGCTTGCTAATAAAAACGCTG-3' (SEQ ID No. 510) AF095646-4
5'-AAGGGTAGGGCTGCACGAAATGC-3' (SEQ ID No. 511) primers to
mutagenize 5'-regulatory sequence AB003322-5
5'-CGCGCGGCGGCGATCGCGCGGGAGAGGCGG-3' (SEQ. ID No. 137) AB003322-6
5'-CTTCTGTTGGACAGTATCAATGAAATC-3' (SEQ. ID No. 138) AB003322-7
5'-ATTTCATCCATGCTACAATTTATTTT-3' (SEQ. ID No. 139) AB003322-8
5'-AGAAGGCCGAGGAGGTCTCCGTGCTC-3' (SEQ. ID No. 140) AB003324-5
5'-TCGAGCGGGAGATCGAGATCGGGCGAGGCA-3' (SEQ. ID No. 149) AB003324-6
5'-AATTCATATTTCAGCATCCTTAAGAC-3' (SEQ. ID No. 150) AB003324-7
5'-ACTGTGCAGATGAGGTCATGCGCTCAT-3' (SEQ. ID No. 151) AB003324-8
5'-AGGCCTACGAGCTGTCCATCCTCTGCGA-3' (SEQ. ID No. 152) AB003324-9
5'-ACGCGTCCGGCCATCGCTGCCTAGCTAG-3' (SEQ. ID No. 153) AB077760-5
5'-GCTGCAGGGGGCGGCCATCGGGAGGGGCAAGATCGA-3'
(SEQ. ID No. 171) AB077760-6 5'-CGCACGGGGATCATCAAGAAGGCCAG-3' (SEQ.
ID No. 172) AB077760-7 5'-TCGCCATCATCATCTTCTCCTCCAC-3' (SEQ. ID No.
173) AB077760-8 5'-ATGAAGAAGGCCAGGGTGCTCACCGTGCTCT-3' (SEQ. ID No.
174) AB095645-5 5'-AGGAGGCGGGATCGGGCGCGGGAAG-3' (SEQ. ID No. 183)
AB095645-6 5'-GAAACGTGGGCCATCGCCATTGAGCGAAA-3' (SEQ. ID No. 184)
AB095645-7 5'-TTCCTCTTCCTCCCATCGCGAACTCCT-3' (SEQ. ID No. 185)
AB095645-8 5'-TGCCGTTTCTGGAGCTGCCCTACTGA-3' (SEQ. ID No. 186)
AF139664-5 5'-AGAACAGGAGGAAGAAGGGGCGGGGCAA-3' (SEQ. ID No. 197)
AF139664-6 5'-TACGCCACCGACTCAAGGTACGTACGTAC-3' (SEQ. ID No. 198)
AF139664-7 5'-TCATTTTTCACAGTATCGACAAAATCCT-3' (SEQ. ID No. 199)
AF139664-8 5'-TGGGCAGGAAAAGGCATGGCCCACCCCCA-3' (SEQ. ID No. 200)
AF139664-9 5'-TGCTTTGATTAATACAGCTCGCAGCGAC-3' (SEQ. ID No. 201)
AF139665-5 5'-CCGCCGACGACGATCGGGAGAGGGCCGGT-3' (SEQ. ID No. 211)
AF139665-6 5'-TCTTCTGTAGTATCGAAGGGATCCTTG-3' (SEQ. ID No. 212)
AF139665-7 5'-TCGCCGCGCGCGCGCGATGGGCGAGCT-3' (SEQ. ID No. 213)
AF141964-5 5'-GGGTTTTTCGGGGCGGGGAAGGCGCGGAGGGGGA-3' (SEQ. ID No.
222) AF141964-6 5'-TGAAGTTGTCCATGCTTACTAATACTCAAA-3' (SEQ. ID No.
223) AF141964-7 5'-ATTGGTTTTCCCATGCGTGTGATGGATATTC-3' (SEQ. ID No.
224) AF141964-8 5'-GACCAAAACCAGCCCATGCTGCATGGTACACTATTAGGCT-3'
(SEQ. ID No. 225) AF141964-9
5'-CCTAATAAGAAACCGATGGGTATAAAATGGAGA-3' (SEQ. ID No. 226)
AF141965-5 5'-GAGAGATCGGGATCGATCGTGCGGGGGA-3' (SEQ. ID No. 236)
AF141965-6 5'-TCCAAGCGCCGGAAAGGCCTCCTCAAGAA-3' (SEQ. ID No. 237)
AF141965-7 5'-GAAAATGGAGGATGCAGAATATATATCCT-3' (SEQ. ID No. 238)
AF141965-8 5'-AACAACTGTTCCAAGGAGCATGTCCAC-3' (SEQ. ID No. 239)
AF141965-9 5'-CTTCTCGCGCGCCCGCGATTTCCGTT-3' (SEQ. ID No. 240)
AF141965-10 5'-TCTCCCCCCGCGGCGGCCTCTACGA-3' (SEQ. ID No. 241)
AF174093-5 5'-GGCGAGGTCGCGTTGGGGCAAGGGAA-3' (SEQ. ID No. 250)
AF174093-6 5'-GAAGATCGAGATCAAGAGGATCGAGGAC-3' (SEQ. ID No. 251)
AF174093-7 5'-CGTGCTGTGCGAAGCGCAGGTCGGC-3' (SEQ ID No. 252)
AF174093-8 5'-AAGAAGGCGAACCAGCTCGCCGTGC-3' (SEQ ID No. 253)
AF204063-5 5'-AGGAGGAGGAAGAAGATCGGGAGGGGGAA-3' (SEQ. ID No. 262)
AF204063-6 5'-TCTCCAGCTCATCTTGGTACGTATAGCA-3' (SEQ. ID No. 263)
AF204063-7 5'-TTCACTTCCAGCATCTACAAAACCTTG-3' (SEQ. ID No. 264)
AF204063-8 5'-ATCTTCTCCGGCCGCCGCCGCCTCTT-3' (SEQ. ID No. 265)
AF204063-9 5'-GTCCAGCTAGCCAAGGATGGATATATTA-3' (SEQ. ID No. 266)
AF345911-5 5'-AGCGGCGAGGATCGGGCGGGGGAAGGT-3' (SEQ. ID No. 275)
AF345911-6 5'-GTTTTCCCTGCAGGATCGACAAAATCCT-3' (SEQ. ID No. 276)
AF345911-7 5'-CGGCCGCTTCCATCGATTTCAGCTGCT-3' (SEQ. ID No. 277)
AF424549-5 5'-AGGGGGCGGCGATGGGGAGGGGCAAGAT-3' (SEQ. ID No. 287)
AF424549-6 5'-CCGCACGGGGATCATCAAGAAGGCCAG-3' (SEQ. ID No. 287)
AF424549-7 5'-AGGTCGCCATCATCAAGTTCTCCTCCA-3' (SEQ. ID No. 289)
AF424549-8 5'-TTGCATCTATCCATCGTTAAAACAAGTC-3' (SEQ. ID No. 290)
AJ293816-5 5'-TGTGTGATCTGATAAGGCTGGCGGCGGCGGT-3' (SEQ. ID No. 299)
AJ293816-6 5'-TACATTGATGAAGCATCGAACAGATTATTG-3' (SEQ. ID No. 300)
AY115556-5 5'-CTTATCTTGATCGATCGCGCGAGGCAAG-3' (SEQ. ID No. 309)
AY115556-6 5'-TGAACAGAACCATCGAGGAGCTG-3' (SEQ. ID No. 310)
AY115556-7 5'-AGCTCTGATATAGCATGGAGGTTAATTG-3' (SEQ. ID No. 311)
AY177695-5 5'-TCGATCGATTGAAGAAGGGGAGAGGGA-3' (SEQ. ID No. 320)
AY177695-6 5'-GACAACACGAAGAACCGGCAGGTGAC-3' (SEQ. ID No. 321)
AY177695-7 5'-CGCGGCGGGCTGATCAAGAAGGCCCGGGAG-3' (SEQ. ID No. 322)
AY177695-8 5'-CTTTTTGTTAGCATCAAATCAATAATA-3' (SEQ. ID No. 323)
AY177695-9 5'-TGAAGAGCAAACCATCGAGCTAACAAACACA-3' (SEQ. ID No. 324)
AY177695-10 5'-TCAGGGATGACAACACGATGGCACCATCAGTCAT-3' (SEQ. ID No.
325) AY177696-5 5'-TAGCGGCGAAGAAGATCGGGAGGGGGAAGA-3' (SEQ. ID No.
334) AY177696-6 5'-GAGGTCGGCCTCAAGATCTTCTCCAG-3' (SEQ. ID No. 335)
AY177696-7 5'-CAAATATGCAGCATCAAGTCAGTTAT-3' (SEQ. ID No. 336)
AY177696-8 5'-CAGCATGGGCCATCGCCAGCTCTTCTCT-3' (SEQ. ID No. 337)
AY177698-5 5'-ACCAACCTGATCAAAGGGGCGTGGGA-3' (SEQ. ID No. 346)
AY177698-6 5'-TCCAAGAGGAGGATCGGGCTGCTCAAGAAA-3' (SEQ. ID No. 347)
AY177698-7 5'-AGGCACTGGCAAGAAGTACGAGTACT-3' (SEQ. ID No. 348)
AY177699-5 5'-ATCGGCCAGAATCGGGAGGGGGCGCA-3' (SEQ. ID No. 360)
AY177700-5 5'-AAGAGTGAAACAACAAGGTCAGAGGA-3' (SEQ. ID No. 370)
AY177700-6 5'-GAAAGGTGCAAATCCGACGAATAGAG-3' (SEQ. ID No. 371)
AY177700-7 5'-ATTAGCTACTAAAGGGTGTGTTTCCA-3' (SEQ. ID No. 372)
AY177700-8 5'-AAAATGCAAGAAATTTGCATAACTTG-3' (SEQ. ID No. 373)
AY177702-5 5'-GAGAGCGAGGAGATCGGGAGGGGGAAGA-3' (SEQ. ID No. 382)
AY177702-6 5'-TTACTTGCATGCTAGCTTGAAAACTGT-3' (SEQ. ID No. 384)
AY177702-7 5'-ATCCTAAATTTTCATCGATGGCATCTAG-3' (SEQ. ID No. 385)
AY177702-8 5'-GATCAGGACCATGCCAGTCTGATGGCCAA-3' (SEQ. ID No. 386)
AY177702-9 5'-GCACAAGGAAAGCCTTGGAACATCTT-3' (SEQ. ID No. 387)
AY224482-5 5'-GAGAGCGAGGAGATCGGGAGGGGGAAGA-3' (SEQ. ID No. 396)
AY224482-6 5'-CTTGCATGCTAGCTTGAAAACTGTGA-3' (SEQ. ID No. 397)
AY224482-7 5'-CTAAATTTTCATGCATGGCATCTAG-3' (SEQ. ID No. 398)
AY224482-8 5'-TGATCAGGACCATCGCAGTCTGATGG-3' (SEQ. ID No. 399)
AY224482-9 5'-CAAGGAAAGCCAAGGAACATCTTTAC-3' (SEQ. ID No. 400)
AY250075-5 5'-AGAGAGAGAGATGAAGGGGAGGGGGAA-3' (SEQ. ID No. 409)
AY250075-6 5'-TATTGATAGTATCGAGCGAATCCTTG-3' (SEQ. ID No. 410)
AY250075-7 5'-CAGTTGGTTGCCAAGGCGCGTTGCCGA-3' (SEQ. ID No. 411)
AY250075-8 5'-GAGCCTCGGCCATGCCCAGCTCCTTCCT-3' (SEQ. ID No. 412)
AY250075-9 5'-CTATTATTTTAAGAAAGCATGGATTATATAG-3' (SEQ. ID No. 413)
AY250075-10 5'-TGAGACAGACCCATGCCTGGGCCTAC-3' (SEQ. ID No. 414)
L37527-5 5'-GAGGAGTTGGATATCGGGCGCGGCAAGAT-3' (SEQ. ID No. 423)
L37527-6 5'-ACCTCCATTCCTTGCATGGCGGCCT-3' (SEQ. ID No. 424) L37527-7
5'-TACTCCACTCCAACGATGGCGTCCAGT-3' (SEQ. ID No. 425) L37528-5
5'-AGCATACCCATCCATCTTGAACATGATGGT-3' (SEQ. ID No. 436) L37528-6
5'-CATCCATCTTGAACATCTTGGTAAATTCTGGCT-3' (SEQ. ID No. 437) U78891-5
5'-AGATCGATCGGGATCGGGAGGGGTCGGGT-3' (SEQ. ID No. 447) U78891-6
5'-GAACTTTGTAGCATCACTAAAACGCT-3' (SEQ. ID No. 448) U78891-7
5'-GTCCATGAAACAAACCGATGGTAATTGC-3' (SEQ. ID No. 449) AF095646-5
5'-CAAAAGTTGAGGGATTGGATCGATCAGA-3' (SEQ ID No. 512) AF095646-6
5'-GATTGGATCGATCAGAGATCGGGAGGGGAAGGGT-3' (SEQ ID No. 513)
AF095646-7 5'-GTTTTGTTTCCTTCGATGGGATGCGTATTC-3' (SEQ ID No. 514)
AF0956461-8 5'-GAAGGCGTACGAGGTCTCCGTGCTCTG-3' (SEQ ID No. 515)
primers to mutagenize 3'-regulatory sequence AB003328-5
5'-CGTATTGTCGTCCATGCATGCGAAATGCTA-3' (SEQ. ID No. 162) AB095645-9
5'-GTAAAATTGCAGATCGATGGATGTCTCCA-3' (SEQ. ID No. 187) AB095645-10
5'-GAACCTCTCCATGCCGTGCACCCCG-3' (SEQ. ID No. 188) AF139664-10
5'-ACCTGACCCGGTCCCGTCGCCTGCTGCT-3' (SEQ. ID No. 202) AF141964-10
5'-AGAGAAAAGAGTTGCCTTGGCCTCTGGCTCTGC-3' (SEQ. ID No. 227)
AF345911-8 5'-AGAACTATTCCAAGGTAATTGTACCATC-3' (SEQ. ID No. 278)
AY177698-8 5'-AGGAGGCCAACCTGCATGGCTACGTTCTT-3' (SEQ. ID No. 349)
AY177698-9 5'-GCTCGTTTGATCCAAGGACAAGATCA-3' (SEQ. ID No. 340)
AY177698-10 5'-TTCCTAAGGACACGATGGAGTCTGGA-3' (SEQ. ID No. 351)
AY177699-6 5'-AGTCGCCCCCGGCCTTTTAAGAGCCGCA-3' (SEQ. ID No. 361)
AY177700-9 5'-TAGGATTCATAACGATGGACATGTTC-3' (SEQ. ID No. 374)
L37527-8 5'-ACGGCAGCGGCCCGTCCGGCAGCTCCGGGA-3' (SEQ. ID No. 426)
L37527-9 5'-GAACCTCTCCATCGCGCCGCCCCGGGGGCT-3' (SEQ. ID No. 427)
L37528-7 5'-ATGTGAGGACCATCGATTGATGCATTG-3' (SEQ. ID No. 438)
U78891-8 5'-ACCTACATGCCCGCATGGCTACCATGAT-3' (SEQ. ID No. 450)
U78891-9 5'-TCGTCGTTTTCTCGATGGCCTTGCAT-3' (SEQ. ID No. 451)
AF095646-9 5'-CCCTTCCTTACCCATGCTGGTAACAAATATG-3' (SEQ ID No. 516)
primers to clone 3'-regulatory sequence in pNOV6901 AB003322-9
5'-TAATAAGAGCTCTGAAAGGAAAAAAAAAGCA-3' (SEQ. ID No. 141) AB003322-10
5'-ATATATGCGGCCGCGGTCCGAATAAATCTGGCAACTGA-3' (SEQ. ID No. 142)
AB003324-10 5'-ATATATGCGGCCGCGGTCCGTGGAGATCGACCACAATTAAATTC-3'
(SEQ. ID No. 154) AB003324-11
5'-TAGGTAGAGCTCCTAGCTAGCTAGCTACCGTTTCA-3' (SEQ. ID No. 155)
AB003328-6 5'-TAATAAGAGCTCGTAACAGGCCAGGAATAAGCT-3' (SEQ. ID No.
163) AB003328-7 5'-ATATATGCGGCCGCGGTCCGATTGCCGGGCTATAGCCTT-3' (SEQ.
ID No. 164) AB077760-11 5'-ATGCATGAGCTCTATATCATCGCCGC-3' (SEQ. ID
No. 175) AB077760-12 5'-ATATATGCGGCCGCGGTCCGTGGTACTGAAATC-3' (SEQ.
ID No. 176) AB095645-13 5'-TAATAAGAGCTCGCTGCTAGGTTGCCCCGCCACT-3'
(SEQ. ID No. 189) AB095645-14
5'-TAATAAGCGGCCGCGGTCCGCACGGCTACCTCTCGCCGGA-3' (SEQ. ID No. 190)
AF139664-11 5'-TAATAAGAGCTCGGAGGCTTCAGATCCATACCA-3' (SEQ. ID No.
203) AF139664-12 5'-ATATATGCGGCCGCGGTCCGGATAAACATTGTGAAGCAAC-3'
(SEQ. ID No. 204) AF139665-8 5'-TGATGAGCGGCCGCAGGCATCTGTTGATCTCA-3'
(SEQ. ID No. 214)
AF139665-9 5'-TATATAACTAGTGCGGTCCGATGAACTCCACCTCGGGAAC-3' (SEQ ID
No. 215) AF141964-11 5'-TGATGAGCGGCCGCAGGAAGCACAGGTGT-3' (SEQ. ID
No. 228) AF141964-12 5'-ATATATCCCGGGCGGTCCGATGAGATACAATCTAGTAC-3'
(SEQ. ID No. 229) AF141965-11 5'-TGATGAGCGGCCGCAGAAGGCCAGCCACA-3'
(SEQ. ID No. 242) AF141965-12
5'-TATATACCCGGGCGGTCCGCCTTATCGAATATTCA-3' (SEQ. ID No. 243)
AF174093-10 5'-TGATGAGAGCTCCTTCCTGGAAGCAGTAG-3' (SEQ. ID No. 254)
AF174093-11 5'-ATATATGCGGCCGCGGTCCGCCCCTTCTCCTCCTCCGGGA-3' (SEQ. ID
No. 255) AF204063-10 5'-TATATACCCGGGCGGTCCGCTGCATATACTTGCAACA-3'
(SEQ. ID No. 267) AF204063-11
5'-TGATGAGCGGCCGCTGTGTGTGTTCAGTTCAG-3' (SEQ. ID No. 268) AF345911-9
5'-TAATAAGCGGCCGCGATGATCATCGTCGTCGT-3' (SEQ. ID No. 279)
AF345911-10 5'-TATATACCCGGGCGGTCCGTTGACTTAAATGGCAAG-3' (SEQ. ID No.
280) AF424549-9 5'-TGATGAGCGGCCGCGATATCATCGCCGCCG-3' (SEQ. ID No.
291) AF424549-10 5'-TATATACCCGGGCGGTCCGTTTTATTATCTTGGCGA-3' (SEQ.
ID No. 292) AJ293816-7 5'-TGATGAGCGGCCGCTGGCTGGAAACTAAAACT-3' (SEQ.
ID No. 301) AJ293816-8 5'-TATATACCCGGGCGGTCCGTTTGAAACATTTCGGCCCT-3'
(SEQ. ID No. 302) AY115556-8 5'-TAATAAGCGGCCGCACTAATAATGGCCTGG-3'
(SEQ. ID No. 312) AY115556-9
5'-TATATACCCGGGCGGTCCGAACTTTGCACGAATG-3' (SEQ. ID No. 313)
AY177695-11 5'-TAATAAGAGCTCCACATGATCAAGTGTTAAAAAC-3' (SEQ ID No.
325) AY177695-12 5'-TATATAGCGGCCGCGGTCCGGCTCGGTCAAACCAGCGT-3' (SEQ.
ID No. 326) AY177696-9 5'-TGATGAGAGCTCCCAGAAAAACATTGTTTTG-3' (SEQ.
ID No. 338) AY177696-10 5'-TATATAGCGGCCGCGGTCCGTTTGGGATTGGAC-3'
(SEQ. ID No. 339) AY177698-11 5'-TGATGAGCGGCCGCTTGCCCTCTTTCCATC-3'
(SEQ. ID No. 325) AY177698-12
5'-TATATACCCGGGCGGTCCGTTAACCTAAAAGAAAT-3' (SEQ. ID No. 353)
AY177699-7 5'-TAGTAGGCGGCCGCAAACAGATGGACGCTTGA-3' (SEQ. ID No. 362)
AY177699-8 5'-TATATACCCGGGCGGTCCGAGGCTTGGTGCAAG-3' (SEQ. ID No.
363) AY177700-10 5'-TAGTAGGCGGCCGCGTCAAAGGACCT-3' (SEQ. ID No. 375)
AY177700-11 5'-TATATACCCGGGCGGTCCGTAATTTTACTAGTACT-3' (SEQ. ID No.
376) AY177702-10 5'-TAATAAGAGCTCGACTATGCCGTACAAGC-3' (SEQ. ID No.
388) AY177702-11 5'-ATATATGCGGCCGCGGTCCGTGGTTGTCACAG-3' (SEQ. ID
No. 389) AY224482-10 5'-TAATAAGAGCTCGACTATGCCGTACAAGC-3' (SEQ. ID
No. 401) AY224482-11 5'-TATATAGCGGCCGCGGTCCGTGGTTGTCACAGA-3' (SEQ.
ID No. 402) AY250075-11 5'-TAGTAGAGCTCAAGATGTTCAGATGAAATG-3' (SEQ.
ID No. 415) AY250075-12 5'-TATATAGCGGCCGCGGTCCGAAAAAAATCACCGGCGT-3'
(SEQ. ID No. 416) L37527-10 5'-TAGTAGAGCTCGCTGCTGAGCACTGCCA-3'
(SEQ. ID No. 428) L37527-11
5'-TATATAGCGGCCGCGGTCCGTGCTGGTGCTCGAGGT-3' (SEQ. ID No. 429)
L37528-8 5'-TAGTAGAGCTCTTTTGGTGTAGACACC-3' (SEQ. ID No. 439)
L37528-9 5'-ATATAGCGGCCGCGGTCCGTGAAAGCTACATTTTAGC-3' (SEQ. ID No.
440) U78891-10 5'-TGATGAGCTCACAGTGCGTGCATGAACA-3' (SEQ. ID No. 452)
U78891-11 5'-TATATAGCGGCCGCGGTCCGATGTTTTTTTTTGTCTTTTGATGC-3' (SEQ.
ID No. 453) AF095646-10
5'-ATATATCTCGAGCGGACCGTGTTGCATGCATGCGTACTGGTGA-3' (SEQ ID No. 517)
AF095646-11 5'-ATATATCCATGGTGGGTTTATTCTGCAAACAAAAATAG-3' (SEQ ID
No. 518) primers to clone 5'-regulatory sequence in pNOV6901
AB003322-10 5'-TATATACCATGGTGGTGATACTGTCCAACAGAAGG-3' (SEQ. ID No.
142) AB003322-11 5'-ATATATGGATCCGGACCGTTGCCCAGCGAGGAGCATG-3' (SEQ.
ID No. 143) AB003324-12
5'-ATATATGGATCCGGACCGTCTAAATAGGGCCCAACATAC-3' (SEQ. ID No. 156)
AB003324-13 5'-TATATACCATGGTGGCTTAAGGATGCTGAAATATGA-3' (SEQ. ID No.
157) AB003328-8 5'-TATATAGGATCCGGACCGAACAAAACCAATAATCTCCAATG-3'
(SEQ. ID No. 165) AB003328-9
5'-TATATACCATGGTGGATCGCCGATGAACCAACCAAC-3' (SEQ. ID No. 166)
AB077760-9 5'-ATATATGTCGACGGACCGATTCGTTGGGGGTGACATGAC-3' (SEQ. ID
No. 177) AB077760-10 5'-TACAGTACCATGGTGGCCCTTGATGCTGCAGCAGGAT-3'
(SEQ. ID No. 178) AB095645-11
5'-GAGAGACCTGCAGGCGGACCGCTCGTCGCCGTCGTTGGCTCGGCGT-3' (SEQ. ID No.
191) AB095645-12 5'-ATATATCCATGGTGGTTGATAGCCTTATACATGTCCC-3' (SEQ.
ID No. 192) AF139664-13
5'-ATATATGGATCCGGACCGTGCATCCTTACAAAGAGACA-3' (SEQ. ID No. 205)
AF139664-14 5'-TATATACCATGGTGGTTGTCCATACTGTGAAAAATG-3' (SEQ. ID No.
206) AF139665-10 5'-ATATATGTCGACGGACCGACCTGGGGTTTGGAAATG-3' (SEQ.
ID No. 216) AF139665-11 5'-TATATACCATGGTGGCCTTCCATACTACAGAAG-3'
(SEQ. ID No. 217) AF141964-13
5'-ATATATCCTGCAGGCGGACCGAAAACACTGATTAAAGGGT-3' (SEQ. ID No. 230)
AF141964-14 5'-ATATATCCATGGTGGCCTTCAATGCTGAAAAAGAAAAC-3' (SEQ. ID
No. 231) AF141965-13 5'-ATATATGGATCCGGACCGTGTTTCTCGATTAGGCTAC-3'
(SEQ. ID No. 244) AF141965-14
5'-ATATATCCATGGTGGTCTGTAGGCTGCAGAACAGACAC-3' (SEQ. ID No. 245)
AF174093-12 5'-TATATCCTGCAGGCGGACCGAAAATTCACAAGTATAATTC-3' (SEQ. ID
No. 256) AF174093-13 5'-TATATACCATGGTGGTCAAGCTTAACAAAGCAT-3' (SEQ.
ID No. 257) AF204063-12 5'-TATATAGGATCCGGACCGACAAATTTGTTGTACCAC-3'
(SEQ. ID No. 269) AF204063-13
5'-ATATATCCATGGTGGTGTACATGCTGGAAGTGA-3' (SEQ. ID No. 270)
AF345911-11 5'-TATATAGTCGACGGACCGAAAATCTCAAGGTTTC-3' (SEQ. ID No.
281) AF345911-12 5'-TATATACCATGGTGGTTGTCGATCCTGCAGGGAA-3' (SEQ. ID
No. 282) AF424549-11 5'-TATATAGTCGACGGACCGTACAAAGTGCTGGAAGT-3'
(SEQ. ID No. 293) AF424549-12
5'-TATATACCATGGTGGTGATGCTGCAGCAGGATGCA-3' (SEQ. ID No. 294)
AJ293816-9 5'-TATATAGTCGACGGACCGCAAACATTTTAAACTTTAAC-3' (SEQ. ID
No. 303) AJ293816-10 5'-TATATACCATGGTGGTGTTCGATGCTTCATCAATGT-3'
(SEQ. ID No. 304) AY115556-10
5'-TATATACCTGCAGGCGGACCGATATAAAAACCTACTTTATG-3' (SEQ. ID No. 314)
AY115556-11 5'-ATATATCCATGGTGGCGATGGTTCTGTTCAAAGAAATC-3' (SEQ. ID
No. 315) AY177695-13 5'-TATATAGGATCCGGACCGTCAGTCCATCATTTCGTC-3'
(SEQ. ID No. 328) AY177695-14
5'-TATATACCATGGTGGATTTGATGCTAACAAAAAGG-3' (SEQ. ID No. 329)
AY177696-11 5'-TATATAGGATCCGGACCGCAAGCAAAGAAACAAATTTCG-3' (SEQ. ID
No. 340) AY177696-12 5'-ATATACCATGGTGGCTTGATGCTGCATATTTG-3' (SEQ.
ID No. 341) AY177698-13 5'-TATATAGGATCCGGACCGAAGATTTGAACTACTGCCT-3'
(SEQ. ID No. 354) AY177698-14 5'-TATATACCATGGTGGTTTGCAATCCTGCCA-3'
(SEQ. ID No. 355) AY177699-9
5'-TATATAGGATCCGGACCGTCCGGTCGGCCCTCGTCCT-3' (SEQ. ID No. 364)
AY177699-10 5'-TATATACCATGGTGGCTCACGCTGCGCCA-3' (SEQ. ID No. 365)
AY177700-12 5'-TATATAGGATCCGGACCGATAAGTATTTCAGAAAG-3' (SEQ. ID No.
377) AY177700-13 5'-TATATACCATGGTGGGCAAATTTCTTGCATTTTG-3' (SEQ. ID
No. 378) AY177702-12 5'-TATATAGTCGACGGACCGTCTTTTCGCAAACTAAAC-3'
(SEQ. ID No. 390) AY177702-13
5'-TATATACCATGGTGGTCAAGCTAGCATGCAAG-3' (SEQ. ID No. 391)
AY224482-12 5'-TATATAGTCGACGGACCGTCTTTTCGCAAACTAAACAAGG-3' (SEQ. ID
No. 403) AY224482-13 5'-TATATACCATGGTGGTTTTCAAGCTAGC-3' (SEQ. ID
No. 404) AY250075-13 5'-TATATACCTGCAGGCGGACCGAAATGGTTTCATTTTGG-3'
(SEQ. ID No. 417) AY250075-14 5'-TATATACCATGGTGGCGCTCGATACTATCA-3'
(SEQ. ID No. 418) L37527-12
5'-TATATACCTGCAGGCGGACCGCCAAGAGAGCCCCCT-3' (SEQ. ID No. 430)
L37527-13 5'-TATATACCATGGTGGTTGACAGCCTAAAACG-3' (SEQ. ID No. 431)
L37528-10 5'-TATATAGGATCCGGACCGACCACCTCAATCTCCACT-3' (SEQ. ID No.
441) L37528-11 5'-TATATACCATGGTGGATCGGTCTGGACA-3' (SEQ. ID No. 442)
U78891-12 5'-TATATAGGATCCGGACCGTCTTCTTCTTGCCTTCATTTG-3' (SEQ. ID
No. 454) U78891-13 5'-TATATACCATGGTGGTTAGTGATGCTACAAAGTTC-3' (SEQ.
ID No. 455) AF095646-12
5'-TATATAGAGCTCAGTCCAAGCTTGCTAATAAAAACGCT-3' (SEQ ID No. 519)
AF095646-13 5'-TATATACCCGGGCGGTCCGAAGGGTAGGGCTGCACGAA-3' (SEQ ID
No. 520)
EXAMPLE 2
[0098] 1. Construction of the assembly vector pNOV6901 containing
the .beta.-glucuronidase (GUS) coding sequence.
[0099] A. Preparation of GUS coding sequence.
[0100] The .beta.-glucuronidase (GUS) coding sequence Narasimhulu,
et al 1996, Plant Cell, 8: 873-886, which includes an engineered
intron, was amplified from pNOV5003 in a Pfuturbo polymerase
(Stratagene, Cat. No. 600250) reaction. The reaction mixture
consisted of 1 .mu.L pNOV5003 miniprep DNA 200 .mu.M dNTPs, 20
.mu.M GUS5 oligonucleotide primer 5'-atggtacgtcctgtagaaacc-3' (SEQ
ID NO 498), 20 .mu.M GUS3 oligonucleotide primer
5'-gatcgagetctcattgtttgcctecctg-3' (SEQ ID NO 499), 5 .mu.L
10.times. cloned Pfu buffer and 2.5 Units of Pfuturbo DNA
polymerase (Stratagene, Cat. No. 600250) in a final volume of 50
.mu.L. The thermocycling program was at 95.degree. C. for 30
seconds then 10 cycles of (95.degree. C. for 5 seconds, 55.degree.
C. for 10 seconds, 72.degree. C. for 2 5 minutes) then 20 cycles of
(95.degree. C. for 5 seconds, 57.degree. C. for 15 seconds,
72.degree. C. for 2.5 minutes) then 72.degree. C. for 2.5 minutes.
The 2.2 kb GUS PCR product was isolated and concentrated using the
QIAEX II kit (Qiagen, Cat. No. 20021). The GUS PCR product was
recovered in 15 .mu.L ddH.sub.2O and subsequently digested in a 20
.mu.L reaction containing 1 .mu.g BSA, 2 .mu.L 10.times.
restriction endonuclease buffer and 1 .mu.L SacI. The reaction was
incubated at 37.degree. C. for 2 hours. The GUS PCR product (SacI)
was resolved on 1.5% TBE agarose and the 2.2 kb GUS PCR product
(SacI) band was excised. The GUS PCR product (SacI) DNA was
recovered from the agarose in 15 .mu.L ddH.sub.2O with the QIAEX II
kit (Qiagen, Cat. No. 20021).
[0101] B. Preparation of the pSP73 Vector.
[0102] An E. coli vector pSP73 (Promega, Cat. No. P2221) miniprep
DNA was prepared. 1 .mu.L of the miniprep DNA was digested in a 20
.mu.L reaction mixture containing 1 .mu.g BSA, 2 .mu.L 10.times.
restriction endonuclease, 1 .mu.L SmaI and 1 .mu.L SacI. The
reaction was incubated at 25.degree. C. for 1.5 hours then
37.degree. C. for 1.5 hours. The pSP73 (SmaI/SacI) DNA was resolved
on 1.5% TBE agarose and the 2.4 kb pSP73 (SmaI/SacI) band was
excised. The pSP73 (SmaI/SacI) DNA was recovered from the agarose
in 15 .mu.L ddH.sub.2O with the QIAEX II kit (Qiagen, Cat No.
20021).
[0103] C. Construction of pSP73-GUS
[0104] 5 .mu.L of pSP73 (SmaI/SacI) was ligated to 5 .mu.L GUS PCR
product (SacI) by mixing with an equal volume of Takara DNA
Ligation Mix, Version II (Cat. No. TAK 6022) and incubating at
16.degree. C. for 30 minutes. 7.5 .mu.L of the ligation mixture was
transformed into 50 .mu.L XL-1 supercompetent cells (Stratagene,
Cat. No. 200236). pSP73-GUS recombinants were verified by digesting
2 .mu.L pSP73-GUS miniprep DNA in a 20 .mu.L reaction containing 2
.mu.g BSA, 2 .mu.L 10.times. restriction endonuclease buffer, 1
.mu.L XbaI and 1 .mu.L SacI and the pSP73-GUS (XbaI/SacI) products
were resolved on 1.5% TBE agarose. The positive pSP73-GUS
recombinants were sequenced.
[0105] D. Addition of Restriction Endonuclease Sites to
pSP73-GUS
[0106] The pSP73-GUS construct lacks flexibility to clone
3'-regulatory sequence just after the GUS coding sequence.
Additional restriction sites were added to the polylinker to
increase flexibility at the 3'-terminus of the GUS coding sequence
by ligating a synthetic adapter to the construct. The adapter
(Synthetic Adaptor I) was made by combining 40 .mu.L of 50 .mu.M
oligonucleotide PL-F 5'-Pccgcgggeggccgcactagteccgggcccat-3' (SEQ ID
NO. 456), 40 .mu.L of 50 .mu.M oligonucleotide PL-R
5'-Pcgatgggccegggactagtgeggccgcccgcggagct-3' (SEQ ID NO.
457)--where P is a 5'-phosphate group--in a 100 .mu.L mixture that
is 25 mM in Tris-HCl (pH 8.0) and 10 mM in MgCl.sub.2. The mixture
was boiled for 5 minutes, removed from heat and naturally cooled to
room temperature (about 60 minutes), yielding a 20 .mu.M Synthetic
Adaptor I solution.
[0107] The pSP73-GUS construct was prepared by digesting 14 .mu.L
of miniprep pSP73-GUS DNA with 1 .mu.L SacI and 1 .mu.L ClaI in a
20 .mu.L reaction mixture containing 2 .mu.g BSA and 2 .mu.L
10.times. restriction endonuclease buffer. The reaction mixture was
incubated at 37.degree. C. for 6 hours, then at 70.degree. C. for
20 minutes. Then 1 .mu.L of the appropriate 10.times. restriction
endonuclease buffer, 1 .mu.L 1 Unit/.mu.L CIP and 8 .mu.L
ddH.sub.2O were added to the reaction and it was further incubated
at 37.degree. C. for 30 minutes. The pSP73-GUS (SacI/ClaI/CIP) DNA
was resolved on 1% TAE agarose, excised, recovered and ethanol
precipitated with glycogen carrier. The pSP73-GUS (SacI/ClaI/CIP)
DNA was recovered by micro centrifugation, washed with 70% ethanol,
dried under vacuum and resuspended in 5 .mu.L ddH.sub.2O.
[0108] 4.5 .mu.L of Synthetic Adaptor I solution was ligated to 2.5
.mu.L pSP73-GUS (SacI/ClaI/CIP) in a 10 .mu.L ligation mixture
containing 1 .mu.L 10.times. T4 DNA ligase buffer and 1 .mu.L T4
DNA ligase and incubated more than 8 hours at 16.degree. C. 4 .mu.L
of the ligation mixture was transformed into 50 .mu.L XL-1
supercompetent cells (Stratagene, Cat. No. 200236). The
pSP73-GUS-mod recombinants were verified by digesting 5 .mu.L
pSP73-GUS-mod miniprep DNA in a 20 .mu.L reaction containing 2
.mu.g BSA, 2 .mu.L 10.times. restriction endonuclease buffer and 1
.mu.L NotI. The digests were resolved on 1.0% TAE agarose, and the
sequence of positive pSP73-GUS-mod recombinants was verified. The
finished clone was designated pNOV6901. [0109] 2. Construction of
pNOV6900
[0110] It was necessary to construct an Abrobacterium binary vector
to facilitate mobilization of expression cassettes constructed in
pNOV6901 into plants. The pNOV2115 vector was modified by inserting
an adaptor that introduces the PacI, SgFI and RsrII restriction
endonuclease recognition sites. pNOV2115 miniprep DNA (14 .mu.L)
was digested with 1 .mu.L KpnI and 1 .mu.L HindIII in a 20 .mu.L
reaction mixture containing 2 .mu.g BSA and 2 .mu.L 10.times.
restriction endonuclease buffer. Digests were incubated at
37.degree. C. for more than 6 hours, then at 70.degree. C. for 20
minutes. Then 1 .mu.L of the appropriate 10.times. restriction
endonuclease buffer, 1 .mu.L 1 Unit/.mu.L CIP and 8 .mu.L
ddH.sub.2O were added to the reaction and it was further incubated
at 37.degree. C. for 30 minutes. pNOV2115 (KpnI/HindIII/CIP) was
resolved on 1% TAE agarose, the 9.2 kb pNOV2115 (KpnI/HindIII/CIP)
DNA band was excised, recovered and ethanol precipitated with
glycogen carrier. The pNOV2115 (KpnI/HindIII/CIP) DNA was recovered
by micro centrifugation, washed with 70% ethanol, dried under
vacuum and resuspended in 5 .mu.L ddH.sub.2O.
[0111] Additional restriction sites were added to pNOV2115
(KpnI/HindIII/CIP) by ligating the vector to Synthetic Adapter II.
The Synthetic Adapter II was made by combining 37 .mu.L of 150
.mu.M oligonucleotide PL1 5'-Pgtaccggaccgcgatcgcttaatta-3' (SEQ ID
NO 458), 37 .mu.L of 150 .mu.M PL2 oligonucleotide
5'-Pagettaattaagcgatcgcggtccg-3' (SEQ ID NO 459)--where P is a
5'-phosphate group--in a 100 .mu.L mixture that is 25 mM in
Tris-HCl (pH 8.0) and 10 mM in MgCl.sub.2. The mixture was boiled
for 5 minutes, removed from heat and naturally cooled to room
temperature (about 60 minutes), yielding a 55 .mu.M Synthetic
Adapter II solution.
[0112] 2.5 .mu.L pNOV2115 (KpnI/HindIII/CIP) was ligated to 2.5
.mu.L 55 .mu.M Synthetic Adapter II solution by mixing with an
equal volume of Takara DNA Ligation Mix, Version II (Cat. No. TAK
6022), and was incubated at 16.degree. C. for 30 minutes. 5.0 .mu.L
of ligation mixture was transformed into 50 .mu.L DH5.alpha.
competent cells (Invitrogen, Cat. No. 18258-012). pNOV2115-mod
recombinants were verified by digesting 2 .mu.L pNOV2115-mod
miniprep DNA with KpnI, HindIII, PacI or RsrII in 10 .mu.L
reactions containing 1 .mu.g BSA and 1 .mu.L 10.times. restriction
endonuclease buffer. The sequence of positive pNOV2115-mod
recombinants was verified. The finished clone was designated
pNOV6900.
EXAMPLE 3
Construction of the OsMADS5 Expression Cassette
[0113] A. Cloning the OsMADS5 5'-Regulatory Sequence
[0114] High-fidelity PCR was used to amplify the OsMADS5
5'-regulatory sequence from rice genomic DNA (gDNA). The 50 .mu.L
reaction mixture consisted of 100 ng rice gDNA, 200 .mu.M dNTPs, 1
.mu.L 20 .mu.M oligonucleotide primer OsMADS5-P3
5'-tgagcaggtagccggcgaccaatcgcgag-3' (SEQ ID NO 460), 1 .mu.L 20
.mu.M oligonucleotide primer OsMADS #5-P2
5'-catactgttacaaaaaagaaaatcagtggaccac-3' (SEQ ID NO 461), 1 .mu.L
10.times. Expand High Fidelity buffer and 1 .mu.L Expand High
Fidelity polymerase. The thermocycling program was at 95.degree. C.
for 2 minutes followed by 40 cycles of (94.degree. C. for 15
seconds, 68.degree. C. for 7.5 minutes) followed by 68.degree. C.
for 10 minutes. The 5.4 kb DNA product, encoding the OsMADS5
5'-regulatory sequence, was cloned with the TOPO XL PCR cloning
kit. The pCR-XL-TOPO-OsMADS5-5'-gDNA recombinants, containing the
OsMADS5 5'-regulatory sequence, were identified by digesting 5
.mu.L pCR-XL-TOPO-OsMADS5-5'-gDNA miniprep DNA with EcoRI in a 20
.mu.L reaction mixture containing 2 .mu.g BSA and 2 .mu.L 10.times.
restriction endonuclease buffer. The reaction mixture was incubated
at 37.degree. C. for 2 hours then the pCR-XL-TOPO-OsMADS5-5'-gDNA
(EcoRI) products were resolved on 1% TAE agarose. Positive
pCR-XL-TOPO-OsMADS5-5'-gDNA clones were sequenced.
[0115] B. Cloning the OsMADS5 3'-Regulatory Sequence
[0116] High-fidelity PCR was used to amplify the OsMADS5
3'-regulatory sequence from rice genomic DNA (gDNA). The 50 .mu.L
reaction mixture consisted of 100 ng rice gDNA, 200 .mu.M dNTPs, 1
.mu.L 20 .mu.M oligonucleotide primer OsMADS #5-T1
5'-atgaattgcttatcacattaatggacatc-3' (SEQ ID NO. 462), 1 .mu.L 20
.mu.M oligonucleotide primer OsMADS #5-T2
5'-caaaactacatcaagagccttggaattggtcc-3' (SEQ ID NO. 463), 1 .mu.L
10.times. Expand High Fidelity buffer and 1 .mu.L Expand High
Fidelity polymerase. The thermocycling program was 95.degree. C.
for 2 minutes followed by 40 cycles of (94.degree. C. for 15
seconds, 60.degree. C. for 30 seconds, 68.degree. C. for 6 minutes)
followed by 68.degree. C. for 15 minutes. The 1.2 kb
OsMADS5-3'-gDNA DNA product, encoding the OsMADS5 3'-regulatory
sequence, was cloned with the Zero Blunt TOPO PCR cloning kit
(Invitrogen, Cat. No. K2875-20). pCR-Blunt II-TOPO-OsMADS5-3'-gDNA
recombinants, with the OsMADS5 3'-regulatory sequence, were
identified by digesting 5 .mu.L pCR-Blunt II-TOPO-OsMADS5-3'-gDNA
miniprep DNA with EcoRI in a 20 .mu.L reaction mixture containing 2
.mu.g BSA and 2 .mu.L 10.times. restriction endonuclease buffer.
The reaction mixture was incubated at 37.degree. C. for 2 hours and
then the pCR-Blunt II-TOPO-OsMADS5-3'-gDNA (EcoRI) products were
resolved on 1% TAE agarose. Positive pCR-Blunt
II-TOPO-OsMADS5-3'-gDNA clones were sequenced.
[0117] C. Construction of the OsMADS5 5'-Regulatory Sequence
[0118] The OsMADS5 5'-regulatory sequence for the expression
cassette was made in several steps. The 3'-half (OsMADS-5Pb, about
3.03 kb) was produced by high-fidelity PCR from the
pCR-XL-TOPO-OsMADS5-5'-gDNA clone described above. The reaction
mixture consisted of 1 .mu.L pCR-XL-TOPO-OsMADS5-5'-gDNA miniprep
DNA, 200 .mu.M dNTPs, 20 .mu.M oligonucleotide primer OsMADS5-C3
5'-cagtgtcgacggggcgagggaaagtagagc-3' (SEQ ID NO 464), 20 .mu.M
oligonucleotide primer OsMADS5-C4
5'-cgatccatggtggatactgttacaaaaaagaaaatcagtg-3' (SEQ ID NO 465), 5
.mu.L 10.times. cloned Pfu buffer and 2.5 Units of Pfuturbo DNA
polymerase (Stratagene, Cat. No. 600252) in a final volume of 50
.mu.L. The thermocycling program was at 95.degree. C. for 30
seconds then 40 cycles of (95.degree. C. for 10 seconds, 50.degree.
C. for 60 seconds, 72.degree. C. for 6 minutes) then 72.degree. C.
for 10 minutes. The OsMADS-5Pb DNA product was recovered using the
QIAquick PCR purification kit (Qiagen, Cat. No. 28106). The
recovered OsMADS-5Pb DNA was ethanol precipitated with glycogen
carrier. The OsMADS-5Pb DNA was recovered by micro centrifugation,
washed with 70% ethanol, dried under vacuum and resuspended in 14
.mu.L ddH.sub.2O. The OsMADS-5Pb was digested in a 20 .mu.L
reaction mixture containing 2 .mu.g BSA, 2 .mu.L 10.times.
restriction endonuclease buffer, 1 .mu.L SalI and 1 .mu.L NcoI. The
reaction mixture was incubated at 37.degree. C. for more than 6
hours. The OsMADS-5Pb (NcoI/SalI) DNA was resolved on 1.0% TAE
agarose and the 3.03 kb OsMADS-5Pb (NcoI/SalI) DNA band was
excised, recovered and ethanol precipitated with glycogen carrier.
The OsMADS-5Pb (NcoI/SalI) DNA was recovered by micro
centrifugation, washed with 70% ethanol, dried under vacuum and
resuspended in 5 .mu.L ddH.sub.2O.
[0119] 2 .mu.g of the pNOV6901 miniprep DNA was digested in a 20
.mu.L reaction mixture containing 2 .mu.g BSA, 2 .mu.L 10.times.
restriction endonuclease buffer, 1 .mu.L SalI and 1 .mu.L NcoI. The
reaction mixture was incubated at 37.degree. C. for more than 6
hours, then at 70.degree. C. for 20 minutes. Then 1 .mu.L of the
appropriate 10.times. restriction endonuclease buffer, 1 .mu.L 1
Unit/.mu.L CIP and 8 .mu.L ddH.sub.2O were added to the reaction
and it was further incubated at 37.degree. C. for 30 minutes. The
pNOV6901 (NcoI/Sa11/CIP) DNA was resolved on 1.0% TAE agarose and
the 4.7 kb pNOV6901 (NcoI/SalI/CIP) band was excised, recovered and
ethanol precipitated with glycogen carrier. The pNOV6901
(NcoI/SalI/CIP) DNA was recovered by micro centrifugation, washed
with 70% ethanol, dried under vacuum and resuspended in 5 .mu.L
ddH.sub.2O.
[0120] 4.0 .mu.L of the OsMADS-5Pb (NcoI/SalI) was ligated to 4.0
.mu.L pNOV6901 (NcoI/SalI/CIP) in a 10 .mu.L reaction mixture
containing 1 .mu.L 10.times. T4 DNA ligase buffer and 1 .mu.L T4
DNA ligase (400 Units/.mu.L) and incubated more than 8 hours at
16.degree. C. 5.0 .mu.L of the ligation mixture was transformed
into 50 .mu.L Top10 competent cells. pNOV6901-OsMADS-5Pb
recombinants were verified by digesting 2 .mu.L pNOV6901-OsMADS-5Pb
miniprep DNA with 0.5 .mu.L SalI, 0.5 .mu.L NcoI in 10 .mu.L
reactions containing 1 .mu.g BSA and 1 .mu.L 10.times. restriction
endonuclease buffer. The digests were incubated at 37.degree. C.
for 2 hours and the pNOV6901-OsMADS-5Pb (NcoI/SalI) DNA was
resolved on 1% TAE agarose. Positive pNOV6901-OsMADS-5Pb
recombinants were sequenced.
[0121] The 5'-half (OsMADS-5Pa, about 2.4 kb) was produced by
high-fidelity PCR from the pCR-XL-TOPO-OsMADS5-5'-gDNA clone
described above. The reaction mixture consisted of 1 .mu.L
pCR-XL-TOPO-OsMADS5-5'-gDNA miniprep DNA, 200 .mu.M dNTPs, 20 .mu.M
oligonucleotide primer OsMADS5-C1
5'-gactctcgaggcgcgcctgagcaggtagccggcgacc-3' (SEQ ID NO 466), 20
.mu.M oligonucleotide primer OsMADS5-C2b
5'-gtgtgtetcgagetctctctagctctctctcgg-3' (SEQ ID NO 467), 5 .mu.L
10.times. cloned Pfu buffer and 2.5 Units of Pfuturbo DNA
polymerase (Stratagene, Cat. No. 600252) in a final volume of 50
.mu.L. The thermocycling program was at 95.degree. C. for 30
seconds then 40 cycles of (95.degree. C. for 10 seconds, 50.degree.
C. for 60 seconds, 72.degree. C. for 6 minutes) then 72.degree. C.
for 10 minutes. The 2.4 kb OsMADS-5Pa DNA product was cloned with
the Zero Blunt TOPO PCR cloning kit (Invitrogen, Cat. No.
K2875-20). pCR-Blunt II-TOPO-OsMADS-5Pa recombinants were
identified by digesting 5 .mu.L pCR-Blunt II-TOPO-OsMADS-5Pa
miniprep DNA with EcoRI in a 20 .mu.L reaction mixture containing 2
.mu.g BSA and 2 .mu.L 10.times. restriction endonuclease buffer.
The digests were incubated at 37.degree. C. for 2 hours then
resolved on 1% TAE agarose. Positive pCR-Blunt II-TOPO-OsMADS-5Pa
recombinants were sequenced.
[0122] 2 .mu.g of the pNOV6901-OsMADS-5Pb miniprep DNA was digested
in a 20 .mu.L reaction mixture containing 2 .mu.g BSA, 2 .mu.L
10.times. restriction endonuclease buffer and 2 .mu.L XhoI. The
digest was incubated at 37.degree. C. for more than 6 hours, then
at 70.degree. C. for 20 minutes. Then 1 .mu.L of the appropriate
10.times. restriction endonuclease buffer, 1 .mu.L 1 Unit/.mu.L CIP
and 8 .mu.L ddH.sub.2O were added to the reaction and it was
further incubated at 37.degree. C. for 30 minutes. 2 .mu.g of the
pCR-Blunt II-TOPO-OsMADS-5Pa miniprep DNA was digested in a 20
.mu.L reaction mixture containing 2 .mu.g BSA, 2 .mu.L 10.times.
restriction endonuclease buffer and 2 .mu.L SalI. The digest was
incubated at 37.degree. C. for more than 6 hours.
[0123] The digested plasmid DNAs, pNOV6901-OsMADS-5Pb (XhoI/CIP)
and pCR-Blunt II-TOPO-OsMADS-5Pa (SalI), were resolved on 1.0% TAE
agarose and the 7.7 kb pNOV6901-OsMADS-5Pb (XhoI/CIP) and the 2.4
kb OsMADS-5Pa (SalI) bands were excised, extracted, recovered and
ethanol precipitated with glycogen. The pNOV6901-OsMADS-5Pb
(XhoI/CIP) and OsMADS-5Pa (SalI) DNA fragments were recovered by
micro centrifugation, washed with 70% ethanol, dried under vacuum
and resuspended each in 5 .mu.L ddH.sub.2O.
[0124] 4.0 .mu.L of the pNOV6901-OsMADS-5Pb (XhoI/CIP) was ligated
to 4.0 .mu.L OsMADS-5Pa (SalI) in a 10 .mu.L reaction mixture
containing 1 .mu.L 10.times. T4 DNA ligase buffer and 1 .mu.L
[0125] T4 DNA ligase (400 U/.mu.L). The reaction mixture was
incubated more than 8 hours at 16.degree. C. 5.0 .mu.L of the
ligation mixture was transformed into 50 .mu.L Top10 competent
cells. The pNOV6901-OsMADS5P recombinants were verified by
digesting 2 .mu.L pNOV6901-OsMADS5P miniprep DNA with 0.5 .mu.L
XhoI, 0.5 .mu.L NcoI in 10 .mu.L reaction mixtures containing 1
.mu.g BSA and 1 .mu.L 10.times. restriction endonuclease buffer.
The digests were incubated at 37.degree. C. for 2 hours then
resolved on 1% TAE agarose. Positive pNOV6901-OsMADS5P recombinants
were sequenced.
[0126] D. Construction of the OsMADS5 3'-Regulatory Sequence
[0127] The OsMADS-5 3'-regulatory sequence for the expression
cassette was produced by high-fidelity PCR from the pCR-Blunt
II-TOPO-OsMADS5-3'-gDNA clone, above. The reaction mixture
consisted of 1 .mu.L pCR-Blunt II-TOPO-OsMADS5-3'-gDNA miniprep
DNA, 200 .mu.M dNTPs, 20 .mu.M oligonucleotide primer OsMADS5T-F
5'-cccgggccatggggggtctagaatgaattgcttatcacattaatgg-3' (SEQ ID NO
468), 20 .mu.M oligonucleotide primer OsMADS5T-R
5'-ccegggcgcgccggatgagaacagctacatcc-3' (SEQ ID NO 469), 5 .mu.L
10.times. cloned Pfu buffer and 2.5 Units of Pfuturbo DNA
polymerase (Stratagene, Cat. No. 600252) in a final volume of 50
.mu.L. The thermocycling program was 95.degree. C. for 30 seconds
then 40 cycles of (95.degree. C. for 10 seconds, 50.degree. C. for
60 seconds, 72.degree. C. for 6 minutes) then 72.degree. C. for 10
minutes. The OsMADS5T DNA product was recovered using the QIAquick
PCR purification kit (Qiagen, Cat. No. 28106) and ethanol
precipitated with glycogen carrier. The OsMADS5T DNA was recovered
by micro centrifugation, washed with 70% ethanol, dried under
vacuum and resuspended in 14 .mu.L ddH.sub.2O. The OsMADS5T DNA was
digested in a 20 .mu.L reaction mixture containing 2 .mu.g BSA, 2
.mu.L 10.times. restriction endonuclease buffer and 2 .mu.L XmaI.
The digest was incubated at 37.degree. C. for more than 6 hours.
The OsMADS5T (XmaI) DNA was resolved on 1.0% TAE agarose and the
1.1 kb OsMADS5T (XmaI) band was excised, recovered and ethanol
precipitated with glycogen carrier. OsMADS5T (XmaI) DNA was
recovered by micro centrifugation, washed with 70% ethanol, dried
under vacuum and resuspended in 5 .mu.L ddH.sub.2O.
[0128] 2 .mu.g pNOV6901-OsMADS5P miniprep DNA was digested in a 20
.mu.L reaction mixture containing 2 .mu.g BSA, 2 .mu.L 10.times.
restriction endonuclease buffer and 2 .mu.L XmaI. The digest was
incubated at 37.degree. C. for more than 6 hours, then at
70.degree. C. for 20 minutes. Then 1 .mu.L of the appropriate
10.times. restriction endonuclease buffer, 1 .mu.L 1 Unit/.mu.L CIP
and 8 .mu.L ddH.sub.2O were added to the reaction and it was
further incubated at 37.degree. C. for 30 minutes. The
pNOV6901-OsMADS5P (XmaI/CIP) DNA was resolved on 1.0% TAE agarose
and the 10.1 kb pNOV6901-OsMADS-5P (XmaI/CIP) band was excised,
recovered and ethanol precipitated with glycogen carrier.
pNOV6901-OsMADS-5P (XmaI/CIP) DNA was recovered by micro
centrifugation, washed with 70% ethanol, dried under vacuum and
resuspended each in 5 .mu.L ddH.sub.2O.
[0129] 4.0 .mu.L pNOV6901-OsMADS5P (XmaI/CIP) was ligated to 4.0
.mu.L OsMADS5T (XmaI) in a 10 .mu.L ligation mixture containing 1
.mu.L 10.times. T4 DNA ligase buffer and 1 .mu.L T4 DNA ligase (400
Units/.mu.L) and incubated more than 8 hours at 16.degree. C. 5.0
.mu.L of ligation mixture was transformed into 50 .mu.L Top10
competent cells. Positive pNOV6901-OsMADS5P/OsMADS5T recombinants
were verified by digesting 2 .mu.L miniprep DNA with 1.0 .mu.L AscI
in 10 .mu.L reaction mixtures containing 1 .mu.g BSA and 1 .mu.L
10.times. restriction endonuclease buffer. The digests were
incubated at 37.degree. C. for 2 hours then resolved on 1% TAE
agarose. Positive pNOV6901-OsMADS5P/OsMADS5T recombinants were
sequenced. The construct was designated pNOV6901-OsMADS5P/OsMADS5T.
The plasmid's QC number is 11084. 11084 contains the complete
OSMADS5 expression cassette depicted by SEQ ID NO. 536.
[0130] E. Mobilization of the OsMADS5 GUS Expression Cassette into
pNOV6900
[0131] 2 .mu.g pNOV6900 was digested in a 20 .mu.L reaction mixture
containing 2 .mu.g BSA, 2 .mu.L 10.times. restriction endonuclease
buffer and 2 .mu.L AscI. The digest was incubated at 37.degree. C.
for more than 6 hours, then at 70.degree. C. for 20 minutes. Then 1
.mu.L of the appropriate 10.times. restriction endonuclease buffer,
1 .mu.L 1 Unit/.mu.L CIP and 8 .mu.L ddH.sub.2O were added to the
reaction and it was further incubated at 37.degree. C. for 30
minutes. 2 .mu.g pNOV6901-OsMADS5P/OsMADS5T miniprep DNA was
digested in a 20 .mu.L reaction mixture containing 2 .mu.g BSA, 2
.mu.L 10.times. restriction endonuclease buffer and 2 .mu.L AscI.
The digest was incubated at 37.degree. C. for more than 6
hours.
[0132] The digested plasmid DNAs, pNOV6900 (AscI/CIP) and
pNOV6901-OsMADS5P/OsMADS5T (AscI), were resolved on 1.0% TAE
agarose and the 9.2 kb pNOV6900 (AscI/CIP) and the 8.7 kb
pNOV6901-OsMADS5P/OsMADS5T (AscI) DNA bands were excised, recovered
and ethanol precipitated with glycogen carrier. The pNOV6900
(AscI/CIP) and pNOV6901-OsMADS5P/OsMADS5T (AscI) DNA fragments were
recovered by micro centrifugation, washed with 70% ethanol, dried
under vacuum and resuspended in 5 .mu.L ddH.sub.2O each.
[0133] 4.0 .mu.L pNOV6900 (AscI/CIP) was ligated to 4.0 .mu.L
pNOV6901-OsMADS5P/OsMADS5T (AscI) in a 10 .mu.L ligation mixture
containing 1 .mu.L 10.times. T4 DNA ligase buffer and 1 .mu.L T4
DNA ligase, which was incubated more than 8 hours at 16.degree. C.
5.0 .mu.L of ligation mixture was transformed into 50 .mu.L Top10
competent cells. pNOV6900-pNOV6901-OsMADS5P/OsMADS5T recombinants
were verified by digesting 7.5 .mu.L
pNOV6900-pNOV6901-OsMADS5P/OsMADS5T miniprep DNA with 1.0 .mu.L
NcoI in 10 .mu.L reactions containing 1 .mu.g BSA and 1 .mu.L
10.times. restriction endonuclease buffer. The digests were
incubated at 37.degree. C. for 2 hours then resolved on 1% TAE
agarose. Positive pNOV6900-pNOV6901-OsMADS5P/OsMADS5T recombinants
were sequenced. The finished clone was designated pNOV6911. The
plasmid's QC number is 11085.
[0134] The engineered alterations in the OsMADS5P sequence include
introduction of an XhoI site followed by an AscI site at the 5'-end
of the OsMADS5P sequence, elimination the natural translation start
codon of the OsMADS5P sequence, elimination of undesired ORFs in
the new leader sequence (5'-UTR) of the OsMADS5P sequence,
insertion a Kozak sequence upstream of the new translation start
codon of the OsMADS5P sequence and insertion of a new translation
start codon downstream of the intron1/exon2 junction as an NcoI
site in the OsMADS5P sequence. The engineered alterations in the
OsMADS5T sequence include introduction of an XmaI site at the
5'-terminus of the OsMADS5T sequence and introduction of an AscI
site at the 3'-terminus of the OsMADS5T sequence. In this
configuration the GUS coding sequence can be replaced with any gene
of interest flanked by NcoI restriction sites.
[0135] The complete cassette can then be excised as an AscI
fragment and cloned into pNOV6900.
[0136] The cassette was transformed into A188 .times. HyII maize
and Kaybonnet rice using standard agrobacterium mediated
methodology.
[0137] GUS Expression in T0 Maize
[0138] Fifteen T0 transgenic maize lines were generated. Tassel
spikelets and leaf punches were harvested just before pollen shed
and histochemically screened for GUS activity. The ear from a plant
containing multiple transgene copies was sacrificed to examine GUS
expression in developing florets. Gus activity localized primarily
to transmitting tissue at the base of each floret, and to a lesser
extent, the vascular bundles in developing ears. GUS activity was
also apparent in developing silks. These data indicate the cassette
drives GUS expression primarily in female reproductive tissue.
[0139] GUS Expression in T0 Rice
[0140] Of forty T0 rice (cv. Kaybonnet) lines containing pNOV6911
(or 11085), eighteen independent transformants were histochemically
stained for GUS expression. Only four events had detectable GUS
activity in leaf tissue. In most events, activity in spikelets was
localized to glume tips, and anthers to a much lesser extent.
[0141] GUS Expression in T1 Maize
[0142] T1 progeny from three events were sown for expression
analysis in vegetative and reproductive tissue. FIGS. 3A and 3B
show GUS activity is restricted to developing ears, particularly
the vasculature along the outer ear and the transmitting tissue
beneath florets. GUS activity is also seen in tissue surrounding
the ovule sac. GUS activity was undetectable in the ear node or the
node beneath it, tassel, leaf or silk. These results provide
further evidence for GUS activity in developing ears. The data show
the pattern established at 5 days before pollination persists up to
2 days after pollination. GUS activity becomes restricted to
transmitting tissue and maternal tissue at the base of developing
kernels during seed development. GUS protein is detectable
throughout ovule and kernel development, up to 20 days after
pollination. There is very light staining in the aerial tissue,
with no GUS activity in the roots
[0143] In summary, the present invention includes expression
cassettes based on the Oryza sativa OsMADS5 gene. These cassettes
consist of the gene's promoter including the first intron, 5'-UTR,
3'-UTR and 3'-nontranscribed sequence. The cassette's design
facilitates replacement of the GUS coding sequence with any gene of
interest. The cassette drives gene expression primarily in maternal
reproductive tissue. Within developing ears, expression localizes
to the outer vasculature along the long axis of the ear, the
transmitting tissue in developing florets and kernels, tissue
surrounding ovules and maternal tissue at the base of developing
kernels. The expression cassettes of the present invention drive
gene expression from a very early point in ovule development,
perhaps from shortly after differentiation.
EXAMPLE 4
Construction of the OsMADS6 Expression Cassette
[0144] A. Cloning of the OsMADS6 5'-Regulatory Sequence
[0145] Used high-fidelity PCR to amplify the OsMADS6 5'-regulatory
sequence from rice genomic DNA (gDNA). The 50 .mu.L reaction
mixture consisted of 100 ng rice gDNA, 200 .mu.M dNTPs, 1 .mu.L 20
.mu.M oligonucleotide primer OsMADS #6-P1
5'-ctaggacgatggtgtgatgtgggaacacg-3' (SEQ ID NO 470), 1 .mu.L 20
.mu.M oligonucleotide primer OsMADS #6-P2
5'-gtacctttctaaagtctttgttatgctgcac-3' (SEQ ID NO 471), 1 .mu.L
10.times. Expand High Fidelity buffer and 1 .mu.L Expand High
Fidelity polymerase. The thermocycling program was at 95.degree. C.
for 2 minutes followed by 40 cycles of (94.degree. C. for 15
seconds, 68.degree. C. for 7.5 minutes) followed by 68.degree. C.
for 10 minutes. Cloned the 4.5 kb OsMADS6-5'-gDNA DNA product with
the TOPO XL PCR cloning kit. pCR-XL-TOPO-OsMADS6-5'-gDNA
recombinants were identified by digesting 5 .mu.L
pCR-XL-TOPO-OsMADS6-5'-gDNA miniprep DNA with EcoRI in a 20 .mu.L
reaction mixture containing 2 .mu.g BSA and 2 .mu.L 10.times.
restriction endonuclease buffer. The digests were incubated at
37.degree. C. for 2 hours then the products were resolved on 1% TAE
agarose. The positive pCR-XL-TOPO-OsMADS6-5'-gDNA clones were
sequenced.
[0146] B. Cloning of the OsMADS6 3'-Regulatory Sequence
[0147] The OsMADS6 3'-regulatory sequence from rice genomic DNA
(gDNA) was amplified using high-fidelity PCR. The 50 .mu.L reaction
mixture consisted of 100 ng rice gDNA, 200 .mu.M dNTPs, 1 .mu.L 20
.mu.M oligonucleotide primer OsMADS #6-T1
5'-gctaagcagccatcgatcagctgtcag-3' (SEQ ID NO 470), 1 .mu.L 20 .mu.M
oligonucleotide primer OsMADS #6-T2
5'-gatgccattgtgtaatgaatggaggagagc-3' (SEQ ID NO 471), 1 .mu.L
10.times. Expand High Fidelity buffer and 1 .mu.L Expand High
Fidelity polymerase. The thermocycling program was at 95.degree. C.
for 2 minutes followed by 40 cycles of (94.degree. C. for 15
seconds, 60.degree. C. for 30 seconds, 68.degree. C. for 6 minutes)
followed by 68.degree. C. for 15 minutes. The 1.2 kb DNA product
was cloned with the Zero Blunt TOPO PCR cloning kit. The
pCR-II-Blunt-OsMADS6-3'-gDNA recombinants were identified by
digesting 5 .mu.L pCR-II-Blunt-OsMADS6-3'-gDNA miniprep DNA with
EcoRI in a 20 .mu.L reaction mixture containing 2 .mu.g BSA and 2
.mu.L 10.times. restriction endonuclease buffer. The digests were
incubated at 37.degree. C. for 2 hours then resolved on 1% TAE
agarose. Positive pCR-II-Blunt-OsMADS6-3'-gDNA clones were
sequenced.
[0148] C. Construction of the OsMADS6 5'-Regulatory Sequence
[0149] The OsMADS6 5'-regulatory sequence for the expression
cassette was made in several steps. The 3'-half (OsMADS-6Pb, about
2.96 kb) was produced by high-fidelity PCR from the OsMADS6 5'-gene
regulatory sequence clone, above. The reaction mixture consisted of
1 .mu.L pCR-XL-TOPO-OsMADS6-5'-gDNA miniprep DNA, 200 .mu.M dNTPs,
20 .mu.M oligonucleotide primer OsMADS6-P3b
5'-cgagtcgacgaggggaagagttgagctgag-3' (SEQ ID NO 474), 20 .mu.M
oligonucleotide primer OsMADS6-P4c
5'-gactccatggtggttatgctgcacaaaaatg-3' (SEQ ID NO 475), 5 .mu.L
10.times. cloned Pfu buffer and 2.5 Units of Pfuturbo DNA
polymerase (Stratagene, Cat. No. 600252) in a final volume of 50
.mu.L. The thermocycling program was at 95.degree. C. for 30
seconds then 40 cycles of (95.degree. C. for 10 seconds, 50.degree.
C. for 60 seconds, 72.degree. C. for 6 minutes) then 72.degree. C.
for 10 minutes. The DNA product was cloned with the Zero Blunt TOPO
PCR cloning kit. The pCR-Blunt-II-TOPO-OsMADS6-Pb recombinants were
identified by digesting 5 .mu.L pCR-Blunt-II-TOPO-OsMADS6-Pb
miniprep DNA with EcoRI in a 20 .mu.L reaction mixture containing 2
.mu.g BSA and 2 .mu.L 10.times. restriction endonuclease buffer.
The digests were incubated at 37.degree. C. for 2 hours then
resolved on 1% TAE agarose. Positive pCR-Blunt-II-TOPO-OsMADS6-Pb
recombinants were sequenced.
[0150] The 5'-half (OsMADS-6Pa, about 1.5 kb) was produced by
high-fidelity PCR from the pCR-XL-TOPO-OsMADS6-5'-gDNA clone,
above. The reaction mixture consisted of 1 .mu.L
pCR-XL-TOPO-OsMADS6-5'-gDNA miniprep DNA, 200 .mu.M dNTPs, 20 .mu.M
oligonucleotide primer OsMADS6-C1b
5'-cagtgcatgcggaccgctaggacgatggtgtgatgtg-3' (SEQ ID NO 476), 20
.mu.M oligonucleotide primer OsMADS6-Paa
5'-cctcgtcgactcgcccgatcgatcgaacg-3' (SEQ ID NO 477), 5 .mu.L
10.times. cloned Pfu buffer and 2.5 Units of Pfuturbo DNA
polymerase in a final volume of 50 .mu.L. The thermocycling program
was at 95.degree. C. for 30 seconds then 40 cycles of (95.degree.
C. for 10 seconds, 50.degree. C. for 60 seconds, 72.degree. C. for
6 minutes) then 72.degree. C. for 10 minutes. The 1.5 kb OsMADS6-Pa
DNA product was cloned with the Zero Blunt TOPO PCR cloning kit.
The pCR-Blunt-II-TOPO-OsMADS6-Pa recombinants were identified by
digesting 5 .mu.L pCR-Blunt-II-TOPO-OsMADS6-Pa miniprep DNA with
EcoRI in a 20 .mu.L reaction mixture containing 2 .mu.g BSA and 2
.mu.L 10.times. restriction endonuclease. The digests were
incubated at 37.degree. C. for 2 hours then resolved on 1% TAE
agarose. Positive pCR-Blunt-II-TOPO-OsMADS6-Pa recombinants were
sequenced.
[0151] 14 .mu.L pCR-Blunt-II-TOPO-OsMADS6-Pb miniprep DNA was
digested in a 20 .mu.L reaction mixture containing 2 .mu.g BSA, 2
.mu.L 10.times. restriction endonuclease buffer, 1 .mu.L SalI and 1
.mu.L NcoI. The digest was incubated at 37.degree. C. for more than
6 hours. The digested DNA was resolved on 1.0% TAE agarose and the
2.96 kb OsMADS6-Pb (SalI/NcoI) DNA band was excised, recovered and
ethanol precipitated with glycogen. The OsMADS6-Pb (SalI/NcoI) DNA
was recovered by micro centrifugation, washed with 70% ethanol,
dried under vacuum and resuspended in 5 .mu.L ddH.sub.2O. 2 .mu.g
pNOV6901 miniprep DNA was digested in a 20 .mu.L reaction mixture
containing 2 .mu.g BSA, 2 .mu.L 10.times. restriction endonuclease
buffer, 1 .mu.L SalI and 1 .mu.L NcoI. The digest was incubated at
37.degree. C. for more than 6 hours, then at 70.degree. C. for 20
minutes. Then 1 .mu.L of the appropriate 10.times. restriction
endonuclease buffer, 1 .mu.L 1 Unit/.mu.L CIP and 8 .mu.L
ddH.sub.2O were added to the reaction and it was further incubated
at 37.degree. C. for 30 minutes. The digested plasmid DNA was
resolved on 1.0% TAE agarose and the 4.7 kb pNOV6901
(SalI/NcoI/CIP) band was excised, recovered and ethanol
precipitated with glycogen. The pNOV6901 (SalI/NcoI/CIP) DNA was
recovered by micro centrifugation, washed with 70% ethanol, dried
under vacuum and resuspended in 5 .mu.L ddH.sub.2O.
[0152] 4.0 .mu.L OsMADS6-Pb (SalI/NcoI) was ligated to 4.0 .mu.L
pNOV6901 (SalI/NcoI/CIP) in a 10 .mu.L ligation mixture containing
1 .mu.L 10.times. T4 DNA ligase buffer and 1 .mu.L T4 DNA ligase
(400 Units/.mu.L). The ligation mixture was incubated for more than
8 hours at 16.degree. C. 5.0 .mu.L of ligation mixture was
transformed into 50 .mu.L Top10 competent cells. The recombinants
were verified by digesting 2 .mu.L pNOV6901-OsMADS6-Pb miniprep DNA
with 0.5 .mu.L SalI, 0.5 .mu.L NcoI in 10 .mu.L reaction mixtures
containing 1 .mu.g BSA and 1 .mu.L 10.times. restriction
endonuclease buffer. The digests were incubated at 37.degree. C.
for 2 hours then resolved on 1% TAE agarose. Positive
pNOV6901-OsMADS6-Pb recombinants were sequenced.
[0153] 2 .mu.g pNOV6901-OsMADS6-Pb miniprep DNA was digested in a
20 .mu.L reaction mixture containing 2 .mu.g BSA, 2 .mu.L 10.times.
restriction endonuclease buffer, 1 .mu.L SalI and 1 .mu.L SphI. The
digest was incubated at 37.degree. C. for more than 6 hours, then
at 70.degree. C. for 20 minutes. Then 1 .mu.L of the appropriate
10.times. restriction endonuclease buffer, 1 .mu.L 1 Unit/.mu.L CIP
and 8 .mu.L ddH.sub.2O were added to the reaction and it was
further incubated at 37.degree. C. for 30 minutes. 2 .mu.g
pCR-Blunt-II-TOPO-OsMADS6-Pa miniprep DNA was digested in a 20
.mu.L reaction containing 2 .mu.g BSA, 2 .mu.L 10.times.
restriction endonuclease buffer, 1 .mu.L SalI and 1 .mu.L SphI. The
digest was incubated at 37.degree. C. for more than 6 hours.
[0154] The digested plasmid DNAs, pNOV6901-OsMADS6-Pb
(SalI/SphI/CIP) and pCR-Blunt-II-TOPO-OsMADS6-Pa (SalI/SphI), were
resolved on 1.0% TAE agarose and the 7.7 kb pNOV6901-OsMADS6-Pb
(SalI/SphI/CIP) and the 1.5 kb OsMADS6-Pa (SalI/SphI) bands were
excised, recovered and ethanol precipitated with glycogen carrier.
The pNOV6901-OsMADS6-Pb (SalI/SphI/CIP) and OsMADS6-Pa (SalI/SphI)
DNA fragments were recovered by micro centrifugation, washed with
70% ethanol, dried under vacuum and each resuspended in 5 .mu.L
ddH.sub.2O.
[0155] 4.0 .mu.L pNOV6901-OsMADS6-Pb (SalI/SphI/CIP) was ligated to
4.0 .mu.L OsMADS6-Pa (SalI/SphI) in a 10 .mu.L reaction mixture
containing 1 .mu.L 10.times. T4 DNA ligase buffer and 1 .mu.L T4
DNA ligase (400 U/.mu.L). The reaction mixture was incubated for
more than 8 hours at 16.degree. C. 5.0 .mu.L of ligation mixture
was transformed into 50 .mu.L Top10 competent cells. The
pNOV6901-OsMADS6P recombinants were verified by digesting 7.5 .mu.L
pNOV6901-OsMADS6P miniprep DNA with 0.5 .mu.L SphI, 0.5 .mu.L NcoI
in 10 .mu.L reactions containing 1 .mu.g BSA and 1 .mu.L 10.times.
restriction endonuclease buffer. Digests were incubated at
37.degree. C. for 2 hours then resolved on 1% TAE agarose. Positive
pNOV6901-OsMADS6P recombinants were sequenced.
[0156] D. Construction of the OsMADS6 3'-Regulatory Sequence
[0157] The OsMADS-6 3'-regulatory sequence for the expression
cassette, about 1.3 kb, was produced by high-fidelity PCR from the
pCR-II-Blunt-OsMADS6-3'-gDNA clone, above. The reaction mixture
consisted of 1 .mu.L pCR-II-Blunt-OsMADS6-3'-gDNA miniprep DNA, 200
.mu.M dNTPs, 20 .mu.M oligonucleotide primer OsMADS6-C4b
5'-acgtgagctcgctaagcagccatcgatcag-3' (SEQ ID NO 478), 20 .mu.M
oligonucleotide primer OsMADS6-C2
5'-actgeggaccgatgccattgtgtaatgaatgg-3' (SEQ ID NO 479), 5 .mu.L
10.times. cloned Pfu buffer and 2.5 Units of Pfuturbo DNA
polymerase in a final volume of 50 .mu.L. The thermocycling program
was at 95.degree. C. for 30 seconds then 40 cycles of (95.degree.
C. for 10 seconds, 50.degree. C. for 60 seconds, 72.degree. C. for
6 minutes) then 72.degree. C. for 10 minutes. The 1.3 kb OsMADS6T
DNA product was recovered and ethanol precipitated with glycogen.
Recovered the OsMADS6T DNA by micro centrifugation, washed with 70%
ethanol, dried under vacuum and resuspended in 14 .mu.L ddH.sub.2O.
The OsMADS6T DNA was digested in a 20 .mu.L reaction mixture
containing 2 .mu.g BSA, 2 .mu.L 10.times. restriction endonuclease
buffer, and 2 .mu.L SmaI. The digest was incubated at 37.degree. C.
for more than 6 hours. The OsMADS6T (SmaI) DNA was resolved on 1.0%
TAE agarose and the 1.3 kb OsMADS6T (Sural) band was excised,
recovered and ethanol precipitated with glycogen carrier. The
OsMADS6T (SmaI) DNA was recovered by micro centrifugation, washed
with 70% ethanol, dried under vacuum and resuspended in 5 .mu.L
ddH.sub.2O.
[0158] 2 .mu.g pNOV6901-OsMADS6P miniprep DNA was digested in a 20
.mu.L reaction mixture containing 2 .mu.g BSA, 2 .mu.L 10.times.
restriction endonuclease buffer and 2 .mu.L SmaI. The digest was
incubated at 37.degree. C. for more than 6 hours, then at
70.degree. C. for 20 minutes. Then 1 .mu.L of the appropriate
10.times. restriction endonuclease buffer, 1 .mu.L 1 Unit/.mu.L CIP
and 8 .mu.L ddH.sub.2O were added to the reaction and it was
further incubated at 37.degree. C. for 30 minutes. The
pNOV6901-OsMADS6P (SmaI/CIP) DNA was resolved on 1.0% TAE agarose
and the 9.7 kb pNOV6901-OsMADS6P (SmaI/CIP) band was excised,
recovered and ethanol precipitated with glycogen carrier. The
pNOV6901-OsMADS6P (SmaI/CIP) DNA fragment was recovered by micro
centrifugation, washed with 70% ethanol, dried under vacuum and
resuspended in 5 .mu.L ddH.sub.2O.
[0159] 4.0 .mu.L pNOV6901-OsMADS6P (SmaI/CIP) was ligated to 4.0
.mu.L OsMADS6T (SmaI) in a 10 .mu.L reaction mixture containing 1
.mu.L 10.times. T4 DNA ligase buffer and 1 .mu.L T4 DNA ligase (400
Units/.mu.L). The reaction mixture was incubated more than 8 hours
at 16.degree. C. 5.0 .mu.L of ligation mixture was transformed into
50 .mu.L Top10 competent cells. The recombinants were verified by
digesting 2 .mu.L pNOV6901-OsMADS6P/OsMADS6T miniprep DNA with 1.0
.mu.L RsrII in 10 .mu.L reaction mixtures containing 1 .mu.g BSA
and 1 .mu.L 10.times. restriction endonuclease buffer. The digests
were incubated at 37.degree. C. for 2 hours then resolved on 1% TAE
agarose. Positive pNOV6901-OsMADS6P/OsMADS6T recombinants were
sequenced. Designated the vector pNOV6901-OsMADS6P/OsMADS6T. The
plasmid's QC number is 11082. 11082 contains the OsMADS6 expression
cassette depicted by SEQ ID NO. 537.
[0160] E. Mobilization of the OsMADS6 GUS Expression Cassette into
pNOV6900
[0161] 2 .mu.g pNOV6900 was digested in a 20 .mu.L reaction mixture
containing 2 .mu.g BSA, 2 .mu.L 10.times. restriction endonuclease
buffer and 2 .mu.L RsrII. The digest was incubated at 37.degree. C.
for more than 6 hours, then at 70.degree. C. for 20 minutes. Then 1
.mu.L of the appropriate 10.times. restriction endonuclease buffer,
1 .mu.L 1 Unit/.mu.L CIP and 8 .mu.L ddH.sub.2O were added to the
reaction and it was further incubated at 37.degree. C. for 30
minutes. 2 .mu.g pNOV6901-OsMADS6P/OsMADS6T miniprep DNA was
digested in a 20 .mu.L reaction mixture containing 2 .mu.g BSA, 2
.mu.L 10.times. restriction endonuclease buffer and 2 .mu.L RsrII.
The digest was incubated at 37.degree. C. for more than 6
hours.
[0162] The pNOV6900 (RsrII/CIP) and the pNOV6901-OsMADS6P/OsMADS6T
(RsrII) plasmid DNAs were resolved on 1.0% TAE agarose, and the 9.2
kb pNOV6900 (RsrII/CIP) and the 8.0 kb pNOV6901-OsMADS6P/OsMADS6T
(RsrII) bands were excised, recovered and ethanol precipitated with
glycogen carrier. The pNOV6900 (RsrII/CIP) and
pNOV6901-OsMADS6P/OsMADS6T (RsrII) DNA fragments were recovered by
micro centrifugation, washed with 70% ethanol, dried under vacuum
and resuspended in 5 .mu.L ddH.sub.2O each.
[0163] 4.0 .mu.L pNOV6900 (RsrII/CIP) was ligated to 4.0 .mu.L
pNOV6901-OsMADS6P/OsMADS6T (RsrII) in a 10 .mu.L ligation mixture
containing 1 .mu.L 10.times. T4 DNA ligase buffer and 1 .mu.L T4
DNA ligase (400 U/.mu.L). The ligation mixture was incubated more
than 8 hours at 16.degree. C. 5.0 .mu.L of ligation mixture was
transformed into 50 .mu.L Top10 competent cells. The
pNOV6900-pNOV6901-OsMADS6P/OsMADS6T recombinants were verified by
digesting 2 .mu.L miniprep DNA with 1.0 .mu.L NcoI in 10 .mu.L
reaction mixtures containing 1 mg BSA and 1 .mu.L 10.times.
restriction endonuclease buffer. The digests were incubated at
37.degree. C. for 2 hours then resolved on 1% TAE agarose. Positive
pNOV6900-pNOV6901-OsMADS6P/OsMADS6T recombinants were sequenced.
The finished clone was designated pNOV6907. The plasmid's QC number
is 11083.
[0164] The engineered alterations in the 5'-regulatory sequence
derived from the OsMADS6 gene include introduction of an SphI site
followed by an RsrII site at the 5'-end of OsMADS6P, elimination of
the natural translation start codon in OsMADS6P, elimination of
undesired open reading frames in the new 5'-untranslated leader
sequence transcribed from OsMADS6P, insertion of a Kozak sequence
upstream of the new translation start codon in OsMADS6P and
insertion of the new translation start codon downstream of the
intron1/exon2 junction in OsMADS6P as an NcoI site. The engineered
alterations in the 3'-gene regulatory sequence derived from the
OsMADS6 gene include introduction of a SacI site at the 5'-terminus
of OsMADS6T and introduction of an RsrII site at the 3'-terminus of
OsMADS6T. In this configuration the GUS coding sequence can be
replaced with any gene of interest flanked by NcoI/NotI or
NcoI/SacI restriction sites. The complete cassette is mobilized, as
an RsrII fragment, to the binary vector pNOV6900.
[0165] The cassette was transformed into A188 .times. HyII maize
and Kaybonnet rice using standard agrobacterium mediated
methodology.
[0166] GUS Expression in T0 Maize
[0167] One hundred-two T0 transgenic maize lines were generated.
Tassel spikelets were histochemically screened for GUS activity.
Sixty-four events were positive for GUS activity in the tassel
glume, and some also stained positive at the spikelet base.
Fifty-six also showed GUS expression in leaf punches. Ears from
several plants were sacrificed to examine GUS expression in
developing florets. GUS activity localizes primarily to vascular
bundles in developing ears, which appears connected to transmitting
tissue in each floret. These data indicate the cassette drives GUS
expression primarily in female reproductive tissue.
[0168] GUS Expression in T0 Rice
[0169] Forty-one T0 rice lines containing pNOV6907 were generated.
Twenty independent transformants were histochemically stained for
GUS expression. Light to strong GUS activity was detected in leaf
tissue. In most events, activity in spikelets localized to glumes.
Staining intensity varied significantly. Seed were collected for
each line, but were not further analyzed.
[0170] GUS Expression in T1 Maize
[0171] T1 progeny from two independent transformants were sown and
analyzed in detail for GUS expression. There was no detectable GUS
expression in silk, leaf and tassel. This indicates tassel and leaf
expression observed in T0 plants may result from tissue culture
associated with the transformation process. Dissected organs from
T1 tassel spikelets had no apparent GUS activity (data not shown).
GUS activity is seen in the ear node and the developing ear shoot.
Residual GUS activity is seen in the central pith, and most
activity in the developing ear shoot. Ear activity is confined to
the node, the outer whorls and the central region. The pith beneath
the ear node has no detectable GUS activity. GUS activity is seen
in ears from 8 to 2 days prior to pollination. As in T0 ears,
activity is confined to the, florets and transmitting tissue.
Post-pollination GUS activity remains confined to the same tissues.
Activity in developing kernels appears restricted to maternal
tissue. This pattern persists through kernel development. No
activity is detected in the endosperm or developing embryo, it
localizes to the placental, funicular and hilar regions of
developing kernels. GUS protein is detectable throughout ovule and
kernel development, up to 20 days after pollination. These data
support the OsMADS6 cassette as a very good candidate for trait
expression in developing florets and kernels. When driven by the
OsMADS6-based expression cassette, genes that facilitate phloem
unloading such as invertase or a sucrose transporter should prove
effective in supporting early ear development by increasing sink
strength. There is very light or no staining in the aerial tissue,
with no GUS activity in the roots
[0172] One embodiment of the invention is an expression cassette
based on the Oryza sativa OsMADS6 gene. The expression cassette
consists of the gene's promoter including the first intron, 5'-UTR,
3'-UTR and 3'-nontranscribed sequence. These components were
assembled into a GUS expression cassette and tested in transgenic
plants. The cassette's design facilitates replacement of the GUS
coding sequence with any gene of interest. The expression cassette
drives gene expression primarily in maternal reproductive tissue.
Within developing ears, expression localizes to florets, maternal
components of developing kernels, the placental or transmitting
tissue and vasculature. The expression cassettes of the present
invention further drive gene expression from a very early point in
ovule development, perhaps from shortly after differentiation.
EXAMPLE 5
Construction of the OsMADS8 Expression Cassette
[0173] A. Cloning of the OsMADS8 5'-Regulatory Sequence
[0174] The OsMADS8 5'-regulatory sequence from rice genomic DNA
(gDNA) was amplified using high-fidelity PCR. The 50 .mu.L reaction
mixture consisted of 100 ng rice gDNA, 200 .mu.M dNTPs, 1 .mu.L 20
.mu.M oligonucleotide primer OsMADS8.P1
5'-ggtatctttccaaagttctggtcatgctgc-3' (SEQ ID NO 522), 1 .mu.L 20
.mu.M oligonucleotide primer OsMADS8.P2
5'-ccattttttgcgaaatgccaaatcctggc-3' (SEQ ID NO 523), 1 .mu.L
10.times. Expand High Fidelity buffer and 1 .mu.L Expand High
Fidelity polymerase. The thermocycling program was at 95.degree. C.
for 2 minutes followed by 40 cycles of (94.degree. C. for 15
seconds, 68.degree. C. for 7.5 minutes) followed by 68.degree. C.
for 10 minutes. The 5.2 kb OsMADS8-5'-gDNA DNA product was cloned
with the TOPO XL PCR cloning kit. The pCR-XL-TOPO-OsMADS8-5'-gDNA
was identified by digesting 5 .mu.L pCR-XL-TOPO-OsMADS8-5'-gDNA
miniprep DNA with EcoRI in a 20 .mu.L reaction mixture containing 2
.mu.g BSA and 2 .mu.L 10.times. restriction endonuclease buffer.
The digests were incubated at 37.degree. C. for 2 hours then
resolved on 1% TAE agarose. Positive pCR-XL-TOPO-OsMADS8-5'-gDNA
clones were sequenced.
[0175] B. Cloning of the OsMADS8 3'-Regulatory Sequence
[0176] The OsMADS8 3'-regulatory sequence from rice genomic DNA
(gDNA) was amplified using high-fidelity PCR. The 50 .mu.L reaction
mixture consisted of 100 ng rice gDNA, 200 .mu.M dNTPs, 1 .mu.L 20
.mu.M oligonucleotide primer OsMADS8.T1
5'-acgtgagetcactectgaaggccgatgcgacaacc-3' (SEQ ID NO 480), 1 .mu.L
20 .mu.M oligonucleotide primer OsMADS8.T2
5'-agtcatcgatcatgacaaaatatcatgtttatttcgagg-3' (SEQ ID NO 481), 1
.mu.L 10.times. Expand High Fidelity buffer and 1 .mu.L Expand High
Fidelity polymerase. The thermocycling program was 95.degree. C.
for 2 minutes followed by 40 cycles of (94.degree. C. for 15
seconds, 60.degree. C. for 30 seconds, 68.degree. C. for 6 minutes)
followed by 68.degree. C. for 15 minutes. Cloned the 2.04 kb
OsMADS8-3'-gDNA DNA product with the Zero Blunt TOPO PCR cloning
kit. The pCR-Blunt-II-OsMADS8-3'-gDNA recombinants were identified
by digesting 5 .mu.L miniprep DNA with EcoRI in a 20 .mu.L reaction
mixture containing 2 .mu.g BSA and 2 .mu.L 10.times. restriction
endonuclease buffer. The digests were incubated at 37.degree. C.
for 2 hours then resolved on 1% TAE agarose. Positive
pCR-Blunt-II-OsMADS8-3'-gDNA clones were sequenced.
[0177] C. Construction of the OsMADS8 5'-Regulatory Sequence
[0178] The OsMADS8 5'-regulatory sequence for the expression
cassette was made in several steps. The 3'-half (OsMADS-8Pb, about
2.8 kb) was produced by high-fidelity PCR from
pCR-XL-TOPO-OsMADS8-5'-gDNA, above. The reaction mixture consisted
of 1 .mu.L pCR-XL-TOPO-OsMADS8-5'-gDNA miniprep DNA, 200 .mu.M dNTP
mixture, 20 .mu.M oligonucleotide primer OsMADS8-Pcc
5'-atcgccatggtggtcaagctgcaagtttcaaaaacac-3' (SEQ ID NO 482), 20
.mu.M oligonucleotide primer OsMADS8-C3
5'-acgtgtcgacgagagggagggtgga-3' (SEQ ID NO 483), 5 .mu.L 10.times.
cloned Pfu buffer and 2.5 Units of Pfuturbo DNA polymerase in a
final volume of 50 .mu.L. The thermocycling program was at
95.degree. C. for 30 seconds then 40 cycles of (95.degree. C. for
10 seconds, 50.degree. C. for 60 seconds, 72.degree. C. for 6
minutes) then 72.degree. C. for 10 minutes. The 2.8 kb OsMADS-8Pb
DNA product was cloned with the Zero Blunt TOPO PCR cloning kit.
The pCR-Blunt-II-TOPO-OsMADS-8Pb recombinants were identified by
digesting 5 .mu.L pCR-Blunt-II-TOPO-OsMADS-8Pb miniprep DNA with
EcoRI in a 20 .mu.L reaction mixture containing 2 .mu.g BSA and 2
.mu.L 10.times. restriction endonuclease buffer. Digests were
incubated at 37.degree. C. for 2 hours then resolved on 1% TAE
agarose. Positive pCR-Blunt-II-TOPO-OsMADS-8Pb clones were
sequenced.
[0179] The 5'-half (OsMADS-8Pa, about 2.4 kb) was produced by
high-fidelity PCR from pCR-XL-TOPO-OsMADS8-5'-gDNA, above. The
reaction mixture consisted of 1 .mu.L pCR-XL-TOPO-OsMADS8-5'-gDNA
miniprep DNA, 200 .mu.M dNTP mixture, 20 .mu.M oligonucleotide
primer OsMADS8-05b 5'-tcctcctcctcctcctccacctcacct-3' (SEQ ID NO
484), 20 .mu.M oligonucleotide primer OsMADS8-C1b
5'-aactaaatcgcctgcaggeggaccgttttttgcgaaatgcc-3' (SEQ ID NO 485), 5
.mu.L 10.times. cloned Pfu buffer and 2.5 Units of Pfuturbo DNA
polymerase in a final volume of 50 .mu.L. The thermocycling program
was at 95.degree. C. for 30 seconds then 40 cycles of (95.degree.
C. for 10 seconds, 50.degree. C. for 60 seconds, 72.degree. C. for
6 minutes) then 72.degree. C. for 10 minutes. The 2.4 kb OsMADS-8Pa
DNA product was cloned with the Zero Blunt TOPO PCR cloning kit.
The pCR-Blunt-II-TOPO-OsMADS-8Pa recombinants were identified by
digesting 5 .mu.L pCR-Blunt-II-TOPO-OsMADS-8Pa miniprep DNA with
EcoRI in a 20 .mu.L reaction mixture containing 2 .mu.g BSA and 2
.mu.L 10.times. restriction endonuclease buffer. Digests were
incubated at 37.degree. C. for 2 hours then resolved on 1% TAE
agarose. Positive pCR-Blunt-II-TOPO-OsMADS-8Pb clones were
sequenced.
[0180] 14 .mu.L pCR-Blunt-II-TOPO-OsMADS-8Pb miniprep DNA was
digested in a 20 .mu.L reaction mixture containing 2 .mu.g BSA, 2
.mu.L 10.times. restriction endonuclease buffer, 1 .mu.L SalI and 1
.mu.L NcoI. The digest was incubated at 37.degree. C. for more than
6 hours. The pCR-Blunt-II-TOPO-OsMADS-8Pb (SalI/NcoI) DNA was
resolved on 1.0% TAE agarose and the 2.96 kb OsMADS-8Pb (SalI/NcoI)
band was excised, recovered and ethanol precipitated with glycogen
carrier. OsMADS-8Pb (SalI/NcoI) DNA was recovered by micro
centrifugation, washed with 70% ethanol, dried under vacuum and
resuspended in 5 .mu.L ddH.sub.2O. 2 .mu.g pNOV6901 miniprep DNA
was digested in a 20 .mu.L reaction mixture containing 2 .mu.g BSA,
2 .mu.L 10.times. restriction endonuclease buffer, 1 .mu.L SalI and
1 .mu.L NcoI. The digest was incubated at 37.degree. C. for more
than 6 hours, then at 70.degree. C. for 20 minutes. Then 1 .mu.L of
the appropriate 10.times. restriction endonuclease buffer, 1 .mu.L
1 Unit/.mu.L CIP and 8 .mu.L ddH.sub.2O were added to the reaction
and it was further incubated at 37.degree. C. for 30 minutes. The
pNOV6901 (SalI/NcoI/CIP) plasmid DNA was resolved on 1.0% TAE
agarose and the 4.7 kb pNOV6901 (SalI/NcoI/CIP) DNA band was
excised, recovered and ethanol precipitated with glycogen carrier.
The pNOV6901 (SalI/NcoI/CIP) DNA was recovered by micro
centrifugation, washed with 70% ethanol, dried under vacuum and
resuspended in 5 .mu.L ddH.sub.2O.
[0181] 4.0 .mu.L OsMADS-8Pb (SalI/NcoI) was ligated to 4.0 .mu.L
pNOV6901 (SalI/NcoI/CIP) in a 10 .mu.L reaction mixture containing
1 .mu.L 10.times. T4 DNA ligase buffer and 1 .mu.L T4 DNA ligase
(400 Units/.mu.L), which was incubated more than 8 hours at
16.degree. C. 5.0 .mu.L of ligation mixture was transformed into 50
.mu.L Top10 competent cells. The pNOV6901-OsMADS-8Pb recombinants
were verified by digesting 2 .mu.L pNOV6901-OsMADS-8Pb miniprep DNA
with 0.5 .mu.L SalI, 0.5 .mu.L NcoI in 10 .mu.L reaction mixtures
containing 1 .mu.g BSA and 1 .mu.L 10.times. restriction
endonuclease buffer. The digests were incubated at 37.degree. C.
for 2 hours then resolved on 1% TAE agarose. Positive
pNOV6901-OsMADS-8Pb recombinants were sequenced.
[0182] An SbfI restriction site was introduced to
pNOV6901-OsMADS-8Pb by ligating Synthetic Adapter III to the
construct. Synthetic Adapter III was made by combining 40 .mu.L of
50 .mu.M oligonucleotide 8PA-1 5'-Pggagatcggg-3' (SEQ ID NO 486),
40 .mu.L of 50 .mu.M oligonucleotide 8PA-2 5'-Ptcgacccgatcacc-3'
(SEQ ID NO 487)--where P is a 5'-phosphate group--in a 100 .mu.L
mixture that is 25 mM in Tris-HCl (pH 8.0) and 10 mM in MgCl.sub.2.
The mixture was boiled for 5 minutes, removed from heat and
naturally cooled to room temperature (about 60 minutes). This
yielded a 20 .mu.M Synthetic Adapter III mixture.
[0183] pNOV6901-OsMADS-8Pb was prepared by digesting 14 .mu.L
pNOV6901-OsMADS-8Pb miniprep DNA with 2 .mu.L SalI in a 20 .mu.L
reaction mixture containing 2 .mu.g BSA and 2 .mu.L 10.times.
restriction endonuclease buffer. The digest was incubated at
37.degree. C. for 6 hours, then at 70.degree. C. for 20 minutes.
Then 1 .mu.L of the appropriate 10.times. restriction endonuclease
buffer, 1 .mu.L 1 Unit/.mu.L CIP and 8 .mu.L ddH.sub.2O were added
to the reaction and it was further incubated at 37.degree. C. for
30 minutes. The pNOV6901-OsMADS-8Pb (SalI/CIP) DNA was resolved on
1% TAE agarose, excised, recovered and ethanol precipitated with
glycogen carrier. The pNOV6901-OsMADS-8Pb (SalI/CIP) DNA was
recovered by micro centrifugation, washed with 70% ethanol, dried
under vacuum and resuspended in 5 .mu.L ddH.sub.2O.
[0184] 4.5 .mu.L Synthetic Adapter III mixture was ligated to 2.5
.mu.L pNOV6901-OsMADS-8Pb (SalI/CIP) in a 10 .mu.L ligation mixture
containing 1 .mu.L 10.times. T4 DNA ligase buffer and 1 .mu.L T4
DNA ligase (400 U/.mu.L), which was incubated more than 8 hours at
16.degree. C. 4 .mu.L of ligation mixture was transformed into 50
.mu.L XL-1 supercompetent cells (Stratagene, Cat. No. 200236). The
pNOV6901-OsMADS-8Pb-SbfI recombinants were verified by digesting
7.5 .mu.L pNOV6901-OsMADS-8Pb-SbfI miniprep DNA in a 10 .mu.L
reaction mixture containing 1 .mu.g BSA, 1 .mu.L 10.times.
restriction endonuclease buffer and 1 .mu.L SalI. The digests were
incubated at 37.degree. C. for 2 hours then resolved on 1.0% TAE
agarose. The pNOV6901-OsMADS-8Pb-SbfI recombinants that lost the
SalI restriction site were digested with SbfI in a 10 .mu.L
reaction mixture containing 1 .mu.g BSA, 1 .mu.L 10.times. SEBuffer
Y restriction endonuclease buffer and 1 .mu.L SbfI (New England
Biolabs). The digests were incubated at 37.degree. C. for 2 hours
then resolved on 1.0% TAE agarose. Positive
pNOV6901-OsMADS-8Pb-SbfI recombinants were sequenced.
[0185] 2 .mu.g pNOV6901-OsMADS-8Pb-SbfI miniprep DNA was digested
in a 20 .mu.L reaction mixture containing 2 .mu.g BSA, 2 .mu.L
10.times. restriction endonuclease buffer and 2 .mu.L SbfI. The
digest was incubated at 37.degree. C. for more than 6 hours, then
at 70.degree. C. for 20 minutes. Then 1 .mu.L of the appropriate
10.times. restriction endonuclease buffer, 1 .mu.L 1 Unit/.mu.L CIP
and 8 .mu.L ddH.sub.2O were added to the reaction and it was
further incubated at 37.degree. C. for 30 minutes. 2 .mu.g
pCR-Blunt-II-TOPO-OsMADS-8Pa miniprep DNA was digested in a 20
.mu.L reaction mixture containing 2 .mu.g BSA, 2 .mu.L 10.times.
restriction endonuclease buffer and 2 .mu.L SbfI. The digest was
incubated at 37.degree. C. for more than 6 hours.
[0186] The digested plasmid DNAs, pNOV6901-OsMADS-8Pb-SbfI
(SbfI/CIP) and pCR-Blunt-II-TOPO-OsMADS-8Pa (SbfI), were resolved
on 1.0% TAE agarose and the 7.5 kb pNOV6901-OsMADS-8Pb (SbfI/CIP)
and the 2.4 kb OsMADS-8Pa (SbfI) bands were excised, recovered and
ethanol precipitated with glycogen carrier. The DNA fragments were
recovered by micro centrifugation, washed with 70% ethanol, dried
under vacuum and each resuspended in 5 .mu.L ddH.sub.2O.
[0187] 4.0 .mu.L pNOV6901-OsMADS-8Pb (SbfI/CIP) was ligated to 4.0
.mu.L OsMADS-8Pa (SbfI) in a 10 .mu.L ligation mixture containing 1
.mu.L 10.times. T4 DNA ligase buffer and 1 .mu.L T4 DNA ligase (400
U/.mu.L), which was incubated for more than 8 hours at 16.degree.
C. 5.0 .mu.L of ligation mixture was transformed into 50 .mu.L
Top10 competent cells. The pNOV6901-OsMADS-8P recombinants were
verified by digesting 7.5 .mu.L pNOV6901-OsMADS-8P miniprep DNA
with 0.5 .mu.L SbfI, 0.5 .mu.L NcoI in 10 .mu.L reaction mixtures
containing 1 .mu.g BSA and 1 .mu.L 10.times. restriction
endonuclease buffer. The digests were incubated at 37.degree. C.
for 2 hours then resolved on 1% TAE agarose. Positive
pNOV6901-OsMADS-8P recombinants were sequenced.
[0188] D. Construction of the OsMADS8 3'-Regulatory Sequence
[0189] The OsMADS-8 3'-regulatory sequence for the expression
cassette, about 2.1 kb, was produced by high-fidelity PCR from the
pCR-Blunt-II-OsMADS8-3'-gDNA clone, above. The reaction mixture
consisted of 1 .mu.L pCR-Blunt-II-OsMADS8-3'-gDNA miniprep DNA, 200
.mu.M dNTP mixture, 20 .mu.M oligonucleotide primer OsMADS8-C2
5'-acgtcccgggcggaccgagtcatcgatcatgac-3' (SEQ ID NO 488), 20 .mu.M
oligonucleotide primer OsMADS8-C4
5'-tcgagcggccgcaggccgatgcgacaaccaataaaaac-3' (SEQ ID NO 489), 5
.mu.L 10.times. cloned Pfu buffer and 2.5 Units of Pfuturbo DNA
polymerase (Stratagene, Cat. No. 600252) in a final volume of 50
.mu.L. The thermocycling program was 95.degree. C. for 30 seconds
then 40 cycles of (95.degree. C. for 10 seconds, 50.degree. C. for
60 seconds, 72.degree. C. for 6 minutes) then 72.degree. C. for 10
minutes. The OsMADS-8T DNA product was recovered using the QIAquick
PCR purification kit. The recovered OsMADS-8T DNA was ethanol
precipitated with glycogen carrier. The OsMADS-8T DNA was recovered
by micro centrifugation, washed with 70% ethanol, dried under
vacuum and resuspended in 14 .mu.L ddH.sub.2O. The OsMADS-8T DNA
was digested in a 20 .mu.L reaction mixture containing 2 .mu.g BSA,
2 .mu.L 10.times. restriction endonuclease buffer, 1 .mu.L NotI and
1 .mu.L XmaI. The digest was incubated at 37.degree. C. for more
than 6 hours. The OsMADS-8T (NotI/XmaI) DNA was resolved on 1.0%
TAE agarose, excised, recovered and ethanol precipitated with
glycogen carrier. The OsMADS-8T (NotI/XmaI) DNA was recovered by
micro centrifugation, washed with 70% ethanol, dried under vacuum
and resuspended in 5 .mu.L ddH.sub.2O.
[0190] 2 .mu.g pNOV6901-OsMADS-8P miniprep DNA was digested in a 20
.mu.L reaction mixture containing 2 .mu.g BSA, 2 .mu.L 10.times.
restriction endonuclease buffer, 1 .mu.L NotI and 1 .mu.L XmaI. The
digest was incubated at 37.degree. C. for more than 6 hours, then
at 70.degree. C. for 20 minutes. Then 1 .mu.L of the appropriate
10.times. restriction endonuclease buffer, 1 .mu.L 1 Unit/.mu.L CIP
and 8 .mu.L ddH.sub.2O were added to the reaction and it was
further incubated at 37.degree. C. for 30 minutes. The
pNOV6901-OsMADS-8P (NotI/XmaI/CIP) plasmid DNA was resolved on 1.0%
TAE agarose and the 9.9 kb pNOV6901-OsMADS-8P band was excised,
recovered and ethanol precipitated with glycogen carrier. The
pNOV6901-OsMADS-8P (NotI/XmaI/CIP) DNA fragment was recovered by
micro centrifugation, washed with 70% ethanol, dried under vacuum
and resuspended in 5 .mu.L ddH.sub.2O.
[0191] 4.0 .mu.L of the pNOV6901-OsMADS-8P (NotI/XmaI/CIP) was
ligated to 4.0 .mu.L OsMADS-8T (NotI/XmaI) in a 10 .mu.L ligation
mixture containing 1 .mu.L 10.times. T4 DNA ligase buffer and 1
.mu.L T4 DNA ligase (400 Units/.mu.L), which was incubated more
than 8 hours at 16.degree. C. 5.0 .mu.L of ligation mixture was
transformed into 50 .mu.L Top10 competent cells.
pNOV6901-OsMADS-8P/OsMADS-8T recombinants were verified by
digesting 2 .mu.L pNOV6901-OsMADS-8P/OsMADS-8T miniprep DNA with
1.0 .mu.L RsrII in 10 .mu.L reaction mixtures containing 1 .mu.g
BSA and 1 .mu.L 10.times. restriction endonuclease buffer. The
digests were incubated at 37.degree. C. for 2 hours then resolved
on 1% TAE agarose. Positive pNOV6901-OsMADS-8P/OsMADS-8T
recombinants were sequenced. The finished clone was designated
pNOV6901-OsMADS-8P/OsMADS-8T. The plasmid's QC number is 11170.
11170 contains the complete OSMADS8 expression cassette depicted by
SEQ ID NO. 538.
[0192] E. Mobilization of the OsMADS8 GUS Expression Cassette into
pNOV6900
[0193] 2 .mu.g pNOV6900 was digested in a 20 .mu.L reaction mixture
containing 2 .mu.g BSA, 2 .mu.L 10.times. restriction endonuclease
buffer and 2 .mu.L RsrII. The digest was incubated at 37.degree. C.
for more than 6 hours, then at 70.degree. C. for 20 minutes. Then 1
.mu.L of the appropriate 10.times. restriction endonuclease buffer,
1 .mu.L 1 Unit/.mu.L CIP and 8 .mu.L ddH.sub.2O were added to the
reaction and it was further incubated at 37.degree. C. for 30
minutes. 2 .mu.g pNOV6901-OsMADS-8P/OsMADS-8T miniprep DNA was
digested in a 20 .mu.L reaction mixture containing 2 .mu.g BSA, 2
.mu.L 10.times. restriction endonuclease buffer and 2 .mu.L RsrII.
The digest was incubated the reaction at 37.degree. C. for more
than 6 hours.
[0194] The digested plasmid DNAs, pNOV6900 (RsrII/CIP) and
pNOV6901-OsMADS-8P/OsMADS-8T (RsrII), were resolved on 1.0% TAE
agarose and the 9.2 kb pNOV6900 (RsrII/CIP) and the 9.5 kb
pNOV6901-OsMADS-8P/OsMADS-8T (RsrII) bands were excised, recovered
and ethanol precipitated with glycogen carrier. The pNOV6900
(RsrII/CIP) and the pNOV6901-OsMADS-8P/OsMADS-8T (RsrII) DNA
fragments were recovered by micro centrifugation, washed with 70%
ethanol, dried under vacuum and each resuspended in 5 .mu.L
ddH.sub.2O.
[0195] 4.0 .mu.L of pNOV6900 (RsrII/CIP) was ligated to 4.0 .mu.L
pNOV6901-OsMADS-8P/OsMADS-8T (RsrII) in a 10 .mu.L ligation mixture
containing 1 .mu.L 10.times. T4 DNA ligase buffer and 1 .mu.L T4
DNA ligase (400 U/.mu.L). The ligation mixture was incubated more
than 8 hours at 16.degree. C. 5.0 .mu.L of ligation mixture was
transformed into 50 .mu.L Top10 competent cells. The
pNOV6900-pNOV6901-OsMADS-8P/OsMADS-8T recombinants were verified by
digesting 7.5 .mu.L pNOV6900-pNOV6901-OsMADS-8P/OsMADS-8T miniprep
DNA with 1.0 .mu.L NcoI in 10 .mu.L reaction mixtures containing 1
.mu.g BSA and 1 .mu.L 10.times. restriction endonuclease buffer.
The digests were incubated at 37.degree. C. for 2 hours then
resolved on 1% TAE agarose. Positive
pNOV6900-pNOV6901-OsMADS-8P/OsMADS-8T recombinants were sequenced.
The finished clone was designated pNOV6909. The plasmid's QC number
is 11171.
[0196] The engineered alterations in the 5'-regulatory sequence
derived from the OsMADS8 gene include introduction of an SbfI site
followed by an RsrII site at the 5'-end of OsMADS8P, elimination of
the natural translation start codon in OsMADS8P, elimination of
undesired open reading frames in the new 5'-untranslated leader
sequence transcribed from OsMADS8P, insertion of a Kozak sequence
upstream of the new translation start codon in OsMADS8P and
insertion of the new translation start codon downstream of the
intron1/exon2 junction in OsMADS8P as an NcoI site. The engineered
alterations in the 3'-gene regulatory sequence derived from the
OsMADS8 gene include introduction of a NotI site at the 5'-terminus
of OsMADS8T and introduction of an RsrII site at the 3'-terminus of
OsMADS8T. In this configuration the GUS coding sequence can be
replaced with any gene of interest flanked by NcoI/NotI restriction
sites. The complete cassette is mobilized, as an RsrII fragment, to
the binary vector pNOV6900.
[0197] The cassette was transformed into A188 .times. HyII maize
and Kaybonnet rice using standard agrobacterium mediated
methodology.
[0198] GUS Expression in T0 Maize
[0199] Forty T0 transgenic maize lines were generated. Tassel
spikelets were histochemically screened for GUS activity.
Twenty-nine events were positive for GUS activity. Thirteen also
showed GUS expression in leaf punches. In general, the pattern
revealed detectable GUS activity in tassels and little of no
activity in leaf punches. The ear from one plant reflecting this
pattern was sacrificed to examine GUS expression. Strong GUS
expression is evident throughout the ear. These data indicate the
cassette drives GUS expression primarily in female reproductive
tissue.
[0200] GUS Expression in T0 Rice
[0201] Of thirty-six T0 rice lines containing pNOV6909, thirty-two
independent transformants were histochemically stained for GUS
expression. No GUS activity was detected in leaf tissue. In most
events, activity localized to panicles and could be seen in anthers
or the carpel base. Staining intensity varied significantly.
[0202] GUS Expression in T1 Maize
[0203] T1 progeny from four independent transformants were sown and
analyzed in detail for GUS expression. There was no detectable GUS
expression in tassels, leaf tissue, developing silk or shoots. This
indicates tassel and leaf expression observed in T0 plants may
result from tissue culture associated with the transformation
process. Dissected organs from T1 tassels indicated no apparent GUS
expression (data not shown). There is no GUS activity in the node
attached to the developing ear shoot. The node below this also has
no detectable GUS activity, but there is distinct activity in
florets on the arrested ear. The expression cassette is activated
very early in floret development. GUS activity is seen in the
central pith and florets of the developing ear before pollination.
This pattern persists from 5 days before pollination to one day
before pollination. Central pith expression persists up to 1 day
before pollination, after which GUS activity is no longer detected
in this zone. There is some GUS activity in the ear's outer
vasculature and the floret's transmitting tissue from the day of
pollination to 1 day after pollination. Afterwards, GUS activity is
detected only in the maternal components of developing kernels. GUS
protein is detectable throughout ovule and kernel development, up
to 20 days after pollination. These data support the OsMADS8
cassette as a very good candidate for trait expression in
developing florets. When driven by the OsMADS8-based expression
cassette, genes that facilitate phloem unloading such as invertase
or a sucrose transporter should prove effective in supporting early
ear development by increasing sink strength. There is very light
staining in the aerial tissue, with no GUS activity in the
roots
[0204] In summary, the present invention includes expression
cassettes based on the Oryza sativa OsMADS8 gene. It consists of
the gene's promoter including the first intron, 5'-UTR, 3'-UTR and
3'-nontranscribed sequence. The cassette's design facilitates
replacement of the GUS coding sequence with any gene of interest.
The cassette targets gene expression primarily to developing
florets and kernels, and the placental tissue beneath each floret.
Post-fertilization, expression is detected in the aleurone, hilar
region and pedicel. Developmentally, the cassette should drive gene
expression from a very early point in ovule development, perhaps
from shortly after differentiation.
EXAMPLE 6
Construction of the OsMADS 13 Expression Cassette
[0205] A. Cloning of the OsMADS13 5'-Regulatory Sequence
[0206] The OsMADS13 5'-regulatory sequence from rice genomic DNA
(gDNA) was amplified using high-fidelity PCR. The 50 .mu.L reaction
mixture consisted of 100 ng rice gDNA, 200 .mu.M dNTPs, 1 .mu.L 20
.mu.M oligonucleotide primer OsMADS13-C1
5'-gactgcatgcggaccgttccaaaattaagcacacacatttg-3' (SEQ ID NO 490), 1
.mu.L 20 .mu.M oligonucleotide primer OsMADS13-C2
5'-gactccatggcttcttgctctcaactgatcaac-3' (SEQ ID NO 491), 1 .mu.L
10.times. Expand High Fidelity buffer and 1 .mu.L Expand High
Fidelity polymerase. The thermocycling program was at 95.degree. C.
for 2 minutes followed by 40 cycles of (94.degree. C. for 15
seconds, 68.degree. C. for 7.5 minutes) followed by 68.degree. C.
for 10 minutes. The 1.9 kb OsMADS13-5'-gDNA DNA fragment was
recovered and ethanol precipitated with glycogen carrier. The
OsMADS13-5'-gDNA fragment was recovered by micro centrifugation,
washed with 70% ethanol, dried under vacuum and resuspended in 14
.mu.L ddH.sub.2O. The OsMADS13-5'-gDNA fragment was digested in a
20 .mu.L reaction mixture containing 2 .mu.g BSA, 2 .mu.L 10.times.
restriction endonuclease buffer and 2 .mu.L NcoI. The digest was
incubated at 37.degree. C. for more than 6 hours. The digest was
resolved on 1.0% TAE agarose and the 1.9 kb OsMADS13-5'-gDNA (NcoI)
DNA band was excised, recovered and ethanol precipitated with
glycogen carrier. The OsMADS13-5'-gDNA (NcoI) DNA as recovered by
micro centrifugation, washed with 70% ethanol, dried under vacuum
and resuspended in 5 .mu.L ddH.sub.2O.
[0207] 2 .mu.g pNOV6901 miniprep DNA was digested in a 20 .mu.L
reaction mixture containing 2 .mu.g BSA, 2 .mu.L 10.times.
restriction endonuclease buffer and 2 .mu.L SphI. The digest was
incubated at 37.degree. C. for more than 6 hours, then at
70.degree. C. for 20 minutes. Then 1 .mu.L of the appropriate
10.times. restriction endonuclease buffer, 1 .mu.L 1 Unit/.mu.L CIP
and 8 .mu.L ddH.sub.2O were added to the reaction and it was
further incubated at 37.degree. C. for 30 minutes. The pNOV6901
(SphI/blunt) DNA was resolved on 1.0% TAE agarose and the 4.7 kb
pNOV6901 (SphI/blunt) band was excised, recovered and ethanol
precipitated with glycogen carrier. The pNOV6901 (SphI/blunt) DNA
was recovered by micro centrifugation, washed with 70% ethanol,
dried under vacuum and resuspended in 14 .mu.L ddH.sub.2O.
[0208] pNOV6901 (SphI/blunt) miniprep DNA was digested in a 20
.mu.L reaction mixture containing 2 .mu.g BSA, 2 .mu.L 10.times.
restriction endonuclease buffer and 2 .mu.L NcoI. The digest was
incubated at 37.degree. C. for more than 6 hours. The pNOV6901
(SphI/blunt/NcoI) plasmid DNA was resolved on 1.0% TAE agarose and
the 4.7 kb pNOV6901 (SphI/blunt/NcoI) band was excised, recovered
and ethanol precipitated with glycogen carrier. The pNOV6901
(SphI/blunt/NcoI) DNA was recovered by micro centrifugation, washed
with 70% ethanol, dried under vacuum and resuspended in 5 .mu.L
ddH.sub.2O.
[0209] 4.0 .mu.L OsMADS13-5'-gDNA (NcoI) was ligated to 4.0 .mu.L
pNOV6901 (SphI/blunt/NcoI) in a 10 .mu.L ligation mixture
containing 1 .mu.L 10.times. T4 DNA ligase buffer and 1 .mu.L T4
DNA ligase (400 Units/.mu.L). The ligation mixture was incubated
more than 8 hours at 16.degree. C. 5.0 .mu.L of the ligation
mixture was transformed into 50 .mu.L Top10 competent cells. The
pNOV6901-OsMADS13P recombinants were verified by digesting 2 .mu.L
pNOV6901-OsMADS13P miniprep DNA with 0.5 .mu.L XhoI, 0.5 .mu.L NcoI
in 10 .mu.L reaction mixtures containing 1 .mu.g BSA and 1 .mu.L
10.times. restriction endonuclease buffer. The digests were
incubated at 37.degree. C. for 2 hours then resolved on 1% TAE
agarose. Positive pNOV6901-OsMADS13P recombinants were
sequenced.
[0210] B. Cloning of the OsMADS13 3'-Regulatory Sequence
[0211] The OsMADS13 3'-regulatory sequence from rice genomic DNA
(gDNA) was amplified using high-fidelity PCR. The 50 .mu.L reaction
mixture consisted of 100 ng rice gDNA, 200 .mu.M dNTPs, 1 .mu.L 20
.mu.M oligonucleotide primer OsMADS13-C3
5'-tcgagcggccgctgacatggatatgatgatcag-3' (SEQ ID NO 492), 1 .mu.L 20
.mu.M oligonucleotide primer OsMADS13-C4
5'-acgtatcgatcggaccgcaacgcacgggcacccaac-3' (SEQ ID NO 493), 1 .mu.L
10.times. Expand High Fidelity buffer and 1 .mu.L Expand High
Fidelity polymerase. The thermocycling program was at 95.degree. C.
for 2 minutes followed by 40 cycles of (94.degree. C. for 15
seconds, 60.degree. C. for 30 seconds, 68.degree. C. for 6 minutes)
followed by 68.degree. C. for 15 minutes. The 1.2 kb
OsMADS13-3'-gDNA DNA fragment was recovered and ethanol
precipitated with glycogen carrier. The OsMADS13-3'-gDNA DNA
fragment was recovered by micro centrifugation, washed with 70%
ethanol, dried under vacuum and resuspended in 14 .mu.L
ddH.sub.2O.
[0212] The OsMADS13-3'-gDNA fragment was digested in a 20 .mu.L
reaction mixture containing 2 .mu.g BSA, 2 .mu.L 10.times.
restriction endonuclease buffer and 2 .mu.L NotI. The digest was
incubated at 37.degree. C. for more than 6 hours then resolved on
1.0% TAE agarose and the 1.2 kb OsMADS13-3'-gDNA (NotI) DNA band
was excised, recovered and ethanol precipitated with glycogen
carrier. The OsMADS13-3'-gDNA (NotI) DNA was recovered by micro
centrifugation, washed with 70% ethanol, dried under vacuum and
resuspended in 5 .mu.L ddH.sub.2O.
[0213] 2 .mu.g pNOV6901-OsMADS13P miniprep DNA was digested in a 20
.mu.L reaction mixture containing 2 .mu.g BSA, 2 .mu.L 10.times.
restriction endonuclease buffer, 1 .mu.L NotI and 1 .mu.L SmaI. The
digest was incubated at 37.degree. C. for more than 6 hours, then
at 70.degree. C. for 20 minutes. Then 1 .mu.L of the appropriate
10.times. restriction endonuclease buffer, 1 .mu.L 1 Unit/.mu.L CIP
and 8 A ddH.sub.2O were added to the reaction and it was further
incubated at 37.degree. C. for 30 minutes. The pNOV6901-OsMADS13P
(NotI/SmaI/CIP) DNA was resolved on 1.0% TAE agarose and the 6.6 kb
band was excised, recovered and ethanol precipitated with glycogen
carrier. The pNOV6901-OsMADS13P (NotI/SmaI/CIP) DNA fragment was
recovered by micro centrifugation, washed with 70% ethanol, dried
under vacuum and resuspended each in 5 .mu.L ddH.sub.2O.
[0214] 4.0 .mu.L pNOV6901-OsMADS13P (NotI/SmaI/CIP) was ligated to
4.0 .mu.L OsMADS13-3'-gDNA (NotI) in a 10 .mu.L ligation mixture
containing 1 .mu.L 10.times. T4 DNA ligase buffer and 1 .mu.L T4
DNA ligase (400 Units/.mu.L). The ligation mixture was incubated
more than 8 hours at 16.degree. C. 5.0 .mu.L of the ligation
mixture was transformed into 50 .mu.L Top10 competent cells. The
pNOV6901-OsMADS13P/OsMADS13T recombinants were verified by
digesting 7.5 .mu.L pNOV6901-OsMADS13P/OsMADS13T miniprep DNA with
1.0 .mu.L NotI in 10 .mu.L reaction mixtures containing 1 .mu.g BSA
and 1 .mu.L 10.times. restriction endonuclease buffer. Digests were
incubated at 37.degree. C. for 2 hours then resolved on 1% TAE
agarose. Positive pNOV6901-OsMADS13P/OsMADS13T recombinants were
sequenced. The finished clone was designated pNOV6904, which is
also the plasmid's QC number. pNOV6904 contains the complete
OSMAD13 expression cassette depicted by SEQ ID NO. 539.
[0215] C. Mobilization of the OsMADS13 GUS Expression Cassette into
pNOV6900
[0216] 2 .mu.g pNOV6900 was digested in a 20 .mu.L reaction mixture
containing 2 .mu.g BSA, 2 .mu.L 10.times. restriction endonuclease
buffer and 2 .mu.L RsrII. The digest was incubated at 37.degree. C.
for more than 6 hours, then at 70.degree. C. for 20 minutes. Then 1
.mu.L of the appropriate 10.times. restriction endonuclease buffer,
1 .mu.L 1 Unit/.mu.L CIP and 8 .mu.L ddH.sub.2O were added to the
reaction and it was further incubated at 37.degree. C. for 30
minutes. 2 .mu.g pNOV6901-OsMADS13P/OsMADS13T miniprep DNA was
digested in a 20 .mu.L reaction mixture containing 2 .mu.g BSA, 2
.mu.L 10.times. restriction endonuclease buffer and 2 .mu.L RsrII.
The digest was incubated the reaction at 37.degree. C. for more
than 6 hours.
[0217] The digested plasmid DNAs, pNOV6900 (RsrII/CIP) and
pNOV6901-OsMADS13P/OsMADS13T (RsrII), were resolved on 1.0% TAE
agarose and the 9.2 kb pNOV6900 (RsrII/CIP) and the 5.3 kb
pNOV6901-OsMADS13P/OsMADS13T (RsrII) bands were excised, recovered
and ethanol precipitated with glycogen carrier. The pNOV6900
(RsrII/CIP) and pNOV6901-OsMADS13P/OsMADS13T (RsrII) DNA fragments
were recovered by micro centrifugation, washed with 70% ethanol,
dried under vacuum and resuspended each in 5 .mu.L ddH.sub.2O.
[0218] 4.0 .mu.L of pNOV6900 (RsrII/CIP) was ligated to 4.0 .mu.L
pNOV6901-OsMADS13P/OsMADS13T (RsrII) in a 10 .mu.L ligation mixture
containing 1 .mu.L 10.times. T4 DNA ligase buffer and 1 .mu.L T4
DNA ligase (400 U/.mu.L). The ligation mixture was incubated more
than 8 hours at 16.degree. C. 5.0 .mu.L of ligation mixture was
transformed into 50 .mu.L Top10 competent cells. The
pNOV6900-pNOV6901-OsMADS13P/OsMADS13T recombinants were verified by
digesting 7.5 .mu.L pNOV6900-pNOV6901-OsMADS13P/OsMADS13T miniprep
DNA with 1.0 .mu.L NcoI in 10 .mu.L reaction mixtures containing 1
.mu.g BSA and 1 .mu.L 10.times. restriction endonuclease buffer.
The digests were incubated at 37.degree. C. for 2 hours then
resolved on 1% TAE agarose. Positive
pNOV6900-pNOV6901-OsMADS13P/OsMADS13T recombinants were sequenced.
The finished clone was designated pNOV6905, which is also the
plasmid's QC number.
[0219] The engineered alterations in the 5'-gene regulatory
sequence derived from the OsMADS13 gene include introduction of an
RsrII site at the 5'-end of OsMADS13P, insertion of a Kozak
sequence upstream of the natural OsMADS13P translation start codon
and modification of the natural OsMADS13P translation start codon
so that it is contained within an NcoI site. The engineered
alterations in the 3'-gene regulatory sequence derived from the
OsMADS13 gene include introduction of a NotI site at the
5'-terminus of OsMADS13T and introduction of an RsrII site at the
3'-terminus of OsMADS13T. In this configuration the GUS coding
sequence can be replaced with any gene of interest flanked by
NcoI/NotI restriction sites. The complete cassette is mobilized, as
an RsrII fragment, to the binary vector pNOV6900.
[0220] The cassette was transformed into A188 .times. HyII maize
and Kaybonnet rice using standard agrobacterium mediated
methodology.
[0221] GUS Expression in T0 Maize
[0222] Sixty-seven T0 transgenic maize lines were generated. Tassel
spikelets were histochemically screened for GUS expression.
Fifty-six were positive for GUS activity. Thirty-five also showed
GUS expression in leaf punches. Ten lines had no detectable GUS
activity in tassels or leaf punches.
[0223] Two T0 lines were selected to analyze GUS expression in
developing ears. Both lines had a GUS signal in tassel spikelets
and no GUS signal in leaf punches. Ears were harvested
approximately 7 days before silking and histochemically stained for
GUS expression. Whole sections showed a strong GUS signal only in
developing florets, whereas GUS activity is absent in surrounding
ear tissue. These data indicate the OsMADS13 expression cassette
functions to drive GUS expression in both male and female spikelets
in T0 maize transformants.
[0224] GUS Expression in T0 Rice
[0225] Thirty-three T0 rice lines were produced. Fourteen
independent transformants were histochemically stained for GUS
expression GUS activity was primarily detected in spikelets. Some
plants also had GUS activity in leaf tissue.
[0226] GUS Expression in T1 Maize
[0227] T1 progeny from three independent transformants were sown
and analyzed in detail for GUS expression. There was no detectable
GUS expression in leaf tissue, developing silk or tassels. This
indicates tassel expression observed in T0 plants may result from
tissue culture associated with the transformation process.
Dissected organs from T1 tassels indicated no apparent GUS
expression (data not shown). GUS activity is seen in the developing
ear harvested about 5 days before pollination. The longitudinal
section showed expression localized to developing ovules and
transmitting or placental tissue. The cross section supports this
and provides further evidence for expression in ear vasculature.
GUS expression is observed in a second transgenic event. It also
localizes to zones where ovules will likely develop. Expression
localizes to vasculature supplying the developing ear.
[0228] These data support the OsMADS13 cassette as a very good
candidate for trait expression in developing ovules. When driven by
the OsMADS13-based expression cassette, genes that facilitate
phloem unloading such as invertase or a sucrose transporter should
prove effective in supporting early ear development by increasing
sink strength.
[0229] The cassette continues to function post-fertilization. The
observed GUS expression pattern at 4 and 6 days after pollination.
Late in kernel development (21 days after pollination) GUS
expression remains localized to the pedicel and hilar regions. It
also appears in the aleurone. GUS protein is detectable throughout
ovule and kernel development, up to 21 days after pollination.
There is very light staining in the aerial tissue, with no GUS
activity in the roots.
[0230] The present invention includes an expression cassette based
on the Oryza sativa OsMADS13 gene. The expression cassette includes
the gene's promoter, including the first intron and the 5'-UTR, the
3'-UTR and the 3'-nontranscribed sequence. These components were
assembled into a GUS expression cassette and tested in transgenic
plants. The cassette's design facilitates replacement of the GUS
coding sequence with any gene of interest. The cassette will target
gene expression to the vasculature within the placental tissue
below the floret of developing ear spikelets. Post-fertilization,
expression is also expected in the aleurone, hilar region and
pedicel. Developmentally, the cassette should drive gene expression
from a very early point, more than 7 days before pollination, in
ovule development.
TABLE-US-00003 TABLE 3 Histochemical staining in Select T1 plant
tissue Promoter OsMADS5-GUS OsMADS6-GUS OsMADS8-GUS OsMADS13-GUS
ZmM8-GUS Leaf negative negative negative negative negative Stalk
negative strong signal negative slight signal negative at ear
branch at ear branch Root negative negative negative negative
negative Seedling negative negative negative negative negative
Tassel negative negative negative negative negative Silk negative
negative negative negative negative Embryo & negative negative
negative negative negative Endosperm Ear modest in strong in strong
in strong in modest in floret and floret and florets and florets
and glume some ear some ear central ear some vasculature
vasculature vasculature vasculature
EXAMPLE 7
Identification of the OsT6PP cDNA Sequence
[0231] The first vascular plant trehalose-6-phosphate phosphatase
genes were cloned from Arabidopsis thaliana by complementation of a
yeast tps2 deletion mutant (Vogel et al. 1998). The genes
designated AtTPPA and AtTPPB (GenBank accessions AF007778 and
AF007779) were shown at that time to have trehalose-6-phosphate
phosphatase activity. The AtTPPA and AtTTPB protein sequences were
used in TBLASTN queries of maize and rice sequence databases.
Sequence alignments organized the hits into individual genes. Three
maize and three rice T6PP homologs were identified. The cDNA
sequences corresponding to the predicted protein sequence for each
gene--ZmT6PP-1, -2 and -3 and OsT6PP-1, -2 and -3--are shown in
global alignment with the Arabidopsis T6PPs in FIG. 10.
[0232] The composition and method of the present invention includes
using the OsMADS6 promoter operably linked to a nucleic acid
molecule that when expressed in a plant cell, increases the
expression of T6PP. By doing so, flux through the trehalose pathway
is increased only in young developing ears where it functions to
increase flux through central carbon metabolism.
[0233] The OsT6PP-3 cDNA sequence (SEQ ID NO. 531) is amplified
using high-fidelity PCR. The 50 .mu.L reaction mixture consists of
1 .mu.L rice cDNA library (prepared from callus mRNA in
Stratagene's Lambda Unizap Vector, primary library size
>1.times.10.sup.6 pfu, amplified library titer
>1.times.10.sup.12 pfu/mL), 200 .mu.M dNTPs, 1 .mu.L 20 .mu.M of
oligonucleotide primer T6PP-EC-5
(5'-catggaccatggatttgagcaatagctcac-3') SEQ ID NO. 528 and 1 .mu.L
20 .mu.M of oligonucleotide primer T6PP-EC-3
(5'-atcgcagagctcacactgagtgcttcttcc-3') SEQ ID NO. 529, 5 .mu.L
10.times. Cloned PFU buffer and 2.5 Units of Pfuturbo DNA
polymerase. The thermocycling program is 95.degree. C. for 2
minutes followed by 40 cycles of (94.degree. C. for 15 seconds,
50.degree. C. for 1 minute, 72.degree. C. for 1 minute) followed by
72.degree. C. for 10 minutes. The OsT6PP-3 product is cloned with
the Zero Blunt TOPO PCR cloning kit. The pCR-Blunt-II-TOPO-OsT6PP-3
is identified by digesting 5 .mu.L pCR-Blunt-II-TOPO-OsT6PP-3
miniprep DNA with EcoRI in a 20 .mu.L reaction containing 2 .mu.g
BSA and 2 .mu.L 10.times. EcoRI restriction endonuclease buffer.
The reaction is incubated at 37.degree. C. for 2 hours and the
pCR-Blunt-II-TOPO-OsT6PP-3 (EcoRI) products are resolved on 1% TAE
agarose. The pCR-Blunt-II-TOPO-OsT6PP-3 clone is then sequenced.
The OsT6PP-3 cDNA is flanked by NcoI/SacI restriction endonuclease
sites.
[0234] To facilitate cloning into 11082, an internal NcoI site in
OsT6PP was silenced using Stratagene's QuikChange Multi
Site-Directed Mutagenesis Kit and the oligonucleotide primer
T6PP-QC (5'-CTTTATTATGCTGGAAGTCATGGTATGGACATAATGGCACC-3') SEQ ID
NO. 530.
EXAMPLE 8
Construction of OsMADS6-T6PP
[0235] A. Construction of the OsMADS6-OsT6PP-3 Expression
Cassette
[0236] The pCR-Blunt-II-TOPO-OsT6PP-3 clone (141.1L) DNA was
digested in a 20 .mu.L reaction mixture containing 2 .mu.g BSA, 2
.mu.L 10.times. restriction endonuclease buffer, 1 .mu.L NcoI and 1
.mu.L SacI. The digest was incubated at 37.degree. C. for more than
6 hours. The pCR-Blunt-II-TOPO-OsT6PP-3 (NcoI/SacI) DNA was
resolved on 1.0% TAE agarose and the 1.3 kb OsT6PP-3 (NcoI/SacI)
band was excised, recovered and ethanol precipitated with glycogen
carrier. The OsT6PP-3 (NcoI/SacI) DNA was recovered by micro
centrifugation, washed with 70% ethanol, dried under vacuum and
resuspended in 5 .mu.L ddH.sub.2O.
[0237] 2 .mu.g 11082 miniprep DNA was digested in a 20 .mu.L
reaction mixture containing 2 .mu.g BSA, 2 .mu.L 10.times.
restriction endonuclease buffer, 1 .mu.L NcoI and 1 .mu.L SacI. The
digest was incubated at 37.degree. C. for more than 6 hours, then
at 70.degree. C. for 20 minutes. Then 1 .mu.L of the appropriate
10.times. restriction endonuclease buffer, 1 .mu.L 1 Unit/.mu.L CIP
and 8 .mu.L ddH.sub.2O were added to the reaction and it was
further incubated at 37.degree. C. for 30 minutes. The 11082
(NcoI/SacI/CIP) DNA was resolved on 1.0% TAE agarose and the 8.1 kb
11082 (NcoI/SacI/CIP) band was excised, recovered and ethanol
precipitated with glycogen carrier. The 11082 (NcoI/SacI/CIP) DNA
fragment was recovered by micro centrifugation, washed with 70%
ethanol, dried under vacuum and resuspended in 5 .mu.L
ddH.sub.2O.
[0238] 4.0 .mu.L 11082 (NcoI/SacI/CIP) was ligated to 4.0 .mu.L
OsT6PP-3 (NcoI/SacI) in a 10 .mu.L reaction mixture containing 1
.mu.L 10.times. T4 DNA ligase buffer and 1 .mu.L T4 DNA ligase (400
Units/.mu.L). The reaction mixture was incubated more than 8 hours
at 16.degree. C. 5.0 .mu.L of ligation mixture was transformed into
50 .mu.L Top10 competent cells. The recombinants were verified by
digesting 7.5 .mu.L 11082-OsT6PP-3 miniprep DNA with 1.0 .mu.L
RsrII in 10 .mu.L reaction mixtures containing 1 .mu.g BSA and 1
.mu.L 10.times. restriction endonuclease buffer. The digests were
incubated at 37.degree. C. for 2 hours then resolved on 1% TAE
agarose. Positive 11082-OsT6PP-3 recombinants were sequenced. The
vector was designated OsMADS6-OsT6PP-Assembly and is shown in FIG.
11A.
[0239] B. Mobilization of the OsMADS6-OsT6PP-Assembly Expression
Cassette into pNOV6900
[0240] 2 .mu.g pNOV6900 was digested in a 20 .mu.L reaction mixture
containing 2 .mu.g BSA, 2 .mu.L 10.times. restriction endonuclease
buffer and 2 .mu.L RsrII. The digest was incubated at 37.degree. C.
for more than 6 hours, then at 70.degree. C. for 20 minutes. Then 1
.mu.L of the appropriate 10.times. restriction endonuclease buffer,
1 .mu.L 1 Unit/.mu.L CIP and 8 .mu.L ddH.sub.2O were added to the
reaction and it was further incubated at 37.degree. C. for 30
minutes. 2 .mu.g pNOV6906-OsT6PP-Assembly miniprep DNA was digested
in a 20 .mu.L reaction mixture containing 2 .mu.g BSA, 2 .mu.L
10.times. restriction endonuclease buffer and 2 .mu.L RsrII. The
digest was incubated at 37.degree. C. for more than 6 hours.
[0241] The pNOV6900 (RsrII/CIP) and the pNOV6906-OsT6PP-Assembly
(RsrII) plasmid DNAs were resolved on 1.0% TAE agarose, and the 9.2
kb pNOV6900 (RsrII/CIP) and the 6.8 kb pNOV6906-OsT6PP-Assembly
(RsrII) bands were excised, recovered and ethanol precipitated with
glycogen carrier. The pNOV6900 (RsrII/CIP) and
pNOV6906-OsT6PP-Assembly (RsrII) DNA fragments were recovered by
micro centrifugation, washed with 70% ethanol, dried under vacuum
and resuspended in 5 .mu.L ddH.sub.2O each.
[0242] 4.0 .mu.L pNOV6900 (RsrII/CIP) was ligated to 4.0 .mu.L
pNOV6906-OsT6PP-Assembly (RsrII) in a 10 .mu.L ligation mixture
containing 1 .mu.L 10.times. T4 DNA ligase buffer and 1 .mu.L T4
DNA ligase (400 U/.mu.L). The ligation mixture was incubated more
than 8 hours at 16.degree. C. 5.0 .mu.L of ligation mixture was
transformed into 50 .mu.L Top10 competent cells. The
pNOV6900-pNOV6906-OsT6PP-Assembly recombinants were verified by
digesting 7.5 .mu.L miniprep DNA with 1.0 .mu.L NcoI in 10 .mu.L
reaction mixtures containing 1 .mu.g BSA and 1 .mu.L 10.times.
restriction endonuclease buffer. The digests were incubated at
37.degree. C. for 2 hours then resolved on 1% TAE agarose. Positive
pNOV6900-pNOV6906-OsT6PP-Assembly recombinants were sequenced. The
finished clone was designated OsMADS6-OsT6PP-Binary and is shown in
FIG. 11B. The plasmid's QC number is 12194.
[0243] The OsMADS6-OsT6PP-3 expression cassette (SEQ ID NO. 533)
was transformed into A188 maize using standard agrobacterium
mediated methodology. Regenerated T0 shoots were screened transgene
copy number and insert integrity using a Taqman.TM. assay. Events
containing a single copy of the OsMADS6-OsT6PP-3 expression
cassette and no other sequence derived from the binary vector were
identified.
[0244] Expression cassette function in each transgenic Event was
verified by RT-PCR. DNA-free total RNA template was prepared from
100 mg of T0 tassel tissue using the RNeasy Plant mini Kit. The
RT-PCR assay was performed using the Qiagen One Step RT-PCR kit
with 100 ng total RNA template, the T6PP-RTPCRF
(5'-gacagaactgacgaagacgctttcaa-3') and 6906-tr
(5'-ctccaacttctgacagctg-3') primers (SEQ ID NOS. 534, 535
respectively). This assay produces a transgene-specific 210 by
fragment.
EXAMPLE 9
Greenhouse Growth Conditions
[0245] Corn seed is sown into 2.5 SVD pots (Classic 600, .about.2
gallon nursery containers) in Universal mix (Sungrow Horticulture,
Pine Bluff, Ark.). Universal mix is 45% Peat moss, 45% bark , 5%
perlite, 5% vermiculite. Environmental conditions for greenhouse
maize cultivation are typically 16 hour days (average light
intensity 600 mmol m.sup.-2s.sup.-2), day time temperature of
80-86.degree. F., night time temperature 70-76.degree. F. and
relative humidity greater than 50%. Plants are placed on 2''
platforms to avoid contact with the greenhouse floor. Plants are
hand watered until daily irrigation as required, then they are
placed on irrigation drip. The irrigation schedule is 4 minutes
every other day. Plants were routinely treated with insecticides to
control pests.
EXAMPLE 10
Evaluation of Transgenic Maize Expressing OsMADS6-OsT6PP-3 in the
Greenhouse
[0246] The greenhouse evaluation is a controlled water-stress
experiment that quantifies ovule viability in water-stressed and
unstressed plants. Data from unstressed plants represent the
genotype's potential to set seed under ideal conditions. Data from
water-stressed plants quantify kernel abortion that results from
drought at the time of flowering. The results of these experiments
can be predictive of field performance. We used this tool to select
transgenic events for field evaluations.
[0247] Transgenic maize segregating for a single copy of the
OsMADS6-T6PP-3 transgene were sown as above. Taqman analysis was
used to divide the progeny into homozygous or hemizygous
(containing OsMADS6-OsT6PP-3) and azygous (lost the
OsMADS6-OsT6PP-3) groups. These individuals were pollinated with
JHAF031 maize pollen to generate hybrid seed
(KPOO188RA.times.JHAF031) for the greenhouse experiment. The hybrid
seed were sown as above. Seedlings were transferred to 600 pots,
above, and maintained using standard greenhouse procedures until
they reached the V6 growth stage (Ritchie et al., 1997). All plants
were treated with the systemic pesticide, Marathon, to reduce
susceptibility to pests. Water stress was gradually imposed, using
salt as the osmoticum (Nuccio et al. 1998). The salt consisted of
sodium chloride/calcium chloride at a 10:1 molar ratio, delivered
in 0.5.times. Hoagland's Solution, to prevent sodium-induced
disruption of potassium uptake. Salt concentration in the irrigant
was increased from 50 mM to 100 mM to 150 mM every three days to
give plants time to adjust to the salt. Plants were maintained on
150 mM salt solution through the flowering period, typically two
weeks, after which pots were thoroughly flushed with water and
plants were returned to normal irrigation. This protocol typically
reduced kernel set by 40-60%, compared to control plants that
received no salt.
[0248] Typically 15-20 seed per transgenic event were sown to
generate a uniform seedling population. Plants were arranged in a
complete, randomized block design consisting of six-eight
replicates per treatment. Developing ears were covered with
pollination bags before silk emergence. Pollen shed and silk
emergence dates were recorded and individual ears were hand
pollinated with donor pollen 5 days after silk emergence.
Pollination bags were removed after completing all pollinations.
Ears were harvested 30 days after pollinations, and dried for 4
days to 15% moisture content. Ears were shelled and the kernels
were counted and weighed.
EXAMPLE 11
Greenhouse Experiment
[0249] Two OsMADS6-T6PP-3 events were studied for their ability to
set seed under water stress. Twenty-four hybrid seed
(A188.times.JHAF031) from each event were germinated. Taqman
analysis was used to establish zygosity in each seedling.
Hemizygotes and azygotes were analyzed using the greenhouse water
stress protocol described above. In this experiment azygote plants
served as the benchmark. In these greenhouse experiments, the
hemizygote plants could not be distinguished from the azygote
plants. On average the water stress reduced kernel set by 42%. The
data in these greenhouse experiments indicate the OsMADS6-T6PP-3
expression cassette does not influence kernel set in maize in these
particular greenhouse experiments and when evaluated by the above
water stress protocols.
EXAMPLE 12
Evaluation of Transgenic Maize Expressing OsMADS-T6PP-3 for Drought
Stress Tolerance in the Field
[0250] Hybrid seed were generated for each transgenic Event at the
Syngenta Seeds field station in Kauai in late 2004. T1 seed
obtained by selfing the T0 plant of the events was sown in four
single-row plots, 12.7 feet long separated by 3 foot alleys with
about 20 plants per row. Taqman analysis was used to divide the
progeny into homozygous or hemizygous (containing OsMADS6-OsT6PP-3)
and azygous (lost the OsMADS6-OsT6PP-3) groups. In two of the
single-row plots, hemizygous and azygous plants were destroyed and
homozygous plants were selfed for seed bulking and also testcrossed
to NP2043BT11 and NP2044BT11. In the other two single-row plots
homozygous and hemizygous plants were destroyed and azygous plants
were selfed and also crossed to NP2043BT11 and NP2044BT11. The
azygous and hemizygous testcross seed of the events was used to
conduct field trials.
[0251] A field evaluation was conducted to test transgene
performance in a controlled drought experiment. The experiment was
conducted at the Syngenta Crop Protection Facility in Visalia,
Calif. in the summer of 2004. The planting site typically gets less
than 3'' of rainfall during the summer The NP2043BT11 testcross
seed, generated above, was used in this study. This population also
contained the BT transgene to control insect pressure. A
split-block design, with watering regime as the main plots arranged
in a randomised complete blocks and replicated three times, events
as the subplots, and in cases where there was seed of the azgous
and hemizygous hybrids, genotype as sub-sub-plots was used. Two
watering regimes were attempted: water-stressed and well-watered.
Each plot consisted of two-rows, 17.5 feet long planted with 40
seeds per row. Alleys between ranges were 2.5 feet. Furrow
irrigation was used to water the fields. Each treatment block had a
dedicated irrigation source situated at one end of the field. The
replication were arranged in such a way that replication one was
closest to and replication three was the furthest from the
irrigation source. After emergence, stand counts were taken and
plots were thinned, as necessary, to establish field
uniformity.
[0252] The well-watered block was thought to have been irrigated
optimally throughout the experiment. The water-stress block was
watered optimally until plants reached approximately V, at which
time water was withheld. Plants were returned to optimal irrigation
after 90% silk emergence.
[0253] After plants transitioned to reproductive development, the
50% pollen shed date, the 50% silk emergence date, and leaf
scrolling at early-, mid- and late-flowering were recorded for each
plot. Plot Barreness was recorded three weeks after silking.
[0254] Plots were combine-harvested and grain yield and grain
moisture were recorded. The data from hemizygous plots were
compared to azygous plots, or wild type plots where necessary, to
gauge the transgene's effect on yield. Results for seven
OsMADS6-T6PP-3 events are shown in FIG. 12. The data show the
OsMADS6-T6PP-3 transgene has a positive effect on yield in four of
the seven Events. The yield gain is evident in both unstressed and
drought-stressed plots. For example in the drought-stressed
treatment block the average yield for 5217 Events containing the
transgene was 73 Bu/acre and the average yield for 5217 Events
lacking the transgene was 54 Bu/acre. Results suggest the transgene
improves kernel set by 25% in drought-stressed conditions. In the
less stressed treatment block the average yield for 5217 Events
containing the transgene was 132 Bu/acre and the average yield for
5217 Events lacking the transgene was 95 Bu/acre. Results suggest
the transgene improves kernel set by nearly 28% in less stressed
plants. The average yield calculated for each plot in the
drought-stressed treatment block was 72 Bu/acre. The average yield
calculated for each plot in the less stressed treatment block was
113 Bu/acre. The yield improvement due to the OsMADS6-T6PP-3 gene
varies from Event to Event. It is observed in four of the seven
Events tested, and is manifest in both less stressed and
drought-stressed plants. Results from this field experiment
demonstrate the effectiveness of the OsMADS-T6PP-3 transgene in
stabilizing kernel set in drought stressed maize.
EXAMPLE 13
Evaluation of Transgenic Maize Expressing OsMADS-T6PP-3 for Yield
in the Field
[0255] Hybrid seed was generated for each transgenic Event at the
Syngenta Seeds field station in Kauai in late 2004. T1 seed
obtained by selfing the T0 plant of the events was sown in four
single-row plots, 12.7 feet long separated by 3 foot alleys with
about 20 plants per row. Taqman analysis was used to divide the
progeny into homozygous or hemizygous (containing OsMADS6-OsT6PP-3)
and azygous (lost the OsMADS6-OsT6PP-3) groups. In two of the
single-row plots, hemizygous and azygous plants were destroyed and
homozygous plants were selfed for seed bulking also testcrossed to
NP2043BT11 and NP2044BT11. In the other two single-row plots
homozygous and hemizygous plants were destroyed and azygous plants
were selfed and also crossed to NP2043BT11 and NP2044BT11. The
azygous and hemizygous testcross seed of the events was used to
conduct field trials. A series of yield trials were conducted in
several mid-West locations to test transgene performance under
conditions typically used by growers. The
XPOO188RA.times.NP2043BT11 material, generated above, was used in
late maturity zones and the XPOO188RA.times.JHAF431B material,
generated above, was used in early maturity zones. These
populations also contained the BT transgene to control insect
pressure. The experimental design consisted of randomised complete
blocks with three replications. Each experimental unit consisted of
two-row plots, 17.5 feet long planted with 34 kernels per row.
Ranges were separated by 3 foot alleys. Events for which there was
seed of both the azygous and the hemizygous hybrids, randomization
was restricted to keep the azygous and hemizygous hybrids of the
events in neighboring plots. Most Events were evaluated in eight to
nine locations. Event 5124 was evaluated in three locations. After
emergence, stand counts were taken and plots were thinned, as
necessary, to establish field uniformity. During the growing season
plots were evaluated for intactness, greensnap, root lodging, heat
units to 50% pollen shed and heat units to 50% silking.
[0256] Plots were Combine-harvested and grain yield and grain
moisture were recorded. The data from hemizygous plots were
compared to azygous plots, or wild type plots where necessary, to
gauge the transgene's effect on yield. The data shows that the
OsMADS6-T6PP-3 transgene does not significantly affect yield in
this experiment. There are two factors to consider. First the
standard deviation for grain yield in this experiment was 15-20% of
the mean. This is not unusual. Second, growth conditions in the
mid-West were ideal for maize in 2004. Depending on location yields
in this experiment averaged from 90 to 130 Bu/acre. Results from
this field experiment indicate the OsMADS-T6PP-3 transgene did not
cause yield drag.
EXAMPLE 14
Transformation
[0257] Once a nucleic acid sequence of the invention has been
cloned into an expression system, it is transformed into a plant
cell. The receptor and target expression cassettes of the present
invention can be introduced into the plant cell in a number of
art-recognized ways. Methods for regeneration of plants are also
well known in the art. For example, Ti plasmid vectors have been
utilized for the delivery of foreign DNA, as well as direct DNA
uptake via electroporation, microinjection, and microprojectiles.
In addition, bacteria from the genus Agrobacterium can be utilized
to transform plant cells. Below are descriptions of representative
techniques for transforming both dicotyledonous and
monocotyledonous plants, as well as a representative plastid
transformation technique.
[0258] A. Transformation of Dicotyledons
[0259] Transformation techniques for dicotyledons are well known in
the art and include Agrobacterium-based techniques and techniques
that do not require Agrobacterium. Non-Agrobacterium techniques
involve the uptake of exogenous genetic material directly by
protoplasts or cells. This can be accomplished by PEG or
electroporation mediated uptake, particle bombardment-mediated
delivery, or microinjection. Examples of these techniques are
described by Paszkowski et al., EMBO J 3: 2717-2722 (1984),
Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich et
al., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature
327: 70-73 (1987). In each case the transformed cells are
regenerated to whole plants using standard techniques known in the
art.
[0260] Agrobacterium-mediated transformation is a preferred
technique for transformation of dicotyledons because of its high
efficiency of transformation and its broad utility with many
different species. Agrobacterium transformation typically involves
the transfer of the binary vector carrying the foreign DNA of
interest (e.g. pCIB200 or pCIB2001) to an appropriate Agrobacterium
strain which may depend on the complement of vir genes carried by
the host Agrobacterium strain either on a co-resident Ti plasmid or
chromosomally (e.g. strain CIB542 for pCIB200 and pCIB2001 (Uknes
et al. Plant Cell 5: 159-169 (1993)). The transfer of the
recombinant binary vector to Agrobacterium is accomplished by a
triparental mating procedure using E. coli carrying the recombinant
binary vector, a helper E. coli strain which carries a plasmid such
as pRK2013 and which is able to mobilize the recombinant binary
vector to the target Agrobacterium strain. Alternatively, the
recombinant binary vector can be transferred to Agrobacterium by
DNA transformation (Hofgen & Willmitzer, Nucl. Acids Res. 16:
9877 (1988)).
[0261] Transformation of the target plant species by recombinant
Agrobacterium usually involves co-cultivation of the Agrobacterium
with explants from the plant and follows protocols well known in
the art. Transformed tissue is regenerated on selectable medium
carrying the antibiotic or herbicide resistance marker present
between the binary plasmid T-DNA borders.
[0262] Another approach to transforming plant cells with a gene
involves propelling inert or biologically active particles at plant
tissues and cells. This technique is disclosed in U.S. Pat. Nos.
4,945,050, 5,036,006, and 5,100,792 all to Sanford et al.
Generally, this procedure involves propelling inert or biologically
active particles at the cells under conditions effective to
penetrate the outer surface of the cell and afford incorporation
within the interior thereof. When inert particles are utilized, the
vector can be introduced into the cell by coating the particles
with the vector containing the desired gene. Alternatively, the
target cell can be surrounded by the vector so that the vector is
carried into the cell by the wake of the particle. Biologically
active particles (e.g., dried yeast cells, dried bacterium or a
bacteriophage, each containing DNA sought to be introduced) can
also be propelled into plant cell tissue.
[0263] B. Transformation of Monocotyledons
[0264] Transformation of most monocotyledon species has now also
become routine. Preferred techniques include direct gene transfer
into protoplasts using PEG or electroporation techniques, and
particle bombardment into callus tissue. Transformations can be
undertaken with a single DNA species or multiple DNA species (i.e.
co-transformation) and both these techniques are suitable for use
with this invention. Co-transformation may have the advantage of
avoiding complete vector construction and of generating transgenic
plants with unlinked loci for the gene of interest and the
selectable marker, enabling the removal of the selectable marker in
subsequent generations, should this be regarded desirable. However,
a disadvantage of the use of co-transformation is the less than
100% frequency with which separate DNA species are integrated into
the genome (Schocher et al. Biotechnology 4: 1093-1096 (1986)).
[0265] Patent Applications EP 0 292 435, EP 0 392 225, and WO
93/07278 describe techniques for the preparation of callus and
protoplasts from an elite inbred line of maize, transformation of
protoplasts using PEG or electroporation, and the regeneration of
maize plants from transformed protoplasts. Gordon-Kamm et al.
(Plant Cell 2: 603-618 (1990)) and Fromm et al. (Biotechnology 8:
833-839 (1990)) have published techniques for transformation of
A188-derived maize line using particle bombardment. Furthermore, WO
93/07278 and Koziel et al. (Biotechnology 11: 194-200 (1993))
describe techniques for the transformation of elite inbred lines of
maize by particle bombardment. This technique utilizes immature
maize embryos of 1.5-2 5 mm length excised from a maize ear 14-15
days after pollination and a PDS-1000He Biolistics device for
bombardment.
[0266] Transformation of rice can also be undertaken by direct gene
transfer techniques utilizing protoplasts or particle bombardment.
Protoplast-mediated transformation has been described for
Japonica-types and Indica-types (Zhang et al. Plant Cell Rep 7:
379-384 (1988); Shimamoto et al. Nature 338: 274-277 (1989); Datta
et al. Biotechnology 8: 736-740 (1990)). Both types are also
routinely transformable using particle bombardment (Christou et al.
Biotechnology 9: 957-962 (1991)). Furthermore, WO 93/21335
describes techniques for the transformation of rice via
electroporation.
[0267] Patent Application EP 0 332 581 describes techniques for the
generation, transformation and regeneration of Pooideae
protoplasts. These techniques allow the transformation of Dactylis
and wheat. Furthermore, wheat transformation has been described by
Vasil et al. (Biotechnology 10: 667-674 (1992)) using particle
bombardment into cells of type C long-term regenerable callus, and
also by Vasil et al. (Biotechnology 11: 1553-1558 (1993)) and Weeks
et al. (Plant Physiol. 102: 1077-1084 (1993)) using particle
bombardment of immature embryos and immature embryo-derived callus.
A preferred technique for wheat transformation, however, involves
the transformation of wheat by particle bombardment of immature
embryos and includes either a high sucrose or a high maltose step
prior to gene delivery. Prior to bombardment, any number of embryos
(0.75-1 mm in length) are plated onto MS medium with 3% sucrose
(Murashiga & Skoog, Physiologia Plantarum 15: 473-497 (1962))
and 3 mg/l 2,4-D for induction of somatic embryos, which is allowed
to proceed in the dark. On the chosen day of bombardment, embryos
are removed from the induction medium and placed onto the osmoticum
(i.e. induction medium with sucrose or maltose added at the desired
concentration, typically 15%). The embryos are allowed to
plasmolyze for 2-3 hours and are then bombarded. Twenty embryos per
target plate is typical, although not critical. An appropriate
gene-carrying plasmid (such as pCIB3064 or pSG35) is precipitated
onto micrometer size gold particles using standard procedures. Each
plate of embryos is shot with the DuPont Biolistics.RTM. helium
device using a burst pressure of 1000 psi using a standard 80 mesh
screen. After bombardment, the embryos are placed back into the
dark to recover for about 24 hours (still on osmoticum). After 24
hrs, the embryos are removed from the osmoticum and placed back
onto induction medium where they stay for about a month before
regeneration. Approximately one month later the embryo explants
with developing embryogenic callus are transferred to regeneration
medium (MS+1 mg/liter NAA, 5 mg/liter GA), further containing the
appropriate selection agent (10 mg/1 basta in the case of pCIB3064
and 2 mg/1 methotrexate in the case of pSOG35). After approximately
one month, developed shoots are transferred to larger sterile
containers known as "GA7s" which contain half-strength MS, 2%
sucrose, and the same concentration of selection agent.
[0268] Transformation of monocotyledons using Agrobacterium has
also been described. See, WO 94/00977 and U.S. Pat. No. 5,591,616,
both of which are incorporated herein by reference. See also,
Negrotto et al., Plant Cell Reports 19: 798-803 (2000),
incorporated herein by reference.
EXAMPLE 15
Use of Expression Cassettes of the Present Invention to Confer
Abiotic Stress Tolerance in Plants
[0269] Once initiated, maize female spikelets are by definition
metabolic sinks. They require a nutrient stream consisting of
carbohydrate, amino acids, cofactors, minerals and other material
from source tissues to fuel development. Source tissues include
leaves, roots, the stalk and other vegetative plant parts. Much of
what arrives at each spikelet is rapidly consumed, being converted
to cell wall material, protein, lipids, nucleic acids etc. Very
little is held in reserve.
[0270] The nutrient stream subsides during periods of abiotic
stress. This stress is imposed by a number of stimuli including
drought, cloud cover, temperature extremes and soil nutrient
depletion. Spikelet development continues despite growing
conditions, relying on reserves for energy and raw material.
Reserves maintain development for, at most, a few days. If the
abiotic stress period is prolonged reserves are depleted and
spikelet development ceases. The result is kernel abortion and
reduced yield.
[0271] The OsMADS expression cassettes of the present invention can
be used to increase the sink strength in female spikelets by fusing
them to genes that function to increase sink strength. These genes
include a sucrose transporter, invertase, and trehalose metabolism
genes. Many of these genes are not highly expressed in early
spikelet development. Early and specific expression in the
reproductive organs of plants, spikelets for example, of any of
these genes will improve spikelet nourishment without detriment to
other plant organs. Improved nutrition will enable spikelets to
complete their developmental cycle and become competent for
fertilization during ideal growth conditions and, importantly,
during prolonged periods of abiotic stress.
[0272] Carbon arrives at developing spikelets as sucrose. Spikelets
have limited ability to utilize sucrose because enzymes
facilitating its entry into metabolism are not highly expressed.
These enzymes include sucrose transporter(s) to aid uptake of
sucrose unloaded from the phloem. The OsMADS expression cassettes
can increase sucrose transporter levels in the transmitting and
other maternal tissue Imported sucrose fuels development and excess
sucrose is incorporated into starch and vacuolar reserves.
Increased starch and sucrose reserves better enable spikelets to
complete development during prolonged periods of abiotic
stress.
[0273] Carbon nutrition can also be enhanced via increased
invertase expression. This enzyme family cleaves sucrose into
glucose and fructose. Both monosaccharides can be accumulated to
high levels and rapidly enter carbon metabolism. The OSMADS
expression cassettes of the present invention can be used to
increase glucose and fructose levels in the apoplastic regions of
spikelet and other maternal tissues via expression of an apoplastic
or cell wall invertase. The monosaccharides enter cells and carbon
metabolism more readily than sucrose. Facilitated sucrose
utilization should increase sucrose unloading from the phloem, and
carbon availability to developing spikelets.
[0274] Similarly, carbon nutrition in the cytosol of developing
spikelets can be enhanced via expression of a cytosolic or neutral
invertase. This enzyme cleaves sucrose in the cytosol, facilitating
entry into carbon metabolism. The OSMADS expression cassettes of
the present invention can increase neutral invertase expression in
developing spikelets. The increased sucrose utilization in the
cytosol, in transmitting and related spikelet tissue increases
sucrose demand and thus, sucrose import from the apoplast.
[0275] Carbon availability and abiotic stress resistance in
developing spikelets also can be enhanced via expression of a
vacuolar or soluble acid invertase. This enzyme cleaves sucrose
into fructose and glucose in the vacuole, making the carbon
available for energy metabolism. Sucrose conversion into glucose
and fructose also increases the solute potential of the cell,
enabling it to maintain water and thus, turgor during periods of
drought. This allows spikelets to continue developing despite
decreased water availability. Again, the OSMADS expression
cassettes of the present invention can increase expression of
vacuolar or soluble acid invertase in developing spikelets for the
purpose of enhancing abiotic stress tolerance.
[0276] The trehalose pathway functions to regulate carbon
partitioning between primary metabolism and starch synthesis.
Up-regulation of this pathway directs carbon towards starch
synthesis. The OsMADS expression cassettes of the present invention
can be used to drive expression of trehalose-6-phosphate synthase,
trehalose-6-phosphate phosphatase and trehalase in developing
spikelets, thereby increasing sink strength and starch synthesis in
those tissues. Maintenance of a large starch pool better enables
developing spikelets to withstand prolonged periods of abiotic
stress and complete their development cycle.
[0277] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced with the scope of the present
invention.
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Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20160222402A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20160222402A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References