U.S. patent application number 13/430869 was filed with the patent office on 2012-09-20 for methods and compositions for modulating flowering and maturity in plants.
This patent application is currently assigned to PIONEER HI BRED INTERNATIONAL INC. Invention is credited to Edward Bruggemann, Bailin Li, Michele Morgante, Xiaomu Niu, Silvio Salvi, Giorgio Sponza, Dwight Tomes, Roberto Tuberosa.
Application Number | 20120240290 13/430869 |
Document ID | / |
Family ID | 35787794 |
Filed Date | 2012-09-20 |
United States Patent
Application |
20120240290 |
Kind Code |
A1 |
Tomes; Dwight ; et
al. |
September 20, 2012 |
Methods and Compositions for Modulating Flowering and Maturity in
Plants
Abstract
The present invention provides compositions and methods for
modulating flowering time in plants. Maize RAP2.7 nucleotide
sequences are disclosed which upon overexpression cause later
flowering and when inhibited cause earlier flowering. Also
disclosed is a DNA sequence which acts as a regulator/enhancer of
RAP2.7, termed VGT1. This sequence does not code for any known
protein, but acts as either a RNAi element or a regulatory DNA or
RNA element that either directly regulates expression of flowering
genes such as Rap2.7 or specifically targets expression of other
genes which control flowering genes such as Rap2.7. This element
this can be used as a sequence--based marker to identify inbred and
hybrids which have altered maturity. Methods for expressing these
nucleotide sequences in a plant for modifying maturity and
flowering in plants are provided as well as expression constructs,
vectors, transformed cells and plants.
Inventors: |
Tomes; Dwight; (Grimes,
IA) ; Salvi; Silvio; (Bologna, IT) ; Morgante;
Michele; (Udine, IT) ; Sponza; Giorgio;
(Bologna, IT) ; Bruggemann; Edward; (West Des
Moines, IA) ; Niu; Xiaomu; (Johnston, IA) ;
Li; Bailin; (Hockessin, DE) ; Tuberosa; Roberto;
(Bologna, IT) |
Assignee: |
PIONEER HI BRED INTERNATIONAL
INC
Johnston
IA
|
Family ID: |
35787794 |
Appl. No.: |
13/430869 |
Filed: |
March 27, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12276961 |
Nov 24, 2008 |
8173867 |
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13430869 |
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11190339 |
Jul 27, 2005 |
7479584 |
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12276961 |
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60592268 |
Jul 29, 2004 |
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Current U.S.
Class: |
800/285 ;
435/320.1; 435/412; 435/419; 435/6.11; 530/376; 536/23.6; 800/290;
800/298 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/827 20130101 |
Class at
Publication: |
800/285 ;
536/23.6; 435/320.1; 435/419; 435/412; 800/298; 800/290; 530/376;
435/6.11 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C12Q 1/68 20060101 C12Q001/68; A01H 5/00 20060101
A01H005/00; C07K 14/415 20060101 C07K014/415; C07H 21/04 20060101
C07H021/04; C12N 5/10 20060101 C12N005/10 |
Claims
1. An isolated nucleic acid molecule that encodes a polypeptide
having RAP2.7-like activity, said nucleic acid molecule being
selected from the group consisting of: (a) a nucleic acid molecule
comprising the sequence set forth in SEQ ID NOS: 1, 3, or 4; (b) a
nucleic acid molecule comprising a sequence encoding the amino acid
sequence set forth in SEQ ID NOS: 2; (c) a nucleic acid molecule
comprising a sequence having at least 80% sequence identity to the
nucleotide sequence set forth in SEQ ID NOS: 1, 3, or 4; and (d) a
nucleic acid molecule comprising a sequence of at least 50
consecutive nucleic acids of any of (a) through (d).
2. A vector comprising the nucleic acid molecule of claim 1.
3. A plant cell having stably incorporated in its genome the
nucleic acid molecule of claim 1.
4. The plant cell of claim 3, wherein said plant cell is from a
monocot plant.
5. The plant cell of claim 4, wherein said monocot plant is
maize.
6. A plant having stably incorporated into its genome the nucleic
acid molecule of claims 1.
7. A method for altering flowering time in a plant comprising:
transforming a plant cell with a nucleic acid molecule operably
linked to a promoter that regulates transcription of said sequence
in a plant cell; wherein said nucleic acid molecule comprises a
nucleotide sequence selected from the group consisting of: (a) a
nucleotide sequence comprising the sequence set forth in SEQ ID
NOS: 1, 3, or 4; (b) a nucleotide sequence encoding the amino acid
sequence of SEQ ID NO:2; (c) a nucleotide sequence having at least
80% sequence identity to the sequence of SEQ ID NOS: 1, 3, or 4,
wherein said nucleotide sequence encodes a protein which regulates
flowering in plants.
8. The method of claim 6, wherein said plant is a monocot.
9. The method of claim 7, wherein said monocot is maize.
10. The method of claim 7 wherein said nucleic acid molecule is a
vector for over-expression of RAP2.7.
11. The method of claim 7 wherein said plant flowers later than a
plant which does not have the overexpression vector incorporated
therein.
12. The method of claim 7 wherein said nucleic acid moleculare is a
vector for inhibiting expression of RAP2.7.
13. The method of claim 12 wherein said vector is an RNA
interference vector.
14. The method of claim 13 wherein said plant flowers earlier than
a plant which does not have an RNA interference vector incorporated
therein.
15. A method for altering maturity of a plant, said method
comprising: transforming said plant with a nucleic acid molecule
comprising a heterologous sequence operably linked to a promoter
that induces transcription of said heterologous sequence in a plant
cell; and regenerating stably transformed plants, wherein said
heterologous sequence comprises a nucleotide sequence selected from
the group consisting of: (a) a nucleotide sequence comprising the
sequence set forth in SEQ ID NOS: 1, 3 or 4; (b) a nucleotide
sequence comprising at least 50 contiguous nucleotides of the
sequence of SEQ ID NOS: 1, 3, or 4, wherein said nucleotide
sequence encodes a protein flowering regulatory activity; and (c) a
nucleotide sequence having at least 80% sequence identity to the
sequence of SEQ ID NOS: 1, 3, or 4.
16. The plant of claim 18, wherein said promoter is a constitutive
promoter.
17. The plant of claim 18, wherein said promoter is a
tissue-preferred promoter.
18. The plant of claim 18, wherein said promoter is an inducible
promoter.
19. The method of 15, wherein said plant is a monocot.
20. The method of 16, wherein said monocot is maize.
21. The method of claim 15 wherein said nucleic acid molecule is a
vector for over-expression of RAP2.7.
22. The method of claim 18 wherein said plant matures earlier than
a plant which does not have the overexpression vector incorporated
therein.
23. The method of claim 15 wherein said nucleic acid molecule is a
vector for inhibiting expression of RAP2.7.
24. The method of claim 20 wherein said vector is an RNA
interference vector.
25. The method of claim 13 wherein said plant matures later than a
plant which does not have an RNA interference vector incorporated
therein.
26. An isolated polypeptide having flowering regulatory activity
and selected from the group consisting of: (a) a polypeptide
comprising the amino acid sequence set forth in SEQ ID NOS: 2; (b)
a polypeptide encoded by a nucleotide sequence comprising the
sequence set forth in SEQ ID NOS: 1, 3, or 4; (c) a polypeptide
encoded by a nucleotide sequence that has at least 80% sequence
identity to the sequence set forth in SEQ ID NOS: 1, 3, or 4. (d) a
polypeptide comprising an amino acid sequence having at least 90%
sequence identity to the sequence set forth in SEQ ID NO:2; and (e)
a polypeptide comprising an amino acid sequence of at least 30
consecutive amino acids of any of (a) through (d).
27. A nucleotide construct comprising: a nucleic acid molecule
encoding an amino acid of claim 23, wherein said nucleic acid
molecule is operably linked to a promoter that drives expression in
a host cell.
28. An isolated nucleic acid molecule that encodes a regulatory
element having regulatory activity for flowering time, said nucleic
acid molecule being selected from the group consisting of: (a) a
nucleic acid molecule comprising the sequence set forth in SEQ ID
NOS: 5, 6, 7, or 8; (b) a nucleic acid molecule comprising a
sequence having at least 80% sequence identity to the nucleotide
sequence set forth in SEQ ID NOS: 5, 6, 7, or 8; and (c) a nucleic
acid molecule comprising a sequence of at least 50 consecutive
nucleic acids of any of (a) through (c).
29. A vector comprising the nucleic acid molecule of claim 28.
30. A plant cell having stably incorporated in its genome the
nucleic acid molecule of claim 27.
31. The plant cell of claim 30, wherein said plant cell is from a
monocot plant.
32. The plant cell of claim 31, wherein said monocot plant is
maize.
33. A plant having stably incorporated in its genome the nucleic
acid molecule of claim 27.
34. A method for changing maturity of a plant, said method
comprising: stably introducing into the genome of a plant, at least
one nucleotide construct comprising a nucleic acid molecule
operably linked to a heterologous promoter that drives
transcription in a plant cell, wherein said nucleic acid molecule
encodes a regulatory element having regulatory activity on
flowering time and is selected from the group consisting of: (a) a
nucleic acid molecule comprising the sequence set forth in SEQ ID
NOS: 5, 6, 7, or 8; (b) a nucleic acid molecule comprising a
sequence having at least 80% sequence identity to the nucleotide
sequence set forth in SEQ ID NOS: 5, 6, 7, or 8; and (c) a nucleic
acid molecule comprising a sequence of at least consecutive nucleic
acids of any of (a) through (c).
35. A method of changing the maturity of a plant comprising,
introducing to a plant cell, a VGT1 expression construct,
comprising, a VGT1 regulatory element which regulates RAP2.7
expression, operably linked to a promoter.
36. A method of identifying a plant with altered maturity time,
comprising: assaying said plant for a VGT1 polymorphism, wherein
said polymorphism is identified in FIG. 8 wherein said VGT1
polymorphism is associated with a earlier maturity in said plant
than the maturity of a plant without said VGT1 polymorphism.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation application of U.S. application Ser.
No. 12/276,961, filed Nov. 24, 2008 which application claims
priority of U.S. application Ser. No. 11/190,339 filed Jul. 27,
2005, now U.S. Pat. No. 7,479,584 issued Jan. 20, 2009 and
Provisional Application Ser. No. 60/592,268 filed Jul. 29, 2004,
all of which are hereby incorporated by reference in their
entirety.
FIELD
[0002] This invention is related to compositions and methods for
affecting flowering time in plants.
BACKGROUND
[0003] Plants have two basic growth modes during their life
cycles--vegetative growth and flower and seed growth. Above ground
vegetative growth of the plant develops from the apical meristem.
This vegetative meristem gives rise to all of the leaves that are
found on the plant. The plant will maintain its vegetative growth
pattern until the apical meristem undergoes a change. This change
actually alters the identity of the meristem from a vegetative to
an inflorescence meristem. The inflorescence meristem produces
small leaves before it next produces floral meristems. It is the
floral meristem from which the flower develops.
[0004] From a genetic perspective, two phenotypic changes that
control vegetative and floral growth are programmed in the plant.
The first genetic change involves the switch from the vegetative to
the floral state. If this genetic change is not functioning
properly, then flowering will not occur. The second genetic event
follows the commitment of the plant to form flowers. The
observation that the organs of the plant develop in a sequential
manner suggests that a genetic mechanism exists in which a series
of genes are sequentially turned on and off.
[0005] Flowering time is an important agronomic trait in cultivated
plant species as it determines in large measure the growing region
of adaptation. Most angiosperm species are induced to flower in
response to environmental stimuli such as day length and
temperature, and internal cues, such as age. Genetic analysis
revealed that there are several types of mutants that alter
flowering time.
[0006] Studies of two distantly related dicotyledons, Arabidopsis
thaliana and Antirrhinum majus, has led to the identification of
three classes of homeotic genes, acting alone or in combination to
determine floral organ identity (Bowman, et al., (1991) Development
112:1; Carpenter and Coen, (1990) Genes Devl. 4:1483;
Schwarz-Sommer, et al., (1990) Science 250:931). Several of these
genes are transcription factors whose conserved DNA-binding domain
has been designated the MADS box (Schwarz-Sommer, et al.,
supra).
[0007] Earlier acting genes that control the identity of flower
meristems have also been characterized. Flower meristems are
derived from inflorescence meristems in both Arabidopsis and
Antirrhinum. Two factors that control the development of
meristematic cells into flowers are known. In Arabidopsis, the
factors are the products of the LEAFY gene (Weigel, et al., (1992)
Cell 69:843) and the APETALA1 gene (Mandel, et al., (1992) Nature
360:273). When either of these genes is inactivated by mutation,
structures combining the properties of flowers and inflorescence
develop (Weigel, et al., supra; Irish and Sussex, (1990) Plant Cell
2:741). In Antirrhinum, the homologue of the Arabidopsis LEAFY gene
is FLORICAULA (Coen, et al., (1990) Cell 63:1311) and that of the
APETALA1 gene is SQUAMOSA (Huijser, et al., (1992) EMBO J.
11:1239). The latter pair contains MADS box domains.
[0008] Genetic studies in Arabidopsis thaliana have identified five
genes (APETALA1 (AP1), APETALA2 (AP2), APETALA3 (AP3), PISTILLATA
(PI) and AGAMOUS (AG)) that are involved in the specification of
floral organ identity. Mutations in these genes result in homeotic
transformation of one organ type into another, much like the
homeotic selector genes in animal development. These five genes act
in spatially localized domains in a flower and in different
combinations to specify the development of the sepals, petals,
stamens and carpels. All five genes encode proteins which appear to
function as transcription factors. Four of these proteins are
members of the MADS domain family of dimeric transcription factors.
MADS domain proteins are found in many organisms including yeast,
mammals, insects and plants. The fifth protein, AP2, is a member of
another class of DNA binding proteins which may be unique to
plants
[0009] APETALA2 (AP2) plays an important role in the control of
Arabidopsis flower and seed development and encodes a putative
transcription factor that is distinguished by a novel DNA binding
motif referred to as the AP2 domain. The AP2 domain containing or
RAP2 (related to AP2) family of proteins is encoded by a minimum of
12 genes in Arabidopsis. The RAP2 genes encode two classes of
proteins, AP2-like and EREBP-like, that are defined by the number
of AP2 domains in each polypeptide as well as by two sequence
motifs referred to as the YRG and RAYD elements that are located
within each AP2 domain. RAP2 genes are differentially expressed in
flower, leaf, inflorescence stem, and root. Moreover, the
expression of at least three RAP2 genes in vegetative tissues are
controlled by AP2. Thus, unlike other floral homeotic genes, AP2 is
active during both reproductive and vegetative development.
[0010] Maize is a monocotyledonous plant species and belongs to the
grass family. It is unusual for a flowering plant as it has
unisexual inflorescences. The male inflorescence (tassel) develops
in a terminal position, whereas the female inflorescences (ears)
grow in the axil of vegetative leaves. The inflorescences, as
typical for grasses, are composed of spikelets. In the case of
maize each spikelet contains two florets (the grass flower)
enclosed by a pair of bracts (inner and outer glume). A number of
genes have been identified which modify flowering time in maize
including Id1 and DLF.
[0011] There is increasing incentive by those working in the field
of plant biotechnology to successfully genetically engineer plants,
including the major crop varieties. One genetic modification that
would be economically desirable would be to accelerate the
flowering time of a plant. Induction of flowering is often the
limiting factor for growing crop plants. One of the most important
factors controlling induction of flowering is day length, which
varies seasonally as well as geographically. There is a need to
develop a method for controlling and inducing flowering in plants,
regardless of the locale or the environmental conditions, thereby
allowing production of crops, at any given time. Since most crop
products (e.g. seeds, grains, fruits), are derived from flowers,
such a method for controlling flowering would be economically
invaluable.
[0012] It is an object of the present invention to provide methods
and compositions for affecting flowering time in plants.
[0013] It is yet another object of the invention to provide novel
nucleotide sequences isolated from maize which encode proteins
which affect flowering time in plants.
[0014] It is yet another object of the invention to provide maize
RAP2.7 genes which affect flowering time and internode length in
maize.
[0015] It is yet another object of the invention to provide DNA
regulatory factors which enhance/inhibit the ability of RAP2.7 to
regulate flowering time.
[0016] It is yet another object of the invention to provide methods
and compositions including nucleotide constructs, vectors,
transgenic cells and plants with altered flowering characteristics
as described herein.
[0017] It is yet another object of the invention to provide markers
for identification of mutant plants which may have altered
flowering time by the presence of marker VGT1 sequences.
SUMMARY
[0018] Compositions and methods involved in the modulation of
flowering in plants are provided. The compositions include nucleic
acid molecules isolated from maize which encode RAP2.7 proteins.
Amino acid sequences of these proteins are also provided. Further,
polynucleotides having nucleic acid sequences encoding maize RAP2.7
proteins are also provided. These proteins and the nucleotide
sequences encoding them provide an opportunity to manipulate
maturity of plants. When polynucleotide sequences encoding the
RAP2.7 gene product are overexpressed flowering time is delayed,
and when the product is inhibited flowering is earlier.
[0019] Typically maturity of a given plant is changed with crossing
of earlier and later maturity plants. Agronomic traits such as
yield or lodging resistance are often lost in the process. The
compositions of the invention provide for the ability to make
significant changes in maturity while keeping other vegetative and
reproductive characteristics similar using a transgenic
approach.
[0020] The invention includes methods for manipulating the maturity
of plants using polynucleotide sequences that were isolated from
maize (Zea mays). These sequences alone, or in combination with
other sequences, can be used to control plant maturity and thus
area of adaptation. In another aspect of the present invention,
nucleotide constructs such as expression cassettes and
transformation vectors comprising the isolated nucleotide sequences
are disclosed. The transformation vectors can be used to transform
plants and express the flower modulation control genes in the
transformed cells. In this manner, the maturity of plants as well
as area of adaptation can be controlled. Transformed cells as well
as regenerated transgenic plants and seeds containing and
expressing the isolated polynucleotide sequences and protein
products are also provided.
[0021] Also according to the invention, a novel DNA sequence termed
(VGT1) has been identified which is a 2 kb region on maize
chromosome 8L. VGT1 acts as a CIS interaction type non-coding RNA
sequence for maize RAP2.7. In mutants with early flowering, VGT1
may interact or repress the expression level of RAP2.7 causing down
regulation of RAP2.7 and early flowering. Thus the invention also
comprises nucleotide sequences encoding a VGT1 DNA factor which
interacts either directly or indirectly with RAP2.7 in modulating
the flowering time in plants. The invention includes nucleotide
sequences, polymorphisms, "expression-type" constructs with the
VGT1 sequences operably linked to promoters regions for
transcription of the same, transgenic cells and plants with altered
flowering time. The VGT1 sequences and the alternate forms thereof
may also be used as markers to identify plants with flowering that
may be different from wild type.
[0022] For any of the sequences disclosed herein, the
polynucleotide of the invention or at least 20 contiguous bases
therefrom may be used as probes to isolate and identify similar
genes in other plant species. The sequences disclosed may also be
used to isolate regulatory elements and promoter sequences that are
natively associated with the polynucleotides disclosed herein to
give spatial and temporal expression of operatively linked
sequences to flowering in plants.
DESCRIPTION OF THE FIGURES
[0023] FIG. 1 is a graph depicting the levels of RAP2.7 expression
at Day: 14, day 20 and day 27.
[0024] FIG. 2 is an illustration of the over-expression vector used
in transformation, showing the portion between the right and left
T-DNA borders (RB, LB). The transformation vector for RAP2.7
over-expression, PHP20922, was created by electroporating the JT
vector PHP20921 into Agrobacterium. The Invitrogen (Carlsbad,
California) Gateway technology was used to create PHP20921.
Specifically, the RAP2.7 coding region was first amplified by PCR
with 5'-primer (ggggacaagtttgtacaaaaaagcaggctatgcagttggatctgaacgt)
and 3' primer (ggggaccactttgtacaagaaagctgggttcagcggggatggtgatg).
These primers contain Gateway attB recombination sites. The PCR
product was confirmed by sequencing and cloned into a Gateway
vector pDONR221 via a BP recombination reaction as described by the
vendor (Invitrogen, Carlsbad, Calif.). This resulted in the entry
clone, PHP20923. Two other entry clones, PHP20830 containing the
rice actin promoter, PHP20234 containing the pinII terminator, have
been previously created using similar cloning strategies. A
destination vector PHP20909 was also created from pDESTR4-R3 vector
(Invitrogen, Carlsbad, Calif.) by inserting an expression cassette
of the CaMV35S promoter driving the herbicide resistance gene Bar
followed by a pinII terminator. The four vectors, PHP20923, 20830,
20234 and 20909, were then used to create the JT vector, PHP20921,
via a LR recombination reaction following vendor's instructions
(Invitrogen, Carlsbad, Calif.).
[0025] FIG. 3 is the deduced amino acid sequence of RAP2.7 from
C22-4 allele. The two putative AP2 domains are highlighted in bold,
whereas the linker region between them is italicized. The well
conserved YRG and RAYD motifs are underlined, although there is an
R to K substitution in the second RAYD motif.
[0026] FIG. 4 is a GAP alignment between the genomic RAP2.7 from
B73 compared to the sequence of RAP2.7 of M017. Gap Weight: 50;
Average Match: 10.000; Length Weight: 3; Average Mismatch: 0.000;
Quality: 59308; Length: 8586; Ratio: 7.730; Gaps: 41; Percent
Similarity: 87.482, Percent Identity: 87.482. Vladutu, et al.,
(1999) Genetics 153:993-1007.
[0027] FIG. 5 is an illustration of construction of the RAP2.7 gene
fragments for the RNA interference vector. Fragment TR1
(Truncated1) was created by PCR using forward
(ggatccgatctgaacgtggccgag) and reverse primers
(gaattcctaggcagctgttcttgtctctttg) corresponding to roughly 2/3 of
the coding sequence starting from 9 bp downstream of ATG. Fragment
IR1 (Inverted1) was generated similarly by PCR with forward
(gcggccgcgatctgaacgtggccgag) and reverse primers
(gaattctgtgggactcccagcggcctgtgc) starting from the same position as
TR1, although IR1 is only half the length of TR1.
[0028] FIG. 6 is an illustration of the vector used in
transformation showing only the portion between the right and left
T-DNA borders (RB, LB). As described in FIG. 5, fragments
corresponding to truncated coding regions to be used for the RNA
interference construct were generated by as TR1, flanked by BamH1
and EcoR1, and IR1, flanked by Notl and EcoR1 restriction sites.
Vector PHP16501 containing the rice actin promoter was linearized
with Notl and BamH1. In a 3-piece ligation, TR1 was cloned in
downstream of Notl followed by IR1 in reverse orientation. The
resulting cassette vector, PHP21767, was then cloned into a JT
vector containing the CaMV35S promoter driving the herbicide
resistance gene Bar. The transformation vector for RAP2.7 down
regulation, PHP21842, was generated by electroporating PHP21798
into Agrobacterium.
[0029] FIG. 7 is the data showing the fine genetic and physical
mapping of VGT1. First row indicates physical distance (in kb) from
Rap2.7, based on sequence derived from the relevant Mo17 BAC clone
from library. Second row indicates the type of molecular marker.
Third row indicates the name of the molecular markers. Rows from 4
to 21 indicate the genotype of parental lines (N28 and C22-4) and
of the 17 segmental QTL-Nearly Isogenic Lines. The VGT1 column
shows where the QTL was mapped. The last two columns provide the
phenotypic scores for DPS (Days to Pollen Shed) and ND (plant node
number).
[0030] FIG. 8 is a graph showing the results of association mapping
for markers developed at the Vgt1-Rap2.7 region and phenotypic data
for flowering time collected in Bologna (2002 and 2003) among a set
of 96 maize inbred lines. Statistical association is expressed as P
from ANOVA tests.
[0031] FIG. 9 is an alignment of the VGT1 sequences from all four
lines, including a consensus sequence line N28 was identical to
B73.
DETAILED DESCRIPTION
[0032] The present invention provides, inter alia, compositions and
methods for manipulating flowering time in plants. As used herein
the term "flowering time" or maturity shall mean the time at which
a plant reaches physiological maturity and is capable of
reproducing.
[0033] The compositions comprise nucleic acid molecules comprising
sequences of plant genes and the polypeptides encoded thereby as
well as regulatory factors which are non-coding. Particularly, the
nucleotide and amino acid sequences for a maize RAP2.7 (related to
AP2 domain containing) gene are provided. Three RAP2.7 encoding
nucleotide sequences are provided at SEQ ID NOS: 1 (cDNA), 3
(genomic) and 4 (genomic) with the corresponding protein at SEQ ID
NO: 2. Three VGT1 nucleotide sequences are provided at SEQ ID NOS:
5, 6 and 7 with the consensus sequence at SEQ ID NO:8. As discussed
in more detail below, the sequences of the invention are involved
in many basic biochemical pathways that regulate flowering time and
maturity in plants. Thus, methods are provided for the expression
of these sequences in a host plant to modulate plant flowering.
Some of the methods involve stably transforming a plant with a
nucleotide sequence capable of modulating plant flowering operably
linked with a promoter capable of driving expression (or
transcription) of a nucleotide sequence in a plant cell.
[0034] Promoter and other regulatory elements which are natively
associated with these genes can be easily isolated using the
sequences and methods described herein with no more than routine
experimentation. These sequences can be used to identify promoter,
enhancer or other signaling signals in the regulatory regions of
RAP2.7 encoding sequences. These regulatory and promoter elements
provide for temporal and spatial expression of operably linked
sequences with flowering in a plant. Methods are provided for the
regulated expression of a nucleotide sequence of interest that is
operably linked to the promoter regulatory sequences disclosed
herein. Nucleotide sequences operably linked to the promoter
sequences are transformed into a plant cell. Exposure of the
transformed plant to a stimulus such as the timing of flowering
induces transcriptional activation of the nucleotide sequences
operably linked to these promoter regulatory sequences.
[0035] By "heterologous nucleotide sequence" is intended a sequence
that is not naturally occurring with the referenced sequence. While
the referenced nucleotide sequence is heterologous to the promoter
sequence or vice versa, it may be homologous, or native, or
heterologous, or foreign, to the plant host.
[0036] By "operably linked" is intended a functional linkage
between a promoter sequence and a second sequence, wherein the
promoter sequence initiates and mediates transcription of the DNA
sequence corresponding to the second sequence. Generally, operably
linked means that the nucleic acid sequences being linked are
contiguous and, where necessary to join two protein coding regions,
contiguous and in the same reading frame. In the case of a DNA
regulatory factor the nucleic acid sequences are transcribed
only.
[0037] A polypeptide is said to have RAP2.7-like activity when it
has one or more of the properties of the native protein. It is
within the skill in the art to assay protein activities obtained
from various sources to determine whether the properties of the
proteins are the same. In so doing, one of skill in the art may
employ any of a wide array of known assays including, for example,
biochemical and/or pathological assays. For example, one of skill
in the art could readily produce a plant transformed with a RAP2.7
polypeptide variant and assay a property of native RAP2.7 protein
in that plant material to determine whether a particular RAP2.7
property was retained by the variant.
[0038] The compositions and methods of the invention are involved
in biochemical pathways and as such may also find use in the
activation or modulation of expression of other genes, including
those involved in other aspects of flowering time.
[0039] Although there is some conservation among these genes,
proteins encoded by members of these gene families may contain
different elements or motifs or sequence patterns that modulate or
affect the activity, subcellular localization, and/or target of the
protein in which they are found. Such elements, motifs or sequence
patterns may be useful in engineering novel enzymes for modulating
gene expression in particular tissues. By "modulating" or
"modulation" is intended that the level of expression of a gene may
be increased or decreased relative to genes driven by other
promoters or relative to the normal or uninduced level of the gene
in question.
[0040] According to the invention, overexpression of maize RAP2.7
caused flowering that was later than normal in plants as well as an
increased number of leaves (nodes) produced prior to flowering
resulting in taller plant stature. Inhibition of maize RAP2.7
caused maturation or flowering that was earlier than normal. Also
the presence of mutant VGT1 correlated with earlier flowering as
well, thus leading to the concept that VGT1 acts as direct or
indirect enhancer/regulator of RAP2.7. Expression of the proteins
encoded by RAP2.7 encoding sequences or transcription of the VGT1
regulatory element can be used to modulate or regulate the
expression of proteins in these flowering pathways and other
directly or indirectly affected pathways. Hence, the compositions
and methods of the invention find use in altering plant flowering
and maturity. In other embodiments, fragments of the sequences are
used to confer desired properties to synthetic constructs for use
in regulating plant maturity and flowering.
[0041] The present invention provides for isolated nucleic acid
molecules comprising nucleotide sequences encoding the amino acid
sequence shown in SEQ ID NO: 2 as well as their conservatively
modified variants. Further provided are polypeptides having an
amino acid sequence encoded by a nucleic acid molecule described
herein, for example those polypeptides comprising the sequences set
forth in SEQ ID NO: 1, 3 and 4, and fragments and variants
thereof.
[0042] The present invention further provides for an isolated
nucleic acid molecule comprising the sequences shown in SEQ ID NO:
1, 3, 4, 5, 6, 7 or 8.
[0043] The invention encompasses isolated or substantially purified
nucleic acid or protein compositions. An "isolated" or "purified"
nucleic acid molecule or protein, or biologically active portion
thereof, is substantially free of other cellular material, or
culture medium when produced by recombinant techniques, or
substantially free of chemical precursors or other chemicals when
chemically synthesized. In some embodiments, an "isolated" nucleic
acid is free of sequences (such as other protein-encoding
sequences) that naturally flank the nucleic acid (i.e., sequences
located at the 5' and 3' ends of the nucleic acid) in the genomic
DNA of the organism from which the nucleic acid is derived. For
example, in various embodiments, the isolated nucleic acid molecule
can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb,
0.4 kb, 0.3 kb, 0.2 kb, or 0.1 kb, or 50, 40, 30, 20 or 10
nucleotides that naturally flank the nucleic acid molecule in
genomic DNA of the cell from which the nucleic acid is derived. A
protein that is substantially free of cellular material includes
preparations of protein having less than about 30%, 20%, 10%, 5%,
(by dry weight) of contaminating protein. When the protein of the
invention or biologically active portion thereof is recombinantly
produced, culture medium may represent less than about 30%, 20%,
10% or 5% (by dry weight) of chemical precursors or
non-protein-of-interest chemicals.
[0044] Fragments and variants of the disclosed nucleotide sequences
which retain the functional properties of the encoded peptide or of
the non-coding RNA are encompassed by the present invention. By
"fragment" is intended a portion of the nucleotide sequence or a
portion of the amino acid sequence and hence protein encoded
thereby. Fragments of a nucleotide sequence may encode protein
fragments that retain the biological activity of the native protein
and hence affect flowering by retaining RAP2.7-like activity or may
include portions of non-coding regulatory element which retain the
RAP2.7 modulating activity of VGT1. Alternatively, fragments of a
nucleotide sequence that are useful as hybridization probes
generally do not encode fragment proteins retaining biological
activity. Thus, fragments of a nucleotide sequence may range from
at least about 20 nucleotides, about 50 nucleotides, about 100
nucleotides and up to the full-length nucleotide sequence encoding
the proteins or regulating RAP2.7 of the invention.
[0045] A fragment of a RAP2.7 nucleotide sequence that encodes a
biologically active portion of a RAP2.7 protein of the invention
will encode at least 12, 25, 30, 50, 75, 100, 125, 150, 175, 200,
225, 250, 275, 300, 325, 350, 375, 400, 425 or 450, contiguous
amino acids, or up to the total number of amino acids present in a
full-length RAP2.7 protein of the invention.
[0046] A fragment of a VGT1 nucleotide sequence that encodes a
biologically active non-transcribed RNA of the invention will
encode at least 12, 25, 30, 50, 75, 100, 125, 150, 175, 200, 225,
250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550,
575, 600, 625, 650, 675 or 680 contiguous nucleotide bases or up to
the total number of nucleotides present in a full-length VGT1
regulatory element of the invention.
[0047] Fragments of a RAP2.7 or VGT1 nucleotide sequence that are
useful as hybridization probes or PCR primers generally need not
encode a biologically active portion of a protein or RNA. Thus, a
fragment of a RAP2.7 or VGT1 nucleotide sequence may encode a
biologically active portion of a RAP2.7 protein or a biologically
active non-coding RNA, or it may be a fragment that can be used as
a hybridization probe or PCR primer using methods disclosed below.
A biologically active portion of a RAP2.7 protein or VGT1
regulatory element can be prepared by isolating a portion of the
RAP2.7 or VGT1 nucleotide sequences of the invention, expressing
the encoded portion of the Rap2.7 or the active portion of the VGT1
regulatory element (e.g., by recombinant expression in vitro), and
assessing the activity of the encoded portion of the RAP2.7 protein
or the regulating ability of the regulatory element on RAP2.7.
Nucleic acid molecules that are fragments of a RAP2.7 or VGT1
nucleotide sequence comprise at least 16, 20, 25, 30, 40, 50, 60,
70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450,
500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,
1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,
2300 or 2400 nucleotides, or up to the number of nucleotides
present in a full RAP2.7 or VGT1 nucleotide sequence disclosed
herein.
[0048] By "variants" is intended substantially similar sequences.
For nucleotide sequences, conservative variants include those
sequences that, because of the degeneracy of the genetic code,
encode the amino acid sequence of one of the polypeptides of the
invention. Naturally occurring allelic variants such as these can
be identified with the use of well-known molecular biology
techniques, as, for example, with polymerase chain reaction (PCR)
and hybridization techniques as outlined below. Variant nucleotide
sequences also include synthetically-derived nucleotide sequences,
such as those generated, for example, by using site-directed
mutagenesis but which still encode a RAP2.7 protein or VGT1
regulatory element of the invention. Generally, variants of a
particular nucleotide sequence of the invention will have at least
40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, or about 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence
identity to that particular nucleotide sequence as determined by
sequence alignment programs described elsewhere herein using
default parameters.
[0049] By "variant" protein is intended a protein derived from the
native protein by deletion (so-called truncation) or addition of
one or more amino acids to the N-terminal and/or C-terminal end of
the native protein; deletion or addition of one or more amino acids
at one or more sites in the native protein or substitution of one
or more amino acids at one or more sites in the native protein.
Variant proteins encompassed by the present invention are
biologically active, that is they continue to possess the desired
biological activity of the native protein, hence they will continue
to possess at least one activity possessed by the native RAP2.7
protein. Such variants may result from, for example, genetic
polymorphism or from human manipulation. Biologically active
variants of a RAP2.7 native protein of the invention will have at
least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, 86%,
87%, 88%, 89%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or more sequence identity to the amino acid sequence for the
native protein as determined by sequence alignment programs
described elsewhere herein using default parameters. A biologically
active variant of a protein of the invention may differ from that
protein by as few as 1-15 amino acid residues, as few as 1-10, as
few as 5, as few as 4, 3, 2 or even 1 amino acid residue. As used
herein, reference to a particular nucleotide or amino acid sequence
(a RAP2.7 or VGT1 sequence) shall include all modified variants as
described supra.
[0050] The proteins of the invention may be altered in various ways
including amino acid substitutions, deletions, truncations, and
insertions. Methods for such manipulations are generally known in
the art. For example, amino acid sequence variants of RAP2.7
proteins can be prepared by mutations in the DNA. Methods for
mutagenesis and nucleotide sequence alterations are well known in
the art. See, for example, Kunkel, (1985) Proc. Nad. Acad. Sci. USA
82:488-492; Kunkel, et al., (1987) Methods in Enzymol. 154:367-382;
U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques
in Molecular Biology (MacMillan Publishing Company, New York) and
the references cited therein. Guidance as to appropriate amino acid
substitutions that do not affect biological activity of the protein
of interest may be found in the model of Dayhoff, et al., (1978)
Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,
Washington, D.C.), herein incorporated by reference. Conservative
substitutions, such as exchanging one amino acid with another
having similar properties, may be made.
[0051] Thus, the nucleotide sequences of the invention include both
naturally occurring sequences as well as mutant forms. Likewise,
the proteins of the invention encompass both naturally-occurring
proteins as well as variations and modified forms thereof. Such
variants whether protein or nucleotide will continue to possess the
desired RAP2.7 or VGT1-like activity. It is recognized that
variants need not retain all of the activities and/or properties of
the native RAP2.7 or VGT1. Obviously, the mutations that will be
made in the DNA encoding the RPA2.7 variant must not place the
sequence out of reading frame and in some embodiments will not
create complementary regions that could produce secondary mRNA
structure. See, EP Patent Application Publication Number
75,444.
[0052] The deletions, insertions and substitutions of the protein
or nucleotide sequences encompassed herein are not expected to
produce radical changes in the characteristics of the RAP 2.7
protein or VGT1 regulatory element. However, when it is difficult
to predict the exact effect of the substitution, deletion or
insertion in advance of doing so, one skilled in the art will
appreciate that the effect will be evaluated by routine screening
assays. That is, the activity of RAP2.7 polypeptides or VGT1 can be
evaluated by either a change in flowering time or maturity when the
encoded protein or regulatory element is altered.
[0053] Variant nucleotide sequences and proteins also encompass
sequences and proteins derived from a mutagenic and recombinogenic
procedure such as DNA shuffling. With such a procedure, one or more
different RAP2.7 or VGT1 coding sequences can be manipulated to
create a new RAP2.7 or VGT1 possessing the desired properties. In
this manner, libraries of recombinant polynucleotides are generated
from a population of related sequence polynucleotides comprising
sequence regions that have substantial sequence identity and can be
homologously recombined in vitro or in vivo. For example, using
this approach, sequence motifs encoding a domain of interest may be
shuffled between the RAP2.7 encoding polynucleotide of the
invention and other known flowering genes to obtain a new gene
coding for a protein with an improved property of interest, such as
an increased K.sub.m in the case of an enzyme. Strategies for such
DNA shuffling are known in the art. See, for example, Stemmer,
(1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer, (1994)
Nature 370:389391; Crameri, et al., (1997) Nature Biotech.
15:436-438; Moore, et al., (1997) J. Mol. Biol. 272:336-347; Zhang,
et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri, et
al., (1998) Nature 391:288-291 and U.S. Pat. Nos. 5,605,793 and
5,837,458.
[0054] The compositions of the invention also include isolated
nucleic acid molecules comprising the promoter nucleotide sequences
natively associated with the RAP2.7 polynucleotides. By "promoter"
is intended a regulatory region of DNA usually comprising a TATA
box capable of directing RNA polymerase II to initiate RNA
synthesis at the appropriate transcription initiation site for a
particular coding sequence. A promoter may additionally comprise
other recognition sequences generally positioned upstream or 5' to
the TATA box, referred to as upstream promoter elements, which
influence the transcription initiation rate.
[0055] The nucleotide sequences of the invention can be used to
isolate corresponding sequences from other organisms, particularly
other plants, more particularly other crop plants. In this manner,
methods such as PCR, hybridization, and the like can be used to
identify such sequences based on their sequence homology to the
sequences set forth herein. Sequences isolated based on their
sequence identity to the nucleotide sequences set forth herein or
to fragments thereof are encompassed by the present invention. Such
sequences include sequences that are orthologs of the disclosed
sequences. By "orthologs" is intended genes derived from a common
ancestral gene and which are found in different species as a result
of speciation. Genes found in different species are considered
orthologs when their nucleotide sequences and/or their encoded
protein sequences share substantial identity as defined elsewhere
herein. Functions of orthologs are often highly conserved among
species. Thus, isolated sequences that have RAP2.7 or VGT1 activity
or encode a RAP2.7 protein and which hybridize under stringent
conditions to RAP2.7 or VGT1 sequences disclosed herein, or to
fragments thereof, are encompassed by the present invention.
[0056] In a PCR approach, oligonucleotide primers can be designed
for use in PCR reactions to amplify corresponding DNA sequences
from cDNA or genomic DNA extracted from any plant of interest.
Methods for designing PCR primers and PCR cloning are generally
known in the art and are disclosed in Sambrook, et al., (1989)
Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor
Laboratory Press Plainview, N.Y.). See also, Innis, et al., eds.
(1990) PCR Protocols: A Guide to Methods and Applications (Academic
Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies
(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR
Methods Manual (Academic Press, New York). Known methods of PCR
include, but are not limited to, methods using paired primers,
nested primers, single specific primers, degenerate primers,
gene-specific primers, vector-specific primers,
partially-mismatched primers, and the like.
[0057] In hybridization techniques, all or part of a known
nucleotide sequence is used as a probe that selectively hybridizes
to other corresponding nucleotide sequences present it a population
of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or
cDNA libraries) from a chosen organism. The hybridization probes
may be genomic DNA fragments, cDNA fragments, RNA fragments or
other oligonucleotides and may be labeled with a detectable group
such as .sup.32P or any other detectable marker. Thus, for example,
probes for hybridization can be made by labeling synthetic
oligonucleotides based on the disease-resistant sequences of the
invention. Methods for preparation of probes for hybridization and
for construction of cDNA and genomic libraries are generally known
in the art and are disclosed in Sambrook, et al., (1989) Molecular
Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Plainview, N.Y.).
[0058] For example, an entire sequence disclosed herein, or one or
more portions thereof, may be used as a probe capable of
specifically hybridizing to corresponding flowering or maturity
regulating sequences, including promoters and messenger RNAs. To
achieve specific hybridization under a variety of conditions, such
probes include sequences that are unique among flowering or
maturity related sequences and may be at least about 10 or 15 or 17
nucleotides in length or at least about 20 or 22 or 25 nucleotides
in length. Such probes may be used to amplify corresponding
sequences from a chosen organism by PCR. This technique may be used
to isolate additional coding sequences from a desired organism or
as a diagnostic assay to determine the presence of coding sequences
in an organism. Hybridization techniques include hybridization
screening of plated DNA libraries (either plaques or colonies; see,
for example, Sambrook, et al., (1989) Molecular Cloning: A
Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,
Plainview, N.Y.).
[0059] Hybridization of such sequences may be carried out under
stringent conditions. By "stringent conditions" or "stringent
hybridization conditions" is intended conditions under which a
probe will hybridize to its target sequence to a detectably greater
degree than to other sequences (e.g., at least 2-fold over
background). Stringent conditions are sequence-dependent and will
be different under different circumstances. By controlling the
stringency of the hybridization and/or washing conditions, target
sequences that are 100% complementary to the probe can be
identified (homologous probing). Alternatively, stringency
conditions can be adjusted to allow some mismatching in sequences
so that lower degrees of similarity are detected (heterologous
probing). Generally, a probe is less than about 1000 nucleotides in
length or less than 500 nucleotides in length.
[0060] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3. Incubation should be at a temperature of least about
30.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at
least about 60.degree. C. for long probes (e.g., greater than 50
nucleotides). Stringent conditions may also be achieved with the
addition of destabilizing agents such as formamide. Exemplary low
stringency conditions include hybridization with a buffer solution
of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate)
at 37.degree. C., and a wash in 1.times. to 2.times.SSC
(20.times.SSC=3.0 M NaCl, 0.3 M trisodium citrate) at 50 to
55.degree. C. Exemplary moderate stringency conditions include
hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at
37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to
60.degree. C. Exemplary high stringency conditions include
hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C.,
and a wash in 0.1.times.SSC at 60 to 65.degree. C. for 20 minutes.
Optionally, wash buffers may comprise about 0.1% to about 1% SDS.
Duration of hybridization is generally less than about 24 hours,
usually about 4 to about 12 hours.
[0061] Specificity is typically a function of post-hybridization
washes, the critical factors being the ionic strength and
temperature of the final wash solution. For DNA-DNA hybrids, the
T.sub.m, can be approximated from the equation of Meinkoth and
Wahl, (1984) Anal. Biochem. 138:267-284: Trn=81.5.degree. C.+16.6
(log M)+0.41 (% GC)--0.61 (% form)-500/L; where M is the molarity
of monovalent cations, % GC is the percentage of guanosine and
cytosine nucleotides in the DNA, % form is the percentage of
formamide in the hybridization solution, and L is the length of the
hybrid in base pairs. The T.sub.m, is the temperature (under
defined ionic strength and pH) at which 50% of a complementary
target sequence hybridizes to a perfectly matched probe. T.sub.m,
is reduced by about 1.degree. C. for each 1% of mismatching; thus,
T.sub.m, hybridization and/or wash conditions can be adjusted to
hybridize to sequences of the desired identity. For example, if
sequences with >90% identity are sought, the T.sub.m, can be
decreased 10.degree. C. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (Tm) for the specific sequence and its complement at a
defined ionic strength and pH. However, severely stringent
conditions can utilize a hybridization and/or wash at 1, 2, 3 or
4.degree. C. lower than the thermal melting point (T.sub.m);
moderately stringent conditions can utilize a hybridization and/or
wash at 6, 7, 8, 9 or 10.degree. C. lower than the thermal melting
point (Tm); low stringency conditions can utilize a hybridization
and/or wash at 11, 12, 13, 14, 15 or 20.degree. C. lower than the
thermal melting point (Tm). Using the equation, hybridization and
wash compositions, and desired T.sub.m, those of ordinary skill
will understand that variations in the stringency of hybridization
and/or wash solutions are inherently described. If the desired
degree of mismatching results in a T.sub.m, of less than 45.degree.
C. (aqueous solution) or 32.degree. C. (formamide solution), the
SSC concentration may be increased so that a higher temperature can
be used. An extensive guide to the hybridization of nucleic acids
is found in Tijssen, (1993) Laboratory Techniques in Biochemistry
and Molecular Biology-Hybridization with Nucleic Acid Probes, Part
I, Chapter 2 (Elsevier, N.Y.); and Ausubel, et al., eds. (1995)
Current Protocols in Molecular Biology, Chapter 2 (Greene
Publishing and Wiley-Interscience, New York). See Sambrook, et al.,
(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring
Harbor Laboratory Press, Plainview, N.Y.).
[0062] In general, sequences that encode a RAP2.7 protein or which
encode a VGT1 regulatory element and which hybridize to the RAP2.7
or VGT1 sequences disclosed herein will be at least about 70%
homologous, and even about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
98%, 99% or more homologous with the disclosed sequences. That is,
the sequence identity of the sequences may be from about 70% or
75%, and even about 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical or higher, so that the sequences may
differ by only 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residue
or by 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,
3, 2 or 1 nucleic acid.
[0063] The following terms are used to describe the sequence
relationships between two or more nucleic acids or polynucleotides:
(a) "reference sequence," (b) "comparison window," (c) "sequence
identity," (d) "percentage of sequence identity" and (e)
"substantial identity."
[0064] (a) As used herein, "reference sequence" is a defined
sequence used as a basis for sequence comparison. A reference
sequence may be a subset or the entirety of a specified sequence;
for example, as a segment of a full-length cDNA or gene sequence or
the complete cDNA or gene sequence.
[0065] (b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. Generally, the
comparison window is at least 20 contiguous nucleotides in length,
and optionally can be 30, 40, 50, 100 or longer. Those of skill in
the art understand that to avoid a high similarity to a reference
sequence due to inclusion of gaps in the polynucleotide sequence a
gap penalty is typically introduced and is subtracted from the
number of matches.
[0066] Methods of alignment of sequences for comparison are well
known in the art. Thus, the determination of percent sequence
identity between any two sequences can be accomplished using a
mathematical algorithm. Non-limiting examples of such mathematical
algorithms are the algorithm of Myers and Miller, (1988) CABIOS
4:11-17; the local homology algorithm of Smith, et al., (1981) Adv.
Appl. Math. 2:482; the homology alignment algorithm of Needleman
and Wunsch, (1970) J. Mol. Biol. 48:443-453; the
search-for-similarity-method of Pearson and Lipman, (1988) Proc.
Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and
Altschul, (1990) Proc. Natl. Acad. Sci. USA 872264, modified as in
Karlin and Altschul, (1993) Proc. Natl. Acad. Sci. USA
90:5873-5877.
[0067] Computer implementations of these mathematical algorithms
can be utilized for comparison of sequences to determine sequence
identity. Such implementations include, but are not limited to:
CLUSTAL in the PC/Gene program (available from Intelligenetics,
Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP,
BESTFIT, BLAST, FASTA and TFASTA in the Wisconsin Genetics Software
Package, Version 8 (available from Genetics Computer Group (GCG),
575 Science Drive, Madison, Wis., USA). Alignments using these
programs can be performed using the default parameters. The CLUSTAL
program is well described by Higgins, et al., (1988) Gene
73:237-244; Higgins, et al., (1989) CABIOS 5:151-153; Corpet, et
al., (1988) Nucleic Acids Res. 16:10881-90; Huang, et al., (1992)
CABIOS 8:155-65 and Pearson, et al., (1994) Meth. Mol. Biol.
24:307-331. The ALIGN program is based on the algorithm of Myers
and Miller, (1988) supra. A PAM120 weight residue table, a gap
length penalty of 12, and a gap penalty of 4 can be used with the
ALIGN program when comparing amino acid sequences. The BLAST
programs of Altschul, et al., (1990) J. Mol. Biol. 215:403 are
based on the algorithm of Karlin and Altschul, (1990) supra. BLAST
nucleotide searches can be performed with the BLASTN program,
score=100, wordlength=12, to obtain nucleotide sequences homologous
to a nucleotide sequence encoding a protein of the invention. BLAST
protein searches can be performed with the BLASTX program,
score=50, wordlength=3, to obtain amino acid sequences homologous
to a protein or polypeptide of the invention. To obtain gapped
alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can
be utilized as described in Altschul, et al., (1997) Nucleic Acids
Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used
to perform an iterated search that detects distant relationships
between molecules. See Altschul, et al., (1997) supra. When
utilizing BLAST, Gapped BLAST or PSI-BLAST, the default parameters
of the respective programs (e.g., BLASTN for nucleotide sequences,
BLASTX for proteins) can be used (see information at
www.ncbi.nlm.nih.gov). Alignment may also be performed manually by
inspection.
[0068] Unless otherwise stated, sequence identity/similarity values
provided herein refer to the value obtained using GAP version 10
using the following parameters: % identity using GAP Weight of 50
and Length Weight of 3; % similarity using Gap Weight of 12 and
Length Weight of 4, or any equivalent program. By "equivalent
program" is intended any sequence comparison program that, for any
two sequences in question, generates an alignment having identical
nucleotide or amino acid residue matches and an identical percent
sequence identity when compared to the corresponding alignment
generated by GAP Version 10. GAP uses the algorithm of Needleman
and Wunsch, (1970) J. Mol. Biol. 48:443-453, to find the alignment
of two complete sequences that maximizes the number of matches and
minimizes the number of gaps. GAP considers all possible alignments
and gap positions and creates the alignment with the largest number
of matched bases and the fewest gaps. It allows for the provision
of a gap creation penalty and a gap extension penalty in units of
matched bases. GAP must make a profit of gap creation penalty
number of matches for each gap it inserts. If a gap extension
penalty greater than zero is chosen, GAP must, in addition, make a
profit for each gap inserted of the length of the gap times the gap
extension penalty. Default gap creation penalty values and gap
extension penalty values in Version 10 of the Wisconsin Genetics
Software Package for protein sequences are 8 and 2, respectively.
For nucleotide sequences the default gap creation penalty is 50
while the default gap extension penalty is 3. The gap creation and
gap extension penalties can be expressed as an integer selected
from the group of integers consisting of from 0 to 200. Thus, for
example, the gap creation and gap extension penalties can be 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65 or greater.
[0069] GAP presents one member of the family of best alignments.
There may be many members of this family, but no other member has a
better quality. GAP displays four figures of merit for alignments:
Quality, Ratio, Identity and Similarity. The Quality is the metric
maximized in order to align the sequences. Ratio is the quality
divided by the number of bases in the shorter segment. Percent
Identity is the percent of the symbols that actually match. Percent
Similarity is the percent of the symbols that are similar. Symbols
that are across from gaps are ignored. A similarity is scored when
the scoring matrix value for a pair of symbols is greater than or
equal to 0.50, the similarity threshold. The scoring matrix used in
Version 10 of the Wisconsin Genetics Software Package is BLOSUM62
(see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA
89:10915).
[0070] (c) As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences makes
reference to the residues in the two sequences that are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity." Means for making
this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif.).
[0071] (d) As used herein, "percentage of sequence identity" means
the value determined by comparing two optimally aligned sequences
over a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0072] (e)(i) The term "substantial identity" of polynucleotide
sequences means that a polynucleotide comprises a sequence that has
at least 70%, 80%, 85%, 90%, 95% or higher sequence identity
compared to a reference sequence using one of the alignment
programs described using standard parameters. One of skill in the
art will recognize that these values can be appropriately adjusted
to determine corresponding identity of proteins encoded by two
nucleotide sequences by taking into account codon degeneracy, amino
acid similarity, reading frame positioning, and the like.
Substantial identity of amino acid sequences for these purposes
normally means sequence identity of at least 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90% or at least 95% or higher sequence
identity.
[0073] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each other
under stringent conditions. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (Trr,) for the specific sequence at a defined ionic strength
and pH. However, stringent conditions encompass temperatures in the
range of about 1.degree. C. to about 20.degree. C. lower than the
T.sub.m, depending upon the desired degree of stringency as
otherwise qualified herein. Nucleic acids that do not hybridize to
each other under stringent conditions are still substantially
identical if the polypeptides they encode are substantially
identical. This may occur, e.g., when a copy of a nucleic acid is
created using the maximum codon degeneracy permitted by the genetic
code. One indication that two nucleic acid sequences are
substantially identical is when the polypeptide encoded by the
first nucleic acid is immunologically cross-reactive with the
polypeptide encoded by the second nucleic acid.
[0074] (e)(ii) The term "substantial identity" in the context of a
peptide indicates that a peptide comprises a sequence with at least
70%, 75%, 80%, 83%, 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98% or 99%
or higher sequence identity to the reference sequence over a
specified comparison window. Preferably, optimal alignment is
conducted using the homology alignment algorithm of Needleman and
Wunsch, (1970) J Mol. Biol. 48:443453. An indication that two
peptide sequences are substantially identical is that one peptide
is immunologically reactive with antibodies raised against the
second peptide. Thus, a peptide is substantially identical to a
second peptide, for example, where the two peptides differ only by
a conservative substitution. Peptides that are "substantially
similar" share sequences as noted above except that residue
positions that are not identical may differ by conservative amino
acid changes.
[0075] Methods for altering flowering time in a plant are provided.
In some embodiments, the methods involve stably transforming a
plant with a DNA construct comprising a nucleotide sequence of the
invention operably linked to a promoter that drives expression (or
transcription) in a plant. While the choice of promoter will depend
on the desired timing and location of expression of the nucleotide
sequences, desirable promoters include constitutive and tissue
specific promoters. These methods may find use in agriculture,
particularly in changing the maturity of a particular crop plant to
alter its area of adaptation. Thus, transformed plants, plant
cells, plant tissues and seeds thereof are provided by the present
invention.
[0076] In another embodiment, the methods of the present invention
involve identifying phenotypes associated with an altered flowering
time by loss of RAP2.7 or VGT1 activity in plants that contain
transposon insertions within the nucleotide sequences herein.
[0077] In some embodiments, the nucleic acid molecules comprising
RAP2.7 or VGT1 sequences of the invention are provided in
expression cassettes or nucleotide constructs for
expression/transcription in the plant of interest. Such cassettes
will include 5' and 3' regulatory sequences operably linked to a
RAP2.7 or VGT1 nucleotide sequence of the invention. By "operably
linked" is intended a functional linkage between a promoter and a
second sequence, wherein the promoter sequence initiates and
mediates transcription of the DNA sequence corresponding to the
second sequence. The cassette may additionally contain at least one
additional nucleotide sequence to be cotransformed into the
organism. Alternatively, the additional sequence(s) can be provided
on multiple expression cassettes or nucleotide construct.
[0078] Such an expression cassette or nucleotide construct is
provided with a plurality of restriction sites for insertion of the
RAP2.7 or VGT1 sequence to be under the transcriptional regulation
of the regulatory regions. The expression cassette or nucleotide
construct may additionally contain selectable marker genes. The
expression cassette will include in the 5'-3' direction of
transcription, a transcriptional and if necessary a translational
initiation region, a RAP2.7 or VGT1 nucleotide sequence of the
invention, and a transcriptional and if necessary, translational
termination region functional in plants. The transcriptional
initiation region, or promoter, may be native or analogous or
foreign or heterologous to the plant host. Additionally, the
promoter may be the natural sequence or alternatively a synthetic
sequence. By "foreign" is intended that the transcriptional
initiation region is not found in the native plant into which the
transcriptional initiation region is introduced. As used herein, a
"chimeric gene" comprises a coding sequence operably linked to a
transcription initiation region that is heterologous to the coding
sequence.
[0079] While it may be preferable to regulate RAP2.7 or VGT1
sequences using heterologous promoters, the native promoter
sequences may be used. Such constructs would change expression
levels of the RAP2.7 or amounts of the VGT1 regulatory element
present in the plant or plant cell. Thus, the phenotype of the
plant or plant cell is altered.
[0080] The termination region may be native with the
transcriptional initiation region, may be native with the operably
linked DNA sequence of interest or may be derived from another
source. Convenient termination regions are available from the
Ti-plasmid of A. tumefaciens, such as the octopine synthase and
nopaline synthase termination regions. See also, Guerineau, et al.,
(1991) Mol. Gen. Genet. 262:141-144; Proudfoot, (1991) Cell
64:671-674; Sanfacon, et al., (1991) Genes Dev. 5:141-149; Mogen,
et al., (1990) Plant Cell 2:1261-1272; Munroe, et al., (1990) Gene
91:151-158; Ballas, et al., (1989) Nucleic Acids Res. 17:7891-7903
and Joshi, et al., (1987) Nucleic Acid Res. 15:9627-9639. Where
appropriate, the gene(s) may be optimized for increased expression
in the transformed plant. That is, the genes can be synthesized
using plant-preferred codons for improved expression. Methods are
available in the art for synthesizing plant-preferred genes. See,
for example, U.S. Pat. Nos. 5,380,831 and 5,436,391 and Murray, et
al., (1989) Nucleic Acids Res. 17:477-498, herein incorporated by
reference.
[0081] Additional sequence modifications are known to enhance gene
expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon-intron
splice site signals, transposon-like repeats and other such well
characterized sequences that may be deleterious to gene expression.
The G-C content of the sequence may be adjusted to enhance
expression in a given host cell. When possible, the sequence is
modified to avoid predicted hairpin secondary mRNA structures.
[0082] The expression cassettes/nucleotide constructs may
additionally contain 5' leader sequences in the expression cassette
construct. Such leader sequences can act to enhance translation.
Translation leaders are known in the art and include: picornavirus
leaders, for example, EMCV leader (Encephalomyocarditis 5'
noncoding region) (Elroy-Stein, et al., (1989) Proc. Nat'l. Acad.
Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader
(Tobacco Etch Virus) (Allison, et al., (1986); MDMV leader (Maize
Dwarf Mosaic Virus); Virology 154:9-20) and human immunoglobulin
heavy-chain binding protein (BiP), (Macejak, et al., (1991) Nature
353:90-94); untranslated leader from the coat protein mRNA of
alfalfa mosaic virus (AMV RNA 4) (Jobling, et al., (1987) Nature
325:622625); tobacco mosaic virus leader (TMV) (Gallie, et al.,
(1989) in Molecular Biology of RNA, ed. Cech, (Liss, New York), pp.
237-256) and maize chlorotic mottle virus leader (MCMV) (Lommel, et
al., (1991) Virology 81:382-385). See also, Della-Cioppa, et al.,
(1987) Plant Physiol. 84:965-968. Other methods known to enhance
translation can also be utilized, for example, introns, and the
like.
[0083] In those instances where it is desirable to have the
expressed product of the heterologous nucleotide sequence of
interest directed to a particular organelle, such as the
chloroplast or mitochondrion, or secreted at the cell's surface or
extracellularly, the expression cassette may further comprise a
coding sequence for a transit peptide. Such transit peptides are
well known in the art and include, but are not limited to, the
transit peptide for the acyl carrier protein, the small subunit of
RUBISCO, plant EPSP synthase, and the like.
[0084] In preparing the expression cassette/nucleotide construct,
the various DNA fragments may be manipulated so as to provide for
the DNA sequences in the proper orientation and, as appropriate, in
the proper reading frame. Toward this end, adapters or linkers may
be employed to join the DNA fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of
superfluous DNA, removal of restriction sites, or the like. For
this purpose, in vitro mutagenesis, primer repair, restriction,
annealing, resubstitutions, e.g., transitions and transversions,
may be involved.
[0085] Generally, the expression cassette/nucleotide construct will
comprise a selectable marker gene for the selection of transformed
cells. Selectable marker genes are utilized for the selection of
transformed cells or tissues. Marker genes include genes encoding
antibiotic resistance, such as those encoding neomycin
phosphotransferase II (NEO) and hygromycin phosphotransferase
(HPT), as well as genes conferring resistance to herbicidal
compounds, such as glufosinate ammonium, bromoxynil,
imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See
generally, Yarranton, (1992) Curr. Opin. Biotech. 3:506-511;
Christopherson, et al., (1992) Proc. Nad. Acad. Sci. USA
89:6314-6318; Yao, et al., (1992) Cell 71:63-72; Reznikoff, (1992)
Mol. Microbiol. 6:2419-2422; Barkley, et al., (1980) in The Operon,
pp. 177-220; Hu, et al., (1987) Cell 48:555-566; Brown, et al.,
(1987) Cell 49:603-612; Figge, et al., (1988) Cell 52:713-722;
Deuschle, et al., (1989) Proc. Nad. Acad. Sci. USA 86:5400-5404;
Fuerst, et al., (1989) Proc. Nad. Acad. Sci. USA 86:2549-2553;
Deuschle, et al., (1990) Science 248:480-483; Gossen, (1993) Ph.D.
Thesis, University of Heidelberg; Reines, et al., (1993) Proc. Nad.
Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) Mol. Cell. Biol.
10:3343-3356; Zambretti, et al., (1992) Proc. Nad. Acad. Sci. USA
89:3952-3956; Baim, et al., (1991) Proc. Nad. Acad. Sci. USA
88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res.
19:4647-4653; Hillenand-Wissman, (1989) Topics Mol. Struc. Biol.
10:143-162; Degenkolb, et al., (1991) Antimicrob. Agents Chemother.
35:1591-1595; Kleinschnidt, et al., (1988) Biochemistry
27:1094-1104; Bonin, (1993) Ph.D. Thesis, University of Heidelberg;
Gossen, et al., (1992) Proc. Nad. Acad. Sci. USA 89:5547-5551;
Oliva, et al., (1992) Antimicrob. Agents Chemother. 36:913-919;
Hlavka, et al., (1985) Handbook of Experimental Pharmacology, Vol.
78 (Springer-Verlag, Berlin); Gill, et al., (1988) Nature
334:721-724. Such disclosures are herein incorporated by
reference.
[0086] The above list of selectable marker genes is not meant to be
limiting. Any selectable marker gene can be used in the present
invention. A number of promoters can be used in the practice of the
invention. The promoters can be selected based on the desired
outcome. That is, the nucleic acids can be combined with
constitutive, tissue-preferred, or other promoters for expression
in plants. Constitutive promoters include, for example, the core
promoter of the Rsyn7 promoter and other constitutive promoters
disclosed in WO 1999/43838 and U.S. Pat. No. 6,072,050; the core
CaMV 35S promoter (Odell, et al., (1985) Nature 313:810-812); rice
actin (McElroy, et al., (1990) Plant Cell 2:163-171); ubiquitin
(Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 and
Christensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU
(Last, et al., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten,
et al., (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No.
5,659,026) and the like. Other constitutive promoters include, for
example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;
5,466,785; 5,399,680; 5,268,463; 5,608,142 and 6,177,611.
[0087] Chemical-regulated promoters can be used to modulate the
expression of a gene in a plant through the application of an
exogenous chemical regulator. Depending upon the objective, the
promoter may be a chemical-inducible promoter, where application of
the chemical induces gene expression, or a chemical-repressible
promoter, where application of the chemical represses gene
expression. Chemical-inducible promoters are known in the art and
include, but are not limited to: the maize In2-2 promoter, which is
activated by benzenesulfonamide herbicide safeners; the maize GST
promoter, which is activated by hydrophobic electrophilic compounds
that are used as pre-emergent herbicides; and the tobacco PR-1 a
promoter, which is activated by salicylic acid. Other
chemical-regulated promoters of interest include steroid-responsive
promoters. See, for example, the glucocorticoid-inducible promoter
in Schena, et al., (1991) Proc. Nad. Acad. Sci. USA 88:10421-10425
and McNellis, et al., (1998) Plant J. 14(2):247-257) and
tetracycline-inducible and tetracycline-repressible promoters (for
example, Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237 and U.S.
Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by
reference.
[0088] Tissue-preferred promoters can be utilized to target
enhanced gene expression within a particular plant tissue.
Tissue-preferred promoters include Yamamoto, et al., (1997) Plant J
12(2):255-265; Kawamata, et al., (1997) Plant Cell Physiol.
38(7):792-803; Hansen, et al., (1997) Mol. Gen. Genet.
254(3):337-343; Russell, et al., (1997) Transgenic Res.
6(2):157-168; Rinehart, et al., (1996) Plant Physiol.
112(3):1331-1341; Van Camp, et al., (1996) Plant Physiol.
112(2):525-535; Canevascini, et al., (1996) Plant Physiol.
112(2):513-524; Yamamoto, et al., (1994) Plant Cell Physiol.
35(5):773-778; Lam, (1994) Results Probl. Cell Differ. 20:181-196;
Orozco, et al., (1993) Plant Mol. Biol. 23(6):1129-1138; Matsuoka,
et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 and
Guevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Such
promoters can be modified, if necessary, for weak expression.
Leaf-specific promoters are known in the art. See, for example,
Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kwon, et al.,
(1994) Plant Physiol. 105:357-67; Yamamoto, et al., (1994) Plant
Cell Physiol. 35(5):773-778; Gotor, et al., (1993) Plant J
3:509-18; Orozco, et al., (1993) Plant Mol. Biol. 23(6):1129-1138
and Matsuoka, et al., (1993) Proc. Nad. Acad. Sci. USA
90(20):9586-9590.
[0089] Where low level expression is desired, weak promoters will
be used. Generally, by "weak promoter" is intended a promoter that
drives expression of a coding sequence at a low level. By low level
is intended at levels of about 1/1000 transcripts to about
1/100,000 transcripts to about 1/500,000 transcripts per cell.
Alternatively, it is recognized that weak promoters also include
promoters that are expressed in only a few cells and not in others
to give a total low level of expression. Where a promoter is
expressed at unacceptably high levels, portions of the promoter
sequence can be deleted or modified to decrease expression levels.
Such weak constitutive promoters include, for example, the core
promoter of the Rsyn7 promoter (WO 1999/43838 and U.S. Pat. No.
6,072,050), the core 35S CaMV promoter, and the like. Other
constitutive promoters include, for example, U.S. Pat. Nos.
5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;
5,268,463 and 5,608,142. See also, U.S. Pat. No. 6,177,611, herein
incorporated by reference.
[0090] As used herein, "vector" refers to a molecule such as a
plasmid, cosmid or bacterial phage for introducing a nucleotide
construct and/or expression cassette into a host cell. Cloning
vectors typically contain one or a small number of restriction
endonuclease recognition sites at which foreign nucleotide
sequences can be inserted in a determinable fashion without loss of
essential biological function of the vector, as well as a marker
gene that is suitable for use in the identification and selection
of cells transformed with the cloning vector. Marker genes
typically include genes that provide tetracycline resistance,
hygromycin resistance or ampicillin resistance.
[0091] The methods of the invention involve introducing a
nucleotide construct into a plant. By "introducing" is intended
presenting to the plant the nucleotide construct in such a manner
that the construct gains access to the interior of a cell of the
plant. The methods of the invention do not depend on a particular
method for introducing a nucleotide construct to a plant, only that
the nucleotide construct gains access to the interior of at least
one cell of the plant. Methods for introducing nucleotide
constructs into plants are known in the art including, but not
limited to, stable transformation methods, transient transformation
methods and virus-mediated methods.
[0092] By "stable transformation" is intended that the nucleotide
construct introduced into a plant integrates into the genome of the
plant and is capable of being inherited by progeny thereof. By
"transient transformation" is intended that a nucleotide construct
introduced into a plant does not integrate into the genome of the
plant.
[0093] The nucleotide constructs of the invention may be introduced
into plants by contacting plants with a virus or viral nucleic
acids. Generally, such methods involve incorporating a nucleotide
construct of the invention within a viral DNA or RNA molecule. It
is recognized that the RAP2.7 protein of the invention may be
initially synthesized as part of a viral polyprotein, which later
may be processed by proteolysis in vivo or in vitro to produce the
desired recombinant protein. Further, it is recognized that
promoters of the invention also encompass promoters utilized for
transcription by viral RNA polymerases. Methods for introducing
nucleotide constructs into plants and expressing a protein encoded
therein, involving viral DNA or RNA molecules, are known in the
art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190,
5,866,785, 5,589,367 and 5,316,931, herein incorporated by
reference.
[0094] A variety of other transformation protocols are contemplated
in the present invention. Transformation protocols as well as
protocols for introducing nucleotide sequences into plants may vary
depending on the type of plant or plant cell, i.e., monocot or
dicot, targeted for transformation. Suitable methods of introducing
nucleotide sequences into plant cells and subsequent insertion into
the plant genome include microinjection (Crossway, et al., (1986)
Biotechniques 4:320-334), electroporation (Riggs, et al., (1986)
Proc. Nad. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated
transformation (Townsend, et al., U.S. Pat. No. 5,563,055; Zhao, et
al., U.S. Pat. No. 5,981,840), direct gene transfer (Paszkowski, et
al., (1984) EMBO J. 3:2717-2722), and ballistic particle
acceleration (see, for example, Sanford, et al., U.S. Pat. No.
4,945,050; Tomes, et al., U.S. Pat. No. 5,879,918; Tomes, et al.,
U.S. Pat. No. 5,886,244; Bidney, et al., U.S. Pat. No. 5,932,782;
Tomes, et al., (1995) "Direct DNA Transfer into Intact Plant Cells
via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ
Culture Fundamental Methods, eds. Gamborg and Phillips
(Springer-Verlag, Berlin); McCabe, et al., (1988) Biotechnology
6:923-926); and Lecl transformation (WO 2000/28058, published May
18, 2000). Also see, Weissinger, et al., (1988) Ann. Rev. Genet.
22:421-477; Sanford, et al., (1987) Particulate Science and
Technology 5:27-37 (onion); Christoul, et al., (1988) Plant
Physiol. 87:671-674 (soybean); McCabe, et al., (1988)
Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In
Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998)
Theor. Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990)
Biotechnology 8:736-740 (rice); Klein, et al., (1988) Proc. Nad.
Acad. Sci. USA 85:43054309 (maize); Klein, et al., (1988)
Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855;
Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes, et
al., (1995) `Direct DNA Transfer into Intact Plant Cells via
Microprojectile Bombardment," in Plant Cell, Tissue, and Organ
Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin)
(maize); Klein, et al., (1988) Plant Physiol. 91:440-444 (maize);
Fromm, et al., (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van
Slogteren, et al., (1984) Nature (London) 311:763-764; Bowen, et
al., U.S. Pat. No. 5,736,369 (cereals); Bytebier, et al., (1987)
Proc. Nad. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet, et al.,
(1985) in The Experimental Manipulation of Ovule Tissues, ed.
Chapman, et al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler, et
al., (1990) Plant Cell Reports 9:415-418 and Kaeppler, et al.,
(1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated
transformation); D'Halluin, et al., (1992) Plant Cell 4:1495-1505
(electroporation); Li, et al., (1993) Plant Cell Reports 12:250-255
and Christou and Ford, (1995) Annals of Botany 75:407-413 (rice);
Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maize via
Agrobacterium tumefaciens), all of which are herein incorporated by
reference.
[0095] The cells that have been transformed may be grown into
plants in accordance with conventional ways. See, for example,
McCormick, et al., (1986) Plant Cell Reports 5:81-84. These plants
may then be grown, and either pollinated with the same transformed
strain or different strains, and the resulting hybrid having
constitutive expression of the desired phenotypic characteristic
identified. Two or more generations may be grown to ensure that
constitutive expression of the desired phenotypic characteristic is
stably maintained and inherited and then seeds harvested to ensure
constitutive expression of the desired phenotypic characteristic
has been achieved.
[0096] The present invention may be used for transformation of any
plant species, including, but not limited to, monocots and dicots.
Examples of plant species of interest include, but are not limited
to, corn (Zea mays), Brassica spp. (e.g., B. napus, B. rapa, B.
juncea), particularly those Brassica species useful as sources of
seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye
(Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare),
millet (e.g., pearl millet (Pennisetum glaucum), proso millet
(Panicum miliaceum), foxtail millet (Setaria italica), finger
millet (Eleusine coracana)), sunflower (Helianthus annuus),
safflower (Carthamus tinctorius), wheat (Triticum aestivum),
soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum
tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium
barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus),
cassaya (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos
nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.),
cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa
spp.), avocado (Persea americana), fig (Ficus casica), guava
(Psidium guajava), mango (Mangifera indica), olive (Olea europaea),
papaya (Carica papaya), cashew (Anacardium occidentale), macadamia
(Macadamia integrifolia), almond (Prunus amygdalus), sugar beets
(Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,
vegetables, ornamentals and conifers.
[0097] Vegetables include tomatoes (Lycopersicon esculentum),
lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris),
lima beans (Phaseolus limensis), peas (Lathyrus spp.) and members
of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C.
cantalupensis), and musk melon (C. melo). Ornamentals include
azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea),
hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa
spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida),
carnation (Dianthus caryophyllus), poinsettia (Euphorbia
pulcherrima) and chrysanthemum.
[0098] Conifers that may be employed in practicing the present
invention include, for example, pines such as loblolly pine (Pinus
taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus
ponderosa), lodgepole pine (Pinus contorta), and Monterey pine
(Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western
hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood
(Sequoia sempervirens); true firs such as silver fir (Abies
amabilis) and balsam fir (Abies balsamea) and cedars such as
Western red cedar (Thujaplicata) and Alaska yellow cedar
(Chamaecyparis nootkatensis). Plants of the present invention may
be crop plants (for example, alfalfa, sunflower, Brassica, cotton,
safflower, peanut, sorghum, wheat, millet, tobacco, etc.), corn or
soybean plants.
[0099] Plants of particular interest include grain plants that
provide seeds of interest, oil-seed plants and leguminous plants.
Seeds of interest include grain seeds, such as corn, wheat, barley,
rice, sorghum, rye, etc. Oil-seed plants include cotton, soybean,
safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc.
Leguminous plants include beans and peas. Beans include guar,
locust bean, fenugreek, soybean, garden beans, cowpea, mungbean,
lima bean, fava bean, lentils, chickpea, etc.
[0100] It is recognized that with these nucleotide sequences, gene
silencing such as antisense constructions complementary to at least
a portion of the messenger RNA (mRNA) for RAP2.7 or VGT1 sequences
can be constructed. Antisense nucleotides are constructed to
hybridize with the corresponding mRNA. Modifications of the
antisense sequences may be made as long as the sequences hybridize
to and interfere with expression of the corresponding mRNA. In this
manner, antisense constructions having 70%, 80%, 85%, 90%, 95% or
more sequence identity to the corresponding antisense sequences may
be used. Furthermore, portions of the antisense nucleotides may be
used to disrupt the expression of the target gene. Generally,
sequences of at least 50 nucleotides, 100 nucleotides, 200
nucleotides or greater may be used.
[0101] Gene silencing refers to posttranscriptional interference
with gene expression. Techniques such as antisense, co-suppression,
and RNA interference (RNAi), for example, have been shown to be
effective in gene silencing. (For reviews, see, Arndt and Rank,
(1997) Genome 40(6):785-797; Turner and Schuch, (2000) Journal of
Chemical Technology and Biotechnology 75(10):869-882; Klink and
Wolniak, (2000) Journal of Plant Growth Regulation
19(4):371-384)
[0102] Antisense technology can be used to control gene expression
through antisense DNA or RNA or through double- or triple-helix
formation. Antisense techniques are discussed, for example, in
Okano, (1991) Neurochem 56:560; OLIGODEOXYNUCLEOTIDES AS ANTISENSE
INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988).
Triple helix formation is discussed in, for instance Lee, et al.,
(1979) Nucleic Acids Research 6:3073; Cooney, et al., (1988)
Science 241:456 and Dervan, et al., (1991) Science 251:1360. The
methods are based on binding of a polynucleotide to a complementary
DNA or RNA. For example, the 5' coding portion of a polynucleotide
that encodes the mature polypeptide of the present invention may be
used to design an antisense RNA oligonucleotide of from about 10 to
40 base pairs in length. A DNA oligonucleotide is designed to be
complementary to a region of the gene involved in transcription,
thereby preventing transcription and the production of cytokinin
biosynthetic enzymes. The antisense RNA oligonucleotide hybridizes
to the mRNA in vivo and blocks translation of the mRNA molecule
into cytokinin biosynthetic enzymes. The oligonucleotides described
above can also be delivered to cells such that the antisense RNA or
DNA may be expressed in vivo to inhibit production of cytokinin
biosynthetic enzymes. The DNAs of this invention may also be
employed to co-suppress or silence the cytokinin metabolic enzyme
genes; for example, as described in PCT Patent Application
Publication WO 1998/36083.
[0103] The RAP 2.7 nucleotide sequence operably linked to the
regulatory elements herein can be an antisense sequence for a
targeted gene. By "antisense DNA nucleotide sequence" is intended a
sequence that is in inverse orientation to the 5'-to-3' normal
orientation of that nucleotide sequence. When delivered into a
plant cell, expression of the antisense DNA sequence prevents
normal expression of the DNA nucleotide sequence for the targeted
gene. The antisense nucleotide sequence encodes an RNA transcript
that is complementary to and capable of hybridizing with the
endogenous messenger RNA (mRNA) produced by transcription of the
DNA nucleotide sequence for the targeted gene. In this case,
production of the native protein encoded by the targeted gene is
inhibited to achieve a desired phenotypic response. Thus the
regulatory sequences disclosed herein can be operably linked to
antisense DNA sequences to reduce or inhibit expression of a native
protein in the plant seed.
[0104] It is also recognized that the level and/or activity of the
polypeptide may be modulated by employing a polynucleotide that is
not capable of directing, in a transformed plant, the expression of
a protein or an RNA. For example, the polynucleotides of the
invention may be used to design polynucleotide constructs that can
be employed in methods for altering or mutating a genomic
nucleotide sequence in an organism. Such polynucleotide constructs
include, but are not limited to, RNA:DNA vectors, RNA:DNA
mutational vectors, RNA:DNA repair vectors, mixed-duplex
oligonucleotides, self-complementary RNA:DNA oligonucleotides and
recombinogenic oligonucleobases. Such nucleotide constructs and
methods of use are known in the art. See, U.S. Pat. Nos. 5,565,350;
5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984, all of
which are herein incorporated by reference. See also, WO
1998/49350, WO 1999/07865, WO 1999/25821 and Beetham, et al.,
(1999) Proc. Natl. Acad. Sci. USA 96:8774-8778; herein incorporated
by reference. It is therefore recognized that methods of the
present invention do not depend on the incorporation of the entire
polynucleotide into the genome, only that the plant or cell thereof
is altered as a result of the introduction of the polynucleotide
into a cell. In one embodiment of the invention, the genome may be
altered following the introduction of the polynucleotide into a
cell. For example, the polynucleotide, or any part thereof, may
incorporate into the genome of the plant. Alterations to the genome
of the present invention include, but are not limited to,
additions, deletions, and substitutions of nucleotides into the
genome. While the methods of the present invention do not depend on
additions, deletions, and substitutions of any particular number of
nucleotides, it is recognized that such additions, deletions or
substitutions comprises at least one nucleotide.
[0105] Methods are provided to reduce or eliminate the activity of
a RAP2.7 polypeptide of the invention by transforming a plant cell
with an expression cassette that expresses a polynucleotide that
inhibits the expression of the RAP2.7 polypeptide. The
polynucleotide may inhibit the expression of the Rap2.7 polypeptide
directly, by preventing translation of the RAP2.7 messenger RNA, or
indirectly, by encoding a polypeptide that inhibits the
transcription or translation of a RAP2.7 gene encoding a RAP2.7
polypeptide. Methods for inhibiting or eliminating the expression
of a gene in a plant are well known in the art, and any such method
may be used in the present invention to inhibit the expression of a
RAP2.7 polypeptide.
[0106] In some embodiments of the present invention, a plant is
transformed with an expression cassette that is capable of
expressing a polynucleotide that inhibits the expression of a
RAP2.7 polypeptide of the invention. The term "expression" as used
herein refers to the biosynthesis of a gene product, including the
transcription and/or translation of said gene product. For example,
for the purposes of the present invention, an expression cassette
capable of expressing a polynucleotide that inhibits the expression
of at least one RAP2.7 polypeptide is an expression cassette
capable of producing an RNA molecule that inhibits the
transcription and/or translation of at least one RAP2.7 polypeptide
of the invention. The "expression" or "production" of a protein or
polypeptide from a DNA molecule refers to the transcription and
translation of the coding sequence to produce the protein or
polypeptide, while the "expression" or "production" of a protein or
polypeptide from an RNA molecule refers to the translation of the
RNA coding sequence to produce the protein or polypeptide.
[0107] Examples of polynucleotides that inhibit the expression of a
RAP2.7 polypeptide are given below.
i. Sense Suppression/Cosuppression
[0108] In some embodiments of the invention, inhibition of the
expression of a RAP2.7 polypeptide may be obtained by sense
suppression or cosuppression. For cosuppression, an expression
cassette is designed to express an RNA molecule corresponding to
all or part of a messenger RNA encoding a RAP2.7 polypeptide in the
"sense" orientation. Over expression of the RNA molecule can result
in reduced expression of the native gene. Accordingly, multiple
plant lines transformed with the cosuppression expression cassette
are screened to identify those that show the greatest inhibition of
RAP2.7 polypeptide expression.
[0109] The polynucleotide used for cosuppression may correspond to
all or part of the sequence encoding the RAP2.7 polypeptide, all or
part of the 5' and/or 3' untranslated region of a RAP2.7
polypeptide transcript, or all or part of both the coding sequence
and the untranslated regions of a transcript encoding a RAP2.7
polypeptide. In some embodiments where the polynucleotide comprises
all or part of the coding region for the RAP2.7 polypeptide, the
expression cassette is designed to eliminate the start codon of the
polynucleotide so that no protein product will be translated.
[0110] Cosuppression may be used to inhibit the expression of plant
genes to produce plants having undetectable protein levels for the
proteins encoded by these genes. See, for example, Broin, et al.,
(2002) Plant Cell 14:1417-1432. Cosuppression may also be used to
inhibit the expression of multiple proteins in the same plant. See,
for example, U.S. Pat. No. 5,942,657. Methods for using
cosuppression to inhibit the expression of endogenous genes in
plants are described in Flavell, et al., (1994) Proc. Natl. Acad.
Sci. USA 91:3490-3496; Jorgensen, et al., (1996) Plant Mol. Biol.
31:957-973; Johansen and Carrington, (2001) Plant Physiol.
126:930-938; Broin, et al., (2002) Plant Cell 14:1417-1432;
Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731; Yu, et
al., (2003) Phytochemistry 63:753-763 and U.S. Pat. Nos. 5,034,323,
5,283,184 and 5,942,657, each of which is herein incorporated by
reference. The efficiency of cosuppression may be increased by
including a poly-dT region in the expression cassette at a position
3' to the sense sequence and 5' of the polyadenylation signal. See,
US Patent Publication Number 2002/0048814, herein incorporated by
reference. Typically, such a nucleotide sequence has substantial
sequence identity to the sequence of the transcript of the
endogenous gene, optimally greater than about 65% sequence
identity, more optimally greater than about 85% sequence identity,
most optimally greater than about 95% sequence identity. See, U.S.
Pat. Nos. 5,283,184 and 5,034,323, herein incorporated by
reference.
ii. Antisense Suppression
[0111] In some embodiments of the invention, inhibition of the
expression of the RAP2.7 polypeptide may be obtained by antisense
suppression. For antisense suppression, the expression cassette is
designed to express an RNA molecule complementary to all or part of
a messenger RNA encoding the RAP2.7 polypeptide. Over expression of
the antisense RNA molecule can result in reduced expression of the
native gene. Accordingly, multiple plant lines transformed with the
antisense suppression expression cassette are screened to identify
those that show the greatest inhibition of RAP2.7 polypeptide
expression.
[0112] The polynucleotide for use in antisense suppression may
correspond to all or part of the complement of the sequence
encoding the RAP2.7 polypeptide, all or part of the complement of
the 5' and/or 3' untranslated region of the RAP2.7 transcript, or
all or part of the complement of both the coding sequence and the
untranslated regions of a transcript encoding the RAP2.7
polypeptide. In addition, the antisense polynucleotide may be fully
complementary (i.e., 100% identical to the complement of the target
sequence) or partially complementary (i.e., less than 100%
identical to the complement of the target sequence) to the target
sequence. Antisense suppression may be used to inhibit the
expression of multiple proteins in the same plant. See, for
example, U.S. Pat. No. 5,942,657. Furthermore, portions of the
antisense nucleotides may be used to disrupt the expression of the
target gene. Generally, sequences of at least 50 nucleotides, 100
nucleotides, 200 nucleotides, 300, 400, 450, 500, 550 or greater
may be used. Methods for using antisense suppression to inhibit the
expression of endogenous genes in plants are described, for
example, in Liu, et al., (2002) Plant Physiol. 129:1732-1743 and
U.S. Pat. Nos. 5,759,829 and 5,942,657, each of which is herein
incorporated by reference. Efficiency of antisense suppression may
be increased by including a poly-dT region in the expression
cassette at a position 3' to the antisense sequence and 5' of the
polyadenylation signal. See, US Patent Publication Number
2002/0048814, herein incorporated by reference.
iii. Double-Stranded RNA Interference
[0113] In some embodiments of the invention, inhibition of the
expression of a RAP2.7 polypeptide may be obtained by
double-stranded RNA (dsRNA) interference. For dsRNA interference, a
sense RNA molecule like that described above for cosuppression and
an antisense RNA molecule that is fully or partially complementary
to the sense RNA molecule are expressed in the same cell, resulting
in inhibition of the expression of the corresponding endogenous
messenger RNA.
[0114] Expression of the sense and antisense molecules can be
accomplished by designing the expression cassette to comprise both
a sense sequence and an antisense sequence. Alternatively, separate
expression cassettes may be used for the sense and antisense
sequences. Multiple plant lines transformed with the dsRNA
interference expression cassette or expression cassettes are then
screened to identify plant lines that show the greatest inhibition
of RAP2.7 polypeptide expression. Methods for using dsRNA
interference to inhibit the expression of endogenous plant genes
are described in Waterhouse, et al., (1998) Proc. Natl. Acad. Sci.
USA 95:13959-13964, Liu, et al., (2002) Plant Physiol.
129:1732-1743 and WO 1999/49029, WO 1999/53050, WO 1999/61631 and
WO 2000/49035, each of which is herein incorporated by
reference.
iv. Hairpin RNA Interference and Intron-Containing Hairpin RNA
Interference
[0115] In some embodiments of the invention, inhibition of the
expression of one or a RAP2.7 polypeptide may be obtained by
hairpin RNA (hpRNA) interference or intron-containing hairpin RNA
(ihpRNA) interference. These methods are highly efficient at
inhibiting the expression of endogenous genes. See, Waterhouse and
Helliwell, (2003) Nat. Rev. Genet. 4:29-38 and the references cited
therein.
[0116] For hpRNA interference, the expression cassette is designed
to express an RNA molecule that hybridizes with itself to form a
hairpin structure that comprises a single-stranded loop region and
a base-paired stem. The base-paired stem region comprises a sense
sequence corresponding to all or part of the endogenous messenger
RNA encoding the gene whose expression is to be inhibited, and an
antisense sequence that is fully or partially complementary to the
sense sequence. Thus, the base-paired stem region of the molecule
generally determines the specificity of the RNA interference. hpRNA
molecules are highly efficient at inhibiting the expression of
endogenous genes, and the RNA interference they induce is inherited
by subsequent generations of plants. See, for example, Chuang and
Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990;
Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731 and
Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4:29-38. Methods
for using hpRNA interference to inhibit or silence the expression
of genes are described, for example, in Chuang and Meyerowitz,
(2000) Proc. Natl. Acad. Sci. USA 97:4985-4990; Stoutjesdijk, et
al., (2002) Plant Physiol. 129:1723-1731; Waterhouse and Helliwell,
(2003) Nat. Rev. Genet. 4:29-38; Pandolfini, et al., BMC
Biotechnology 3:7 and US Patent Application Publication Number
2003/0175965, each of which is herein incorporated by reference. A
transient assay for the efficiency of hpRNA constructs to silence
gene expression in vivo has been described by Panstruga, et al.,
(2003) Mol. Biol. Rep. 30:135-140, herein incorporated by
reference.
[0117] For ihpRNA, the interfering molecules have the same general
structure as for hpRNA, but the RNA molecule additionally comprises
an intron that is capable of being spliced in the cell in which the
ihpRNA is expressed. The use of an intron minimizes the size of the
loop in the hairpin RNA molecule following splicing, and this
increases the efficiency of interference. See, for example, Smith,
et al., (2000) Nature 407:319-320. In fact, Smith, et al., show
100% suppression of endogenous gene expression using
ihpRNA-mediated interference. Methods for using ihpRNA interference
to inhibit the expression of endogenous plant genes are described,
for example, in Smith, et al., (2000) Nature 407:319-320; Wesley,
et al., (2001) Plant J. 27:581-590; Wang and Waterhouse, (2001)
Curr. Opin. Plant Biol. 5:146-150; Waterhouse and Helliwell, (2003)
Nat. Rev. Genet. 4:29-38; Helliwell and Waterhouse, (2003) Methods
30:289-295 and US Patent Application Publication Number
2003/0180945, each of which is herein incorporated by
reference.
[0118] The expression cassette for hpRNA interference may also be
designed such that the sense sequence and the antisense sequence do
not correspond to an endogenous RNA. In this embodiment, the sense
and antisense sequence flank a loop sequence that comprises a
nucleotide sequence corresponding to all or part of the endogenous
messenger RNA of the target gene. Thus, it is the loop region that
determines the specificity of the RNA interference. See, for
example, WO 2002/00904, herein incorporated by reference.
v. Amplicon-Mediated Interference
[0119] Amplicon expression cassettes comprise a plant virus-derived
sequence that contains all or part of the target gene but generally
not all of the genes of the native virus. The viral sequences
present in the transcription product of the expression cassette
allow the transcription product to direct its own replication. The
transcripts produced by the amplicon may be either sense or
antisense relative to the target sequence (i.e., the messenger RNA
for the RAP2.7 polypeptide). Methods of using amplicons to inhibit
the expression of endogenous plant genes are described, for
example, in Angell and Baulcombe, (1997) EMBO J. 16:3675-3684,
Angell and Baulcombe, (1999) Plant J. 20:357-362 and U.S. Pat. No.
6,646,805, each of which is herein incorporated by reference.
vi. Ribozymes
[0120] In some embodiments, the polynucleotide expressed by the
expression cassette of the invention is catalytic RNA or has
ribozyme activity specific for the messenger RNA of the RAP2.7
polypeptide. Thus, the polynucleotide causes the degradation of the
endogenous messenger RNA, resulting in reduced expression of the
RAP2.7 polypeptide.
[0121] This method is described, for example, in U.S. Pat. No.
4,987,071, herein incorporated by reference.
vii. Small Interfering RNA or Micro RNA
[0122] In some embodiments of the invention, inhibition of the
expression of a RAP2.7 polypeptide may be obtained by RNA
interference by expression of a gene encoding a micro RNA (miRNA).
miRNAs are regulatory agents consisting of about 22
ribonucleotides. miRNA are highly efficient at inhibiting the
expression of endogenous genes. See, for example Javier, et al.,
(2003) Nature 425:257-263, herein incorporated by reference.
[0123] For miRNA interference, the expression cassette is designed
to express an RNA molecule that is modeled on an endogenous miRNA
gene. The miRNA gene encodes an RNA that forms a hairpin structure
containing a 22-nucleotide sequence that is complementary to
another endogenous gene (target sequence). For suppression of
RAP2.7 expression, the 22-nucleotide sequence is selected from a
RAP2.7 transcript sequence and contains 22 nucleotides of said
RAP2.7 sequence in sense orientation and 21 nucleotides of a
corresponding antisense sequence that is complementary to the sense
sequence. miRNA molecules are highly efficient at inhibiting the
expression of endogenous genes, and the RNA interference they
induce is inherited by subsequent generations of plants.
[0124] The availability of reverse genetics systems, which are
well-known in the art, makes the generation and isolation of
down-regulated or null mutants feasible, given the availability of
a defined nucleic acid sequence, as provided herein. One such
system (the Trait Utility System for Corn, i.e., TUSC) is based on
successful systems from other organisms (Ballinger, et al., (1989)
Proc. Natl. Acad. Sci. USA 86:9402-9406; Kaiser, et al., (1990)
Proc. Natl. Acad. Sci. USA 87:1686-1690 and Rushforth, et al.,
(1993) Mol. Cell. Biol. 13:902-910). The central feature of the
system is to identify Mu transposon insertions within a DNA
sequence of interest in anticipation that at least some of these
insertion alleles will be mutants. See, U.S. Pat. Nos. 6,300,542
and 5,962,764. To develop the system, DNA was collected from a
large population of Mutator transposon stocks that were then
self-pollinated to produced F2 seed. To find Mu transposon
insertions within a specific DNA sequence, the collection of DNA
samples is screened via PCR using a gene-specific primer and a
primer that anneals to the inverted repeats of Mu transposons. A
PCR product is expected only when the template DNA comes from a
plant that contains a Mu transposon insertion within the target
gene. Once such a DNA sample is identified, F2 seed from the
corresponding plant is screened for a transposon insertion allele.
Transposon insertion mutations of the an1 gene have been obtained
via the TUSC procedure (Bensen, et al., (1995)). This system is
applicable to other plant species, at times modified in accordance
with knowledge and skills reasonably attributed to ordinary
artisans.
[0125] The use of the term "nucleotide constructs" herein is not
intended to limit the present invention to nucleotide constructs
comprising DNA. Those of ordinary skill in the art will recognize
that nucleotide constructs, particularly polynucleotides and
oligonucleotides, comprised of ribonucleotides and combinations of
ribonucleotides and deoxyribonucleotides may also be employed in
the methods disclosed herein. Thus, the nucleotide constructs of
the present invention encompass all nucleotide constructs that can
be employed in the methods of the present invention for
transforming plants including, but not limited to, those comprised
of deoxyribonucleotides, ribonucleotides and combinations thereof.
Such deoxyribonucleotides and ribonucleotides include both
naturally occurring molecules and synthetic analogues. The
nucleotide constructs of the invention also encompass all forms of
nucleotide constructs including, but not limited to,
single-stranded forms, double-stranded forms, hairpins,
stem-and-loop structures, and the like.
[0126] Furthermore, it is recognized that the methods of the
invention may employ a nucleotide construct that is capable of
directing, in a transformed plant, the expression of at least one
protein, or at least one RNA, such as, for example, an antisense
RNA that is complementary to at least a portion of an mRNA.
Typically such a nucleotide construct is comprised of a coding
sequence for a protein or an RNA operably linked to 5' and 3'
transcriptional regulatory regions. Alternatively, it is also
recognized that the methods of the invention may employ a
nucleotide construct that is not capable of directing, in a
transformed plant, the expression of a protein or an RNA.
[0127] In addition, it is recognized that methods of the present
invention do not depend on the incorporation of the entire
nucleotide construct into the genome. Rather, the methods of the
present invention only require that the plant or cell thereof is
altered as a result of the introduction of the nucleotide construct
into a cell. In one embodiment of the invention, the genome may be
altered following the introduction of the nucleotide construct into
a cell. For example, the nucleotide construct, or any part thereof,
may incorporate into the genome of the plant. Alterations to the
genome of the present invention include, but are not limited to,
additions, deletions, and substitutions of nucleotides in the
genome. While the methods of the present invention do not depend on
additions, deletions or substitutions of any particular number of
nucleotides, it is recognized that such additions, deletions or
substitutions comprise at least one nucleotide.
[0128] In certain embodiments the nucleic acid sequences of the
present invention can be used in combination ("stacked") with other
polynucleotide sequences of interest in order to create plants with
a desired phenotype. The combinations generated can include
multiple copies of any one or more of the polynucleotides of
interest. The polynucleotides of the present invention may be
stacked with any gene or combination of genes to produce plants
with a variety of desired trait combinations, including but not
limited to traits desirable for animal feed such as high oil genes
(e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g.,
hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802 and
5,703,409); barley high lysine (Williamson, et al., (1987) Eur. J.
Biochem. 165:99-106 and WO 1998/20122) and high methionine proteins
(Pedersen, et al., (1986) J. Biol. Chem. 261:6279; Kirihara, et
al., (1988) Gene 71:359 and Musumura, et al., (1989) Plant Mol.
Biol. 12:123)); increased digestibility (e.g., modified storage
proteins (U.S. patent application Ser. No. 10/053,410, filed Nov.
7, 2001) and thioredoxins (U.S. patent application Ser. No.
10/005,429, filed Dec. 3, 2001)), the disclosures of which are
herein incorporated by reference. The polynucleotides of the
present invention can also be stacked with traits desirable for
insect, disease or herbicide resistance (e.g., Bacillus
thuringiensis toxic proteins (U.S. Pat. Nos. 5,366,892; 5,747,450;
5,737,514; 5723,756; 5,593,881; Geiser, et al., (1986) Gene
48:109); lectins (Van Damme, et al., (1994) Plant Mol. Biol.
24:825); fumonisin detoxification genes (U.S. Pat. No. 5,792,931);
avirulence and disease resistance genes (Jones, et al., (1994)
Science 266:789; Martin, et al., (1993) Science 262:1432;
Mindrinos, et al., (1994) Cell 78:1089); acetolactate synthase
(ALS) mutants that lead to herbicide resistance such as the S4
and/or Hra mutations; inhibitors of glutamine synthase such as
phosphinothricin or basta (e.g., bar gene) and glyphosate
resistance (EPSPS gene)) and traits desirable for processing or
process products such as high oil (e.g., U.S. Pat. No. 6,232,529);
modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No.
5,952,544; WO 1994/11516)); modified starches (e.g., ADPG
pyrophosphorylases (AGPase), starch synthases (SS), starch
branching enzymes (SBE) and starch debranching enzymes (SDBE)) and
polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;
beta-ketothiolase, polyhydroxybutyrate synthase and acetoacetyl-CoA
reductase (Schubert, et al., (1988) J. Bacteriol. 170:5837-5847)
facilitate expression of polyhydroxyalkanoates (PHAs)), the
disclosures of which are herein incorporated by reference. One
could also combine the polynucleotides of the present invention
with polynucleotides affecting agronomic traits such as male
sterility (e.g., see, U.S. Pat. No. 5,583,210), stalk strength,
flowering time or transformation technology traits such as cell
cycle regulation or gene targeting (e.g., WO 1999/61619; WO
2000/17364; WO 1999/25821), the disclosures of which are herein
incorporated by reference.
[0129] These stacked combinations can be created by any method,
including but not limited to cross breeding plants by any
conventional or TopCross methodology or genetic transformation. If
the traits are stacked by genetically transforming the plants, the
polynucleotide sequences of interest can be combined at any time
and in any order. For example, a transgenic plant comprising one or
more desired traits can be used as the target to introduce further
traits by subsequent transformation. The traits can be introduced
simultaneously in a co-transformation protocol with the
polynucleotides of interest provided by any combination of
transformation cassettes. For example, if two sequences will be
introduced, the two sequences can be contained in separate
transformation cassettes (trans) or contained on the same
transformation cassette (cis). Expression of the sequences of
interest can be driven by the same promoter or by different
promoters. In certain cases, it may be desirable to introduce a
transformation cassette that will suppress the expression of a
polynucleotide of interest. This may be accompanied by any
combination of other suppression cassettes or overexpression
cassettes to generate the desired combination of traits in the
plant.
[0130] The nucleotide constructs of the invention also encompass
nucleotide constructs that may be employed in methods for altering
or mutating a genomic nucleotide sequence in an organism,
including, but not limited to, chimeric vectors, chimeric
mutational vectors, chimeric repair vectors, mixed-duplex
oligonucleotides, self-complementary chimeric oligonucleotides and
recombinogenic oligonucleobases. Such nucleotide constructs and
methods of use, such as, for example, chimeraplasty, are known in
the art. Chimeraplasty involves the use of such nucleotide
constructs to introduce site-specific changes into the sequence of
genomic DNA within an organism, e.g., U.S. Pat. Nos. 5,565,350;
5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984, all of
which are herein incorporated by reference. See also, WO
1998/49350, WO 1999/07865, WO 1999/25821, and Beetham, et al.,
(1999) Proc. Nad. Acad. Sci. USA 96:8774-8778, herein incorporated
by reference. The following examples are offered by way of
illustration and not by way of limitation. All references cited
herein are hereby expressly incorporated in their entirety herein
by reference.
[0131] Other embodiments of the invention include the use of VGT1
and its alternate forms described herein as markers to screen for
and identify plants which may have altered maturity. The invention
thus relates to genetic markers for plants with altered maturity.
The markers represent polymorphic variants of the non-coding
regulatory element VGT1 that are associated with RAP2.7 regulation
and thus provides a method of genotyping plants to determine those
more likely to have flowering time that is altered from
wildtype.
[0132] Thus, the invention relates to genetic markers and methods
of identifying those markers in plants, whereby the plant is more
likely to have a maturity that is earlier than normal by means of a
mutant VGT1 which does not down regulate RAP2.7 appropriately.
[0133] Any method of identifying the presence or absence of these
markers may be used, including, for example, single-strand
conformation polymorphism (SSCP) analysis, base excision sequence
scanning (BESS), RFLP analysis, heteroduplex analysis, denaturing
gradient gel electrophoresis and temperature gradient
electrophoresis, allelic PCR, ligase chain reaction direct
sequencing, mini sequencing, nucleic acid hybridization,
micro-array-type detection of the VGT1 regulatory element.
[0134] The following is a general overview of techniques which can
be used to assay for the polymorphisms of the invention.
[0135] In the present invention, a sample of genetic material is
obtained from a plant.
[0136] Isolation and Amplification of Nucleic Acid
[0137] Samples of genomic DNA are isolated from any convenient
source including any suitable cell or tissue sample with intact
interphase nuclei or metaphase cells. The cells can be obtained
from solid tissue as from a fresh or preserved plant part or from a
tissue sample. The sample can contain compounds which are not
naturally intermixed with the biological material such as
preservatives, anticoagulants, buffers, fixatives, nutrients,
antibiotics, or the like.
[0138] Methods for isolation of genomic DNA from these various
sources are described in, for example, Kirby, DNA Fingerprinting,
An Introduction, W.H. Freeman & Co. New York (1992). Genomic
DNA can also be isolated from cultured primary or secondary cell
cultures or from transformed cell lines derived from any of the
aforementioned tissue samples.
[0139] Samples of RNA can also be used. RNA can be isolated from
tissues as described in Sambrook, et al., supra. RNA can be total
cellular RNA, mRNA, poly A+RNA, or any combination thereof. For
best results, the RNA is purified, but can also be unpurified
cytoplasmic RNA. RNA can be reverse transcribed to form DNA which
is then used as the amplification template, such that the PCR
indirectly amplifies a specific population of RNA transcripts. See,
e.g., Sambrook, supra, Kawasaki, et al., Chapter 8 in PCR
Technology, (1992) supra, and Berg, et al., (1990) Hum. Genet.
85:655-658.
[0140] PCR Amplification
[0141] The most common means for amplification is polymerase chain
reaction (PCR), as described in U.S. Pat. Nos. 4,683,195; 4,683,202
and 4,965,188, each of which is hereby incorporated by reference.
Tissues should be roughly minced using a sterile, disposable
scalpel and a sterile needle (or two scalpels) in a 5 mm Petri
dish. Procedures for removing paraffin from tissue sections are
described in a variety of specialized handbooks well known to those
skilled in the art.
[0142] To amplify a target nucleic acid sequence in a sample by
PCR, the sequence must be accessible to the components of the
amplification system. Kits for the extraction of high-molecular
weight DNA for PCR include a Genomic Isolation Kit A.S.A.P.
(Boehringer Mannheim, Indianapolis, Ind.), Genomic DNA Isolation
System (GIBCO BRL, Gaithersburg, Md.), Elu-Quik DNA Purification
Kit (Schleicher & Schuell, Keene, N.H.), DNA Extraction Kit
(Stratagene, LaJolla, Calif.), TurboGen Isolation Kit (Invitrogen,
San Diego, Calif.), and the like. Use of these kits according to
the manufacturer's instructions is generally acceptable for
purification of DNA prior to practicing the methods of the present
invention.
[0143] The concentration and purity of the extracted DNA can be
determined by spectrophotometric analysis of the absorbance of a
diluted aliquot at 260 nm and 280 nm. After extraction of the DNA,
PCR amplification may proceed. The first step of each cycle of the
PCR involves the separation of the nucleic acid duplex formed by
the primer extension. Once the strands are separated, the next step
in PCR involves hybridizing the separated strands with primers that
flank the target sequence. The primers are then extended to form
complementary copies of the target strands. For successful PCR
amplification, the primers are designed so that the position at
which each primer hybridizes along a duplex sequence is such that
an extension product synthesized from one primer, when separated
from the template (complement), serves as a template for the
extension of the other primer. The cycle of denaturation,
hybridization and extension is repeated as many times as necessary
to obtain the desired amount of amplified nucleic acid.
[0144] In a particularly useful embodiment of PCR amplification,
strand separation is achieved by heating the reaction to a
sufficiently high temperature for a sufficient time to cause the
denaturation of the duplex but not to cause an irreversible
denaturation of the polymerase (see, U.S. Pat. No. 4,965,188,
incorporated herein by reference). Typical heat denaturation
involves temperatures ranging from about 80.degree. C. to
105.degree. C. for times ranging from seconds to minutes. Strand
separation, however, can be accomplished by any suitable denaturing
method including physical, chemical, or enzymatic means. Strand
separation may be induced by a helicase, for example, or an enzyme
capable of exhibiting helicase activity. For example, the enzyme
RecA has helicase activity in the presence of ATP. The reaction
conditions suitable for strand separation by helicases are known in
the art (see, Kuhn Hoffman-Berling, (1978) CSH-Quantitative
Biology, 43:63-67 and Radding, (1982) Ann. Rev. Genetics
16:405-436, each of which is incorporated herein by reference).
[0145] Template-dependent extension of primers in PCR is catalyzed
by a polymerizing agent in the presence of adequate amounts of four
deoxyribonucleotide triphosphates (typically dATP, dGTP, dCTP and
dTTP) in a reaction medium comprised of the appropriate salts,
metal cations and pH buffering systems. Suitable polymerizing
agents are enzymes known to catalyze template-dependent DNA
synthesis. In some cases, the target regions may encode at least a
portion of a protein expressed by the cell. In this instance, mRNA
may be used for amplification of the target region. Alternatively,
PCR can be used to generate a cDNA library from RNA for further
amplification, the initial template for primer extension is RNA.
Polymerizing agents suitable for synthesizing a complementary,
copy-DNA (cDNA) sequence from the RNA template are reverse
transcriptase (RT), such as avian myeloblastosis virus RT, Moloney
murine leukemia virus RT or Thermus thermophilus (Tth) DNA
polymerase, a thermostable DNA polymerase with reverse
transcriptase activity marketed by Perkin Elmer Cetus, Inc.
Typically, the genomic RNA template is heat degraded during the
first denaturation step after the initial reverse transcription
step leaving only DNA template. Suitable polymerases for use with a
DNA template include, for example, E. coli DNA polymerase I or its
Klenow fragment, T4 DNA polymerase, Tth polymerase, and Taq
polymerase, a heat-stable DNA polymerase isolated from Thermus
aquaticus and commercially available from Perkin Elmer Cetus, Inc.
The latter enzyme is widely used in the amplification and
sequencing of nucleic acids. The reaction conditions for using Taq
polymerase are known in the art and are described in Gelfand,
(1989) PCR Technology, supra.
[0146] Allele Specific PCR
[0147] Allele-specific PCR differentiates between target regions
differing in the presence of absence of a variation or
polymorphism. PCR amplification primers are chosen which bind only
to certain alleles of the target sequence. This method is described
by Gibbs, Nucleic Acid Res. 17:12427-2448 (1989).
[0148] Allele Specific Oligonucleotide Screening Methods
[0149] Further diagnostic screening methods employ the
allele-specific oligonucleotide (ASO) screening methods, as
described by Saiki et al., Nature 324:163-166 (1986).
Oligonucleotides with one or more base pair mismatches are
generated for any particular allele. ASO screening methods detect
mismatches between variant target genomic or PCR amplified DNA and
non-mutant oligonucleotides, showing decreased binding of the
oligonucleotide relative to a mutant oligonucleotide.
Oligonucleotide probes can be designed so that under low
stringency, they will bind to both polymorphic forms of the allele,
but at high stringency, bind to the allele to which they
correspond. Alternatively, stringency conditions can be devised in
which an essentially binary response is obtained, i.e., an ASO
corresponding to a variant form of the target gene will hybridize
to that allele, and not to the wild-type allele.
[0150] Ligase Mediated Allele Detection Method
[0151] Target regions of a test subject's DNA can be compared with
target regions in unaffected and affected family members by
ligase-mediated allele detection. See Landegren et al., Science
241:107-1080 (1988). Ligase may also be used to detect point
mutations in the ligation amplification reaction described in Wu et
al., Genomics 4:560-569 (1989). The ligation amplification reaction
(LAR) utilizes amplification of specific DNA sequence using
sequential rounds of template dependent ligation as described in
Wu, supra, and Barany, Proc. Nat. Acad. Sci. 88:189-193 (1990).
[0152] Denaturing Gradient Gel Electrophoresis
[0153] Amplification products generated using the polymerase chain
reaction can be analyzed by the use of denaturing gradient gel
electrophoresis. Different alleles can be identified based on the
different sequence-dependent melting properties and electrophoretic
migration of DNA in solution. DNA molecules melt in segments,
termed melting domains, under conditions of increased temperature
or denaturation. Each melting domain melts cooperatively at a
distinct, base-specific melting temperature (T.sub.m). Melting
domains are at least 20 base pairs in length, and may be up to
several hundred base pairs in length.
[0154] Differentiation between alleles based on sequence specific
melting domain differences can be assessed using polyacrylamide gel
electrophoresis, as described in Chapter 7 of Erlich, ed., PCR
Technology, "Principles and Applications for DNA Amplification",
W.H. Freeman and Co., New York (1992), the contents of which are
hereby incorporated by reference.
[0155] Generally, a target region to be analyzed by denaturing
gradient gel electrophoresis is amplified using PCR primers
flanking the target region. The amplified PCR product is applied to
a polyacrylamide gel with a linear denaturing gradient as described
in Myers et al., Meth. Enzymol. 155:501-527 (1986), and Myers et
al., in Genomic Analysis, A Practical Approach, K. Davies Ed. IRL
Press Limited, Oxford, pp. 95-139 (1988), the contents of which are
hereby incorporated by reference. The electrophoresis system is
maintained at a temperature slightly below the Tm of the melting
domains of the target sequences.
[0156] In an alternative method of denaturing gradient gel
electrophoresis, the target sequences may be initially attached to
a stretch of GC nucleotides, termed a GC clamp, as described in
Chapter 7 of Erlich, supra. Preferably, at least 80% of the
nucleotides in the GC clamp are either guanine or cytosine.
Preferably, the GC clamp is at least 30 bases long. This method is
particularly suited to target sequences with high T.sub.m's.
[0157] Generally, the target region is amplified by the polymerase
chain reaction as described above. One of the oligonucleotide PCR
primers carries at its 5' end, the GC clamp region, at least 30
bases of the GC rich sequence, which is incorporated into the 5'
end of the target region during amplification. The resulting
amplified target region is run on an electrophoresis gel under
denaturing gradient conditions as described above. DNA fragments
differing by a single base change will migrate through the gel to
different positions, which may be visualized by ethidium bromide
staining.
[0158] Temperature Gradient Gel Electrophoresis
[0159] Temperature gradient gel electrophoresis (TGGE) is based on
the same underlying principles as denaturing gradient gel
electrophoresis, except the denaturing gradient is produced by
differences in temperature instead of differences in the
concentration of a chemical denaturant. Standard TGGE utilizes an
electrophoresis apparatus with a temperature gradient running along
the electrophoresis path. As samples migrate through a gel with a
uniform concentration of a chemical denaturant, they encounter
increasing temperatures. An alternative method of TGGE, temporal
temperature gradient gel electrophoresis (TTGE or tTGGE) uses a
steadily increasing temperature of the entire electrophoresis gel
to achieve the same result. As the samples migrate through the gel
the temperature of the entire gel increases, leading the samples to
encounter increasing temperature as they migrate through the gel.
Preparation of samples, including PCR amplification with
incorporation of a GC clamp, and visualization of products are the
same as for denaturing gradient gel electrophoresis.
[0160] Single-Strand Conformation Polymorphism Analysis
[0161] Target sequences or alleles at the VGT1 loci can be
differentiated using single-strand conformation polymorphism
analysis, which identifies base differences by alteration in
electrophoretic migration of single-stranded PCR products, as
described in Orita et al., Proc. Nat. Acad. Sci. 85:2766-2770
(1989). Amplified PCR products can be generated as described above,
and heated or otherwise denatured, to form single-stranded
amplification products. Single-stranded nucleic acids may refold or
form secondary structures which are partially dependent on the base
sequence. Thus, electrophoretic mobility of single-stranded
amplification products can detect base-sequence difference between
alleles or target sequences.
[0162] Chemical or Enzymatic Cleavage of Mismatches
[0163] Differences between target sequences can also be detected by
differential chemical cleavage of mismatched base pairs, as
described in Grompe et al., Am. J. Hum. Genet. 48:212-222 (1991).
In another method, differences between target sequences can be
detected by enzymatic cleavage of mismatched base pairs, as
described in Nelson et al., Nature Genetics 4:11-18 (1993).
Briefly, genetic material from a plant and an affected family
member may be used to generate mismatch free heterohybrid DNA
duplexes. As used herein, "heterohybrid" means a DNA duplex strand
comprising one strand of DNA from one plant, and a second DNA
strand from another plant, usually a plant differing in the
phenotype for the trait of interest. Positive selection for
heterohybrids free of mismatches allows determination of small
insertions, deletions or other polymorphisms that may be associated
with VGT1 polymorphisms.
[0164] Non-Gel Systems
[0165] Other possible techniques include non-gel systems such as
TAQMAN.TM. (Perkin Elmer). In this system, oligonucleotide PCR
primers are designed that flank the mutation in question and allow
PCR amplification of the region. A third oligonucleotide probe is
then designed to hybridize to the region containing the base
subject to change between different alleles of the gene. This probe
is labeled with fluorescent dyes at both the 5' and 3' ends. These
dyes are chosen such that while in this proximity to each other the
fluorescence of one of them is quenched by the other and cannot be
detected. Extension by Taq DNA polymerase from the PCR primer
positioned 5' on the template relative to the probe leads to the
cleavage of the dye attached to the 5' end of the annealed probe
through the 5' nuclease activity of the Taq DNA polymerase. This
removes the quenching effect allowing detection of the fluorescence
from the dye at the 3' end of the probe. The discrimination between
different DNA sequences arises through the fact that if the
hybridization of the probe to the template molecule is not
complete, i.e., there is a mismatch of some form, the cleavage of
the dye does not take place. Thus, only if the nucleotide sequence
of the oligonucleotide probe is completely complimentary to the
template molecule to which it is bound will quenching be removed. A
reaction mix can contain two different probe sequences each
designed against different alleles that might be present thus
allowing the detection of both alleles in one reaction.
[0166] Yet another technique includes an Invader Assay, which
includes isothermic amplification that relies on a catalytic
release of fluorescence. See Third Wave Technology at world wide
web at twt.com.
[0167] Non-PCR Based DNA Screening
[0168] Hybridization probes are generally oligonucleotides which
bind through complementary base pairing to all or part of a target
nucleic acid. Probes typically bind target sequences lacking
complete complementarity with the probe sequence depending on the
stringency of the hybridization conditions. The probes are
preferably labeled directly or indirectly, such that by assaying
for the presence or absence of the probe, one can detect the
presence or absence of the target sequence. Direct labeling methods
include radioisotope labeling, such as with P.sup.32 or S.sup.35.
Indirect labeling methods include fluorescent tags, biotin
complexes which may be bound to avidin or streptavidin, or peptide
or protein tags. Visual detection methods include
photoluminescents, Texas red, rhodamine and its derivatives, red
leuco dye and 3,3',5,5'-tetramethylbenzidine (TMB), fluorescein,
and its derivatives, dansyl, umbelliferone and the like or with
horse radish peroxidase, alkaline phosphatase and the like.
[0169] Hybridization probes include any nucleotide sequence capable
of hybridizing to the chromosome where VGT1 resides, and thus
defining a genetic marker linked to VGT1, including a restriction
fragment length polymorphism, a hypervariable region, repetitive
element, or a variable number tandem repeat. Further suitable
hybridization probes include exon fragments or portions of cDNAs or
genes known to map to the relevant region of the chromosome.
[0170] Preferred tandem repeat hybridization probes for use
according to the present invention are those that recognize a small
number of fragments at a specific locus at high stringency
hybridization conditions, or that recognize a larger number of
fragments at that locus when the stringency conditions are
lowered.
[0171] One or more additional restriction enzymes and/or probes
and/or primers can be used. Additional enzymes, constructed probes,
and primers can be determined by routine experimentation by those
of ordinary skill in the art and are intended to be within the
scope of the invention.
[0172] Although the methods described herein may be in terms of the
use of a single restriction enzyme and a single set of primers, the
methods are not so limited. One or more additional restriction
enzymes and/or probes and/or primers can be used, if desired.
Indeed, in some situations it may be preferable to use combinations
of markers giving specific haplotypes. Additional enzymes,
constructed probes and primers can be determined through routine
experimentation, combined with the teachings provided and
incorporated herein.
[0173] The sequences surrounding the polymorphism will facilitate
the development of alternate PCR tests in which a primer of about
4-30 contiguous bases taken from the sequence immediately adjacent
to the polymorphism is used in connection with a polymerase chain
reaction to greatly amplify the region before treatment with the
desired restriction enzyme. The primers need not be the exact
complement; substantially equivalent sequences are acceptable. The
design of primers for amplification by PCR is known to those of
skill in the art and is discussed in detail in Ausubel (ed.), Short
Protocols in Molecular Biology, 4th Edition, John Wiley and Sons
(1999).
[0174] The following is a brief description of primer design.
[0175] Primer Design Strategy
[0176] Increased use of polymerase chain reaction (PCR) methods has
stimulated the development of many programs to aid in the design or
selection of oligonucleotides used as primers for PCR. Four
examples of such programs that are freely available via the
Internet are: PRIMER by Mark Daly and Steve Lincoln of the
Whitehead Institute (UNIX, VMS, DOS, and Macintosh),
Oligonucleotide Selection Program (OSP) by Phil Green and LaDeana
Hiller of Washington University in St. Louis (UNIX, VMS, DOS, and
Macintosh), PGEN by Yoshi (DOS only), and Amplify by Bill Engels of
the University of Wisconsin (Macintosh only). Generally these
programs help in the design of PCR primers by searching for bits of
known repeated-sequence elements and then optimizing the T.sub.m by
analyzing the length and GC content of a putative primer.
Commercial software is also available and primer selection
procedures are rapidly being included in most general sequence
analysis packages.
[0177] Sequencing and PCR Primers
[0178] Designing oligonucleotides for use as either sequencing or
PCR primers requires selection of an appropriate sequence that
specifically recognizes the target, and then testing the sequence
to eliminate the possibility that the oligonucleotide will have a
stable secondary structure. Inverted repeats in the sequence can be
identified using a repeat-identification or RNA-folding program
such as those described above. If a possible stem structure is
observed, the sequence of the primer can be shifted a few
nucleotides in either direction to minimize the predicted secondary
structure. The sequence of the oligonucleotide should also be
compared with the sequences of both strands of the appropriate
vector and insert DNA. Obviously, a sequencing primer should only
have a single match to the target DNA. It is also advisable to
exclude primers that have only a single mismatch with an undesired
target DNA sequence. For PCR primers used to amplify genomic DNA,
the primer sequence should be compared to the sequences in the
GenBank database to determine if any significant matches occur. If
the oligonucleotide sequence is present in any known DNA sequence
or, more importantly, in any known repetitive elements, the primer
sequence should be changed.
[0179] The following examples serve to better illustrate the
invention and are not intended to limit the scope of the invention
in any way. All references and patents disclosed herein are
specifically incorporated herein in their entirety by reference.
[0180] Salvi S., Morgante M., Fengler K., Meeley R., Ananiev E.,
Svitashev S., Bruggemann E., Niu X., Li B., Tingey S V., Tomes D.,
Miao G.-H., Phillips R L., Tuberosa R. Progress in the positional
cloning of Vgt1, a QTL controlling flowering time in maize (2003).
Proceedings of 57.sup.th Corn and Sorghum and 32.sup.nd Soybean
Seed Research Conference. Dec. 11-13, 2002, Chicago. [0181]
Phillips R. L., Kim T. S., Kaeppler S. M., Parentoni S. N., Shaver
D. L., Stucker R. I. and Openshaw S. J. 1992. Genetic dissection of
maturity using RFLPs. Proc. 47th Ann. Corn and Sorghum Res. Conf.
47:135-150. [0182] Vladutu, C., McLaughlin J. and Phillips R. L.
1999. Fine Mapping and Characterization of Linked Quantitative
Trait Loci Involved in the Transition of the Maize Apical Meristem
From Vegetative to Generative Structures. Genetics 153: 993-1007.
[0183] Salvi S., Tuberosa R., Chiapparino E., Maccaferri M.,
Veillet S., van Beuningen L., Isaac P., Edward K. J., Phillips R.
L. (2002). Toward positional cloning of Vgt1, a QTL controlling the
transition from the vegetative to the reproductive phase in maize.
Plant Mol Biol 48:601-613.
EXAMPLES
Example 1
RAP2.7 Expression Level is Associated with Differences in
Maturity
[0184] It was determined that Rap2.7 gene expression level
determines the transition to flowering in plants and that vgt1 is a
cis-element that regulates RAP2.7 transcription. Two plants with
different maturities (N28 and C22-4) were then screened to identify
if the RAP2.7 expression levels differ between them.
[0185] RNA was synthesized from N28 and C22-4 from different
tissues and stages of development, and RAP2.7 expression was
measured by RT-PCR.
[0186] Tissue Types:
[0187] Mature leaves--exposed, blades+sheaths
[0188] Immature leaves--in whorl, blades+sheaths
[0189] Shoot apical meristems
[0190] Roots--whole, including root apical meristems
[0191] Stalks--leftovers
[0192] Developmental Stages
TABLE-US-00001 Sample Number Genotype 1 2 3 4 5 N28 (late) veg veg
veg trans rep C22-4 (early) veg trans rep rep rep
[0193] Gene Expression Assay
[0194] First total RNA was prepared from frozen tissue samples.
cDNA was then made by reverse transcriptase and the PAR2.7 region
was amplified using PCR with agarose gel and ethidium bromide
staining. Band fluorescence was quantified and tubulin was used as
an internal control, and to normalize expression levels.
[0195] FIG. 1 shows the levels of RAP2.7 expression at Day: 14
before transition, C22-4 on transition at 20 days, and N28 at
transition at 27 days. Last two dates have 3 samples of each. P
values are significantly different at the first two sampling dates,
but not at the latest date. There is RAP2.7 signal from meristems,
and other tissues. RAP2.7 is a repressor of flowering, and must be
below a particular level for flowering to occur.
[0196] The results indicate that RAP2.7 is expressed in every
tissue type. RAP2.7 expression levels are lower in C22-4 than in
N28 before the reproductive transition (mature leaves), and RAP2.7
expression levels decrease during development in both C22-4 and
N28. Thus, overexpression of RAP2.7 will delay transition from
vegetative state to flowering.
Example 2
Over-Expression of Maize Rap2.7 Under a Moderate-Strength
Constitutive Promoter
[0197] The cDNA sequences of RAP2.7 were obtained from a RT-PCR
experiment. Specifically, total RNA from C22-4 leaves was isolated
and used as template in RT-PCR with gene-specific primers. The
gene-specific primers were designed based on RAP2.7 genomic
sequences from B73 genotype. The primer sequences are sense
-ATGCAGTTGGATCTGAACGT (SEQ ID NO: 9) and antisense
-GCCATCACCATCCCCGCTGA (SEQ ID NO:10).
[0198] The RT-PCR amplified fragment of RAP2.7, including the
entire 1371-bp sequence including the ATG start codon and the TAG
stop codon, was fused to the rice actin promoter and pinII
terminator to produce an expression cassette. This expression
cassette was then linked to a selectable marker cassette containing
a bar gene driven by CaMV 35S promoter and a pinII terminator in
FIG. 2.
[0199] Transgenic maize plants were produced by transforming
Immature GS3 maize embryos with this expression cassette, using the
Agrobacterium-based transformation method described as below.
[0200] For Agrobacterium-mediated transformation of maize with the
expression cassette comprising the rice actin promoter operably
linked to the maize RAP2.7 gene, the method of Zhao was employed
(U.S. Pat. No. 5,981,840, and PCT patent publication WO98/32326;
the contents of which are hereby incorporated by reference). While
the method below is described for the transformation of maize
plants with the actin promoter--RAP2.7 expression cassette, those
of ordinary skill in the art recognize that this method can be used
to produce transformed maize plants with any nucleotide construct
or expression cassette of the invention that comprises a promoter
operably linked to maize RAP2.7 gene for expression in a plant.
[0201] Briefly, immature embryos were isolated from maize and the
embryos contacted with a suspension of Agrobacterium, where the
bacteria are capable of transferring the ACTIN promoter-RAP2.7
expression cassette (illustrated above) to at least one cell of at
least one of the immature embryos (step 1: the infection step). In
this step the immature embryos were immersed in an Agrobacterium
suspension for the initiation of inoculation. The embryos were
co-cultured for a time with the Agrobacterium (step 2: the
co-cultivation step). The immature embryos were cultured on solid
medium following the infection step. Following this co-cultivation
period an optional "resting" step was included. In this resting
step, the embryos were incubated in the presence of at least one
antibiotic known to inhibit the growth of Agrobacterium without the
addition of a selective agent for plant transformants (step 3:
resting step). The immature embryos were cultured on solid medium
with antibiotic, but without a selecting agent, for elimination of
Agrobacterium and for a resting phase for the infected cells. Next,
inoculated embryos were cultured on medium containing a selective
agent and growing transformed callus was recovered (step 4: the
selection step). Preferably, the immature embryos were cultured on
solid medium with a selective agent resulting in the selective
growth of transformed cells. The resulting calli were then
regenerated into plants by culturing the calli on solid, selective
medium (step 5: the regeneration step).
[0202] The transformation produced 15 events in GS3XGaspe flint, an
early-flowering genotype. Transformation in GS3XHC69, a normal
maturity genotype, was problematic during shoot regeneration
process and only produced 5 events. The problem was presumed to be
related to the transgene. Two of the 5 events had delays in
flowering time for up to 30 days. In GS3XGaspe flint genotype,
majority (16 out of 20) of the events had various degrees of delay
in flowering time, from 16 days to 36 days. In almost all events
with delayed flowering, there was significant change in plant
architecture, mostly increase in plant height and internode
length.
[0203] Ectopic expression of Rap2.7 with the rice actin promoter
resulted in over expression of the gene, and significantly delayed
flowering as measured by the number of leaves (nodes) produced
prior to flowering. According to the invention, vectors for
transformation were produced in two corn genetic backgrounds (GS3 X
HC69, and GS3 X GF, gaspe flint) using the Rap2.7 structural gene
with the rice actin promoter.
[0204] A brief summary of the T0 phenotype from over-expressing
RAP2.7 in maize is shown in the following tables. As a reference,
GS3XGaspe plants coming out of tissue culture get pollinated
(exuding silks) within 45-60 days, and have 10 or less leaves.
Since these are T0 plants, accurate counts for leaf number and days
to flowering are not possible. The plants that were late in
flowering also had substantial increase in internode elongation,
resulting in increase in plant height. These plants also had
delayed senescence.
TABLE-US-00002 Events GS3XHC69 GS3XGaspe Late 2 16 Total 5 20
TABLE-US-00003 Days to Pollination GS3XGaspe Events .sup. <60
days 4 61-70 days 5 71-80 days 7 .sup. >81 days 1 no ear 3
TABLE-US-00004 Leaf Number (estimated) GS3XGaspe Events <10 4
10-12 10 13-14 4 >15 2
Example 3
Down-Regulation of Maize Rap2.7 by RNA Interference
[0205] Two fragments from the cDNA sequences of RAP2.7 from
genotype C22-4 were cloned by PCR to create an inverted repeat as
illustrated below. Specifically, a 955-bp fragment starting at 9-bp
downstream from ATG was cloned by PCR and designated as ZM-RAP2.7
(TR1) as a truncated form. Another fragment, 499-bp in length
starting from the same position, was cloned and designated as
ZM-RAP2.7 (IR1) for inverted repeat FIG. 5.
[0206] FIG. 5 Illustration of construction of the RAP2.7 gene
fragments for RNA interference vector. TR1 fragment was then
ligated to IR1, with IR1 in reverse orientation. The ligated
2-piece fragment was then linked to rice actin promoter with actin
5'-UTR and actin intron1 to create a full expression cassette. This
expression cassette was then linked to a selectable marker cassette
containing a bar gene driven by CaMV 35S promoter and a pinII
terminator.
[0207] FIG. 6 is an illustration of the vector used in
transformation. Note only the portion between the right and left
T-DNA borders (RB, LB) is shown. Transgenic maize plants were
produced by transforming Immature GS3 maize embryos with this
expression cassette, using the Agrobacterium-based transformation
method described for the RAP2.7 over-expression study. The
transformation produced 20 transgenic events from GS3XHC69, a
genotype with a normal maturity; and 15 events from GS3XGaspe
flint, an early-flowering genotype. Based on preliminary
observation on TO plants, all 15 events from the GS3XGaspe flint
background showed no visible change in flowering time. However, 9
out of the 20 GS3XHC69 transgenic events had various degrees of
early flowering phenotype. The earliest event flowered
approximately 2 week earlier compared to other events with the same
construct, and to other unrelated transgenic plants in the same
greenhouse room. All plants had normal plant architecture.
[0208] The T0 data shows that if you down regulate RAP2.7 earlier
flowering is observed. The following table shows the next (T1)
generation of plants. We can see from this data that the phenotype
is heritable and stable and the initial trend of decreased days to
pollination is still observed.
TABLE-US-00005 PHP21842 T1 Phenotype Event- Plant Transgene Leaf #
Days to Pollination 1-1 + 15 58 1-2 + 13 55 1-3 + 15 57 2-1 + 14 57
2-2 + 14 54 2-3 + 13 53 3-1 + 14 57 3-2 + 13 57 3-3 + 14 59 Control
+ 19.2 66 average (n = 20)
Example 4
Positional Cloning of Vgt1
[0209] Positional cloning was completed after mapping Vgt1 in a
1.3-cM interval flanked by two AFLP markers (Salvi et al., 2002),
in a cross between two nearly isogenic lines, N28 and its early
derivative C22-4 (Vladutu et al., 1999). Following BAC library
screening and the analysis of the relevant BAC contig, further
development of new markers and genetic mapping allowed for the
delimiting of Vgt1 within a ca. 2.7-kb region (FIG. 7.)
[0210] Sequencing of the relevant BAC clone and of the
corresponding DNA region from the parental lines involved in the
cross showed that Vgt1 lies within an intergenic region, ca. 75 kb
upstream of an Ap-2 like gene (Rap2.7) and ca. 10 kb downstream of
a RAD51-like gene. The 2.7 kb region found to be completely
associated with Vgt1 is essentially a low-copy region with a number
of polymorphisms between N28 and C22-4 (one of the polymorphisms is
caused by the insertion of a MITE transposable element in
C22-4).
[0211] Surprisingly, this sequence does not code for any known
protein. It is hypothesized to either be a RNAi element or a
regulatory RNA or DNA element that either directly regulates
expression of flowering genes such as Rap2.7 or specifically
targets expression of other genes which control flowering genes
such as Rap2.7.
Example 5
Vgt1 is Associated with Maturity Shift in Inbred Lines
[0212] The information gathered with the positional cloning study
allowed the testing of the 2.7 kb region as candidate for
controlling flowering time through association mapping. Several
SNPs and other polymorphisms identified at and around Vgt1 were
screened on a panel of ca. 100 lines representative of cultivated
maize germplasm (Remington et al., 2001) and a panel of elite
proprietary lines.
[0213] Linkage disequilibrium at the Vgt1 region was quickly
dissipated over distance of ca. 1 kb within the panel of 100 lines.
Association analysis showed that among the genes and sequence at
and around Vgt1, the only DNA region statistically associated with
flowering time was a sub-region of ca. 2 kb within the same 2.7 kb
region identified by positional cloning (FIG. 8.).
[0214] This element thus can be used as a sequence-based marker to
identify inbred and hybrids which have altered maturity.
Sequence CWU 1
1
1411371DNAZea mays 1atgcagttgg atctgaacgt ggccgaggcg ccgccgccgg
tggagatgga ggcgagcgac 60tcggggtcgt cggtgctgaa cgcgtcggaa gcggcgtcgg
cgggcggcgc gcccgcgccg 120gcggaggagg gatctagctc aacgccggcc
gtgctggagt tcagcatcct catccggagc 180gatagcgacg cggccggcgc
ggacgaggac gaggacgcca cgccatcgcc tcctcctcgc 240caccgccacc
agcaccagca gcagctcgtg acccgcgagc tgttcccggc cggcgccggt
300ccgccggccc cgacgccgcg gcattgggcc gagctcggct tcttccgcgc
cgacctgcag 360cagcaacagg cgccgggccc caggatcgtg ccgcacccac
acgccgcgcc gccgccggcc 420aagaagagcc gccgcggccc gcgctcccgc
agctcgcagt accgcggcgt caccttctac 480cgccgcacag gccgctggga
gtcccacatc tgggattgcg gcaagcaggt gtacctaggt 540ggattcgaca
ccgctcacgc cgctgcaagg gcgtacgacc gggcggcgat caagttccgc
600ggcgtcgacg ccgacatcaa cttcaacctc agcgactacg aggacgacat
gaagcagatg 660gggagcctgt ccaaggagga gttcgtgcac gtcctgcgcc
gtcagagcac cggcttctcg 720agaggcagct ccaggtacag aggcgtcacc
ctgcacaagt gcggccgctg ggaggcgcgc 780atggggcagt tcctcggcaa
gaaagcttac gacaaggccg ccatcaaatg caatggcaga 840gaggccgtga
caaacttcga gccgagcacg tatcacgggg agctgccgac tgaagttgct
900gatgtcgatc tgaacctgag catatctcag ccgagccccc aaagagacaa
gaacagctgc 960ctaggtctgc agctccacca cggaccattc gagggctccg
aactgaagaa aaccaagatc 1020gacgatgctc cctctgagct cccgggccgc
cctcgtcggc tgtctcctgt cgtggctgag 1080catccgccgg cctggcctgc
gcagccgcct caccccttct tcgtcttcac aaaccatgag 1140atgagtgcat
caggagatct ccacaggagg cctgcagggg ctgttcccag ctgggcatgg
1200caggtggcag cagcagctcc tcctcctgcc gccctgccgt cgtccgctgc
agcatcatca 1260ggattctcca acaccgccac gacagctgcc accgccgccc
catcggcctc ctccctccgg 1320tactgcccac cgccgccgcc gccgccgtcg
agccatcacc atccccgctg a 13712456PRTZea mays 2Met Gln Leu Asp Leu
Asn Val Ala Glu Ala Pro Pro Pro Val Glu Met1 5 10 15Glu Ala Ser Asp
Ser Gly Ser Ser Val Leu Asn Ala Ser Glu Ala Ala 20 25 30Ser Ala Gly
Gly Ala Pro Ala Pro Ala Glu Glu Gly Ser Ser Ser Thr 35 40 45Pro Ala
Val Leu Glu Phe Ser Ile Leu Ile Arg Ser Asp Ser Asp Ala 50 55 60Ala
Gly Ala Asp Glu Asp Glu Asp Ala Thr Pro Ser Pro Pro Pro Arg65 70 75
80His Arg His Gln His Gln Gln Gln Leu Val Thr Arg Glu Leu Phe Pro
85 90 95Ala Gly Ala Gly Pro Pro Ala Pro Thr Pro Arg His Trp Ala Glu
Leu 100 105 110Gly Phe Phe Arg Ala Asp Leu Gln Gln Gln Gln Ala Pro
Gly Pro Arg 115 120 125Ile Val Pro His Pro His Ala Ala Pro Pro Pro
Ala Lys Lys Ser Arg 130 135 140Arg Gly Pro Arg Ser Arg Ser Ser Gln
Tyr Arg Gly Val Thr Phe Tyr145 150 155 160Arg Arg Thr Gly Arg Trp
Glu Ser His Ile Trp Asp Cys Gly Lys Gln 165 170 175Val Tyr Leu Gly
Gly Phe Asp Thr Ala His Ala Ala Ala Arg Ala Tyr 180 185 190Asp Arg
Ala Ala Ile Lys Phe Arg Gly Val Asp Ala Asp Ile Asn Phe 195 200
205Asn Leu Ser Asp Tyr Glu Asp Asp Met Lys Gln Met Gly Ser Leu Ser
210 215 220Lys Glu Glu Phe Val His Val Leu Arg Arg Gln Ser Thr Gly
Phe Ser225 230 235 240Arg Gly Ser Ser Arg Tyr Arg Gly Val Thr Leu
His Lys Cys Gly Arg 245 250 255Trp Glu Ala Arg Met Gly Gln Phe Leu
Gly Lys Lys Ala Tyr Asp Lys 260 265 270Ala Ala Ile Lys Cys Asn Gly
Arg Glu Ala Val Thr Asn Phe Glu Pro 275 280 285Ser Thr Tyr His Gly
Glu Leu Pro Thr Glu Val Ala Asp Val Asp Leu 290 295 300Asn Leu Ser
Ile Ser Gln Pro Ser Pro Gln Arg Asp Lys Asn Ser Cys305 310 315
320Leu Gly Leu Gln Leu His His Gly Pro Phe Glu Gly Ser Glu Leu Lys
325 330 335Lys Thr Lys Ile Asp Asp Ala Pro Ser Glu Leu Pro Gly Arg
Pro Arg 340 345 350Arg Leu Ser Pro Val Val Ala Glu His Pro Pro Ala
Trp Pro Ala Gln 355 360 365Pro Pro His Pro Phe Phe Val Phe Thr Asn
His Glu Met Ser Ala Ser 370 375 380Gly Asp Leu His Arg Arg Pro Ala
Gly Ala Val Pro Ser Trp Ala Trp385 390 395 400Gln Val Ala Ala Ala
Ala Pro Pro Pro Ala Ala Leu Pro Ser Ser Ala 405 410 415Ala Ala Ser
Ser Gly Phe Ser Asn Thr Ala Thr Thr Ala Ala Thr Ala 420 425 430Ala
Pro Ser Ala Ser Ser Leu Arg Tyr Cys Pro Pro Pro Pro Pro Pro 435 440
445Pro Ser Ser His His His Pro Arg 450 45537236DNAZea mays
3caagacttga gctcgaaaag tagcacagag agttcacaac tcgaacggag ctcaaatcac
60taacacaatc gatcaaatgc gaggaggcgg agtgtgggag tcttagaatg cttagtggat
120gcttagatgt ttcctccatg cgcctagagg tcccttttat agccccaaga
cacctaagag 180ccgttggaga tcaacatgga atgctatcct tgccttctgt
cgagtggcgc accggacagg 240tcctgtagat tgttcggtgc gcgatctcct
tccaaatttg gcatatccga ccgttgctcc 300tctgggctga ttggcgcacc
ggacacagtc cggtgcacac cggacagtcc ggtgcaccag 360ctgaccgttg
gagcagtcca cgtgtcgcgc gaagattgcg tggccgaccg ttgctcaggc
420gaccgttggc tcaccggaca gtccggtgca ccaccggaca gtccggtgaa
ttatagtcgt 480acgccgccgt cgaaacccga gagcggcgag ttcacagtgg
accagcctgg cgcaccggac 540actgtccggt gcaccattgg acattgtccg
gtgcaccacc ggacagtccc gtgtgcgaga 600ccgagcacaa gattggctgc
acagagccaa gcttttccct ttttctccct tcttttttag 660tcactgtttc
tagcacttgg ataaccatgt tagtacataa aacaattcac caagtctaga
720aacatacctt ttgccttgat tttcacttct cactttattt ggcacataag
aacttaatta 780aacgtgttgg gcacttaatc accaaaacat tatagaaatg
gcccaaaggc acatttccct 840ttcagtaata agttaatacc tctttatatc
attatttgaa cactgtgcaa tgatgttcat 900ttatgtaatc gttgtgtacg
tcagttctaa ttctagcacg tacatggttc acatccaatt 960tgtcttctaa
aaacgaatgt gacataatgt catatgtatg tgataatgct ttttgttggg
1020gtccttcgtc tttcaaaggt cctcaaaaac acatttaacc attggttgtt
agcacatcct 1080taagtgttgc aggagctttg gtattgaata ccttcggagc
aggacatgga ggaagacgaa 1140gatgttagct tcgtcataac aacacaagga
aacgaaggca gaagtggaac aaggccggga 1200tatggtgttt tcaagactct
gtatccaaag caaaaaagac agaaagacga tactgccctt 1260acataatttg
taaactatgt gaacaagttt tatggacatg tttgtaactt tacacgaaat
1320tgtaccacca cactataaat agataaatag tgccctgcat gaggcgcctc
ttgggaacaa 1380tgaggaacaa ctctgtataa tcctttttct tctaagtacc
ttcgggtttt ctcctcatca 1440aaaagccgaa ggtactattg taaattcgtt
tcatataaag aaagaaatcc caagttgttt 1500gagataagta atcttatcta
gctttgttat agccatgtgt gtaatcttta tctttatcct 1560ctgacaatcc
tatatattat atataataac cttcgtactt tacttggatg tcccgaagga
1620caaactcttt aagtacgaag gataacatct tttttaataa tgtgttgcct
tgttttttat 1680tgtgtacaac aattaaaaac gagtgaccaa cattttcatg
tcagggtatg gggacccatt 1740ggagactcga tatccaaatg aggatgagta
tatgatgaat cctataccta tgatgagtat 1800aagtatgaga atcaggatga
gtataacttc atcagaatag gtgcgggggc gtccttgtgg 1860gcgtgcctac
cgtgcgatcg cacaaggcct ccaaaaccat agggccccaa aatttataac
1920aatctttata tacaatataa ataaaaaata ttattttata taaaatattt
accaacatat 1980agcatagaat cgtaaaagcg ttgaaatcga tctgttctta
ttgttattca aactatttac 2040ctccagcaca ttgtagtcat tagataaaaa
agattgagat cttattgtca ctatcttaag 2100aagacacagt taaaagaggt
agacaatatg tcaatatgct ttgatgcaag tgaccaattc 2160gtgacgttga
gtttcctcta agattttgtt taaaaaattg ctatgttgac attctaaatt
2220ttataaagca gaggagcaaa actgagtaaa atcgcattta atgataaaaa
tgtggaaagt 2280gacaaaacta agaatacaat tttaaatagt ccaatatttt
tttactatct tttgcacagg 2340gcctctcaac ttgggaggac gcttctgggt
gtgggtttac aaattcgatg aaaaattccc 2400cattgacaaa cgataggagg
atatttttct cccagcacaa aatagcatag ccataaggca 2460acaaggcatg
gcaaaggatc gtatcatcgt catccgagac ccattgcttt ctctctctct
2520cctcgtgctt tcattactgg ggtgggggtg gagtggacca gtggagtgga
gaaatgacaa 2580atccaggccc gcaggcagcc ccacccacca aatcggccga
gcagggtgcc caaatcagga 2640aggattttaa ggttaaccgg ctgccaccgc
ccaccgccgg tgaccccagt ctctcttcta 2700tctatatatt acccgcctcc
ttttctcctc tctctccgcc ccaccctcct tcctcagctc 2760cgttgcgcac
cgccaccgcc ggccggccag ccgccggagc accgaaagac ccccgttctt
2820tcctgtaaaa aaaaacccgc cgcctttagc tagctaaccg gtcgtcctct
tcacccccta 2880gctttgctag ctctagctag gaacgaaaga aattaaagga
taactgagat tgctgattgg 2940tggtccgggt acggtgttct tgagtcgtga
agcgacagta cagtggctag ggtcgtgccg 3000cccctgcagt ctccggggtt
gcgtgcagga tggtcgtcag ggatcgggag tgaggaggca 3060tcagctctcg
cggtcgtgga gcctaaatgt accgcaacaa cgactcggca ctctcctgct
3120tctacctctt cctcctctgg ttcttcttct tgaagtagac accaccagtt
cgccaggtag 3180ttagcagccc agttgcgact ggggatcggt ggcgggctgc
cgcttgcgag ttgtaagctt 3240ggaggggagg ggagcaggag caggagatgc
agttggatct gaacgtggcc gaggcgccgc 3300cgccggtgga gatggaggcg
agcgactcgg ggtcgtcggt gctgaacgcg tcggaagcgg 3360cgtcggcggg
cggcgcgccc gcgccggcgg aggagggatc tagctcaacg ccggccgtgc
3420tggagttcag catcctcatc cggagcgata gcgacgcggc cggcgcggac
gaggacgagg 3480acgccacgcc atcgcctcct cctcgccacc gccaccagca
ccagcagcag ctcgtgaccc 3540gcgagctgtt cccggccggc gccggtccgc
cggccccgac gccgcggcat tgggccgagc 3600tcggcttctt ccgcgccgac
ctgcagcagc aacaggcgcc gggccccagg atcgtgccgc 3660acccacacgc
cgcgccgccg ccggccaaga agagccgccg cggcccgcgc tcccgcagct
3720cgcagtaccg cggcgtcacc ttctaccgcc gcacaggccg ctgggagtcc
cacatctggt 3780cagtactacc actgtctaca actagccaca ccacaccgat
tgcttccgac tctcattaat 3840ttctgacaca aactctccgt cttcctcctc
ttctcccgcg acgcagggat tgcggcaagc 3900aggtgtacct aggtgagcaa
gagcagatct cttttgcgtt cccaaagatt tttccccttt 3960tagttcctta
tcccatccca tctcgaatgg cctagctaac cgattcagtg gtggtccggc
4020tgctggccga tatacgcagg tggattcgac accgctcacg ccgctgcaag
gcacgcactg 4080gactggacgc ccagaattct tcgtcatgtg agtctctgac
cgaattgatt gattaacgag 4140tctctggctc ctggaactcg cagggcgtac
gaccgggcgg cgatcaagtt ccgcggcgtc 4200gacgccgaca tcaacttcaa
cctcagcgac tacgaggacg acatgaagca gatggggagc 4260ctgtccaagg
aggagttcgt gcacgtcctg cgccgtcaga gcaccggctt ctcgagaggc
4320agctccaggt acagaggcgt caccctgcac aagtgcggcc gctgggaggc
gcgcatgggg 4380cagttcctcg gcaagaagta agaaccaacc aacgcttctt
ttctttttct tttttttata 4440gcatgcagtg atgattcaac cttagttgtg
cctttcctcc taatcctata tatgtaggat 4500ttagtactgg ttgactatat
aagtatatat gtattgttca gtaaaagtat acataggtta 4560gctgcatgtt
tatgtatgta gctggttgtt tcaatcagaa gataaaaaaa aagggaagta
4620gtggctaggg aattcctcca atcctcaccg gtgggaacgc cgtgcttggg
tgcaggtaca 4680tataccttgg gctattcgac agcgaagtag aggctgcaag
gttcttcatc ttggattctg 4740ccgttcatat atgcataatc atgtctttta
atttccaaag ggttgagtac cgactcgatt 4800cctcttcgtg tcttttttct
ttctttcttc gaaatccaga gcctacgaca aggccgccat 4860caaatgcaat
ggcagagagg ccgtgacgaa cttcgagccg agcacgtatc acggggagct
4920gccgactgaa ggtacgtatt ttctttctgc atatatatat cttcaggtat
tattggctat 4980taaactgctt ggatcttact gcttcttctg cagttgctga
tgtcgatctg aacctgagca 5040tatctcagcc gagcccccaa agagacaaga
acagctgcct aggtctgcag ctccaccacg 5100gaccattcga gggctccgaa
ctgaagaaaa ccaaggcaag cgctaacgat agatatacct 5160tgacaagcta
gtatcaaaca aaaccagtaa aaaaagttta ctttcttgtc gaatttcatt
5220gcctacctga tgtacgtact tgtgcttctg cacaaaataa cgaaatcctt
ttgccctctg 5280atgatgatgc agatcgacga tgctccctct gagctaccgg
gccgccctcg tcagctgtct 5340cctctcgtgg ctgagcatcc gccggcctgg
cctgcgcagc cgcctcaccc cttcttcgtc 5400ttcacaaacc atgaggttag
gtgacagcta ctgatcgaga tgcagcagca gttcaaacct 5460gtctgttcca
aggaccttta ggccggatta ccaaatcatc ggtcaactgt cctgtctgtt
5520atatatttat gtgttaattt ataatacaag tgtgactatt tttcaaacct
tccttcaaaa 5580tgcatgaaaa gagttttttt taacgaaagg cgaaaagaaa
atatgatact tgggacagga 5640gcaagcttgg atcatcagaa agtattatta
attaggatca ctgagctgtt cattttgttc 5700ttgagtcaat cctaatcgta
ctatgtcagt gaatgaactt gtgttgcacc aatgcagatg 5760agtgcatcag
gagatctcca caggaggcct gcaggggctg ttcccagctg ggcatggcag
5820gtggcagcag cagctcctcc tcctgccgcc ctgccgtcgt ccgctgcagc
atcatcagga 5880ttctccaaca ccgccacgac agctgccacc accgccccat
cggcctcctc cctccggtac 5940tgcccgccgc cgccgccgcc gtcgagccat
caccatcccc gctgagagaa tcaagaagcc 6000gcactgtaaa tctgccggga
agctagcatt ttccccccgg cccctccccc tctccgggcg 6060ttgcgacttt
ttcagttttg cgccgccggc cggggtggtg gtttcttgta gccgatcgat
6120tggattcctc gtattactgc tgcttacact cccaattaag tgaaaaaaaa
acgctcctct 6180actctttaca ctacacacac tgttagctga tcgattggac
gtacttgcta gctgctgttg 6240ctgctgctag cttgagattg actaacttca
gcacttggat tgatctatat ctatatgact 6300atatagacga cacattgtgt
acgtgtagat aatatttctt cttttcctga ccgccataaa 6360actgtttact
ctggccattt tgaactaaag gctagctaca aatgagtgtc cttctcggcc
6420ttctacatgt tctggtcatg gacatcgaga gatcaaactt ctctgtcctg
cttactagat 6480acgtactaga tttacttagc ctagatagat tccgttccaa
actcgaggcc aggcgcatcg 6540agatccgaga acttcatcca ctcgtcgctc
atcatgctgc atgcatgatg gtctcaactc 6600tgaggcatgc aaacgcagtg
agacgaactg ggaggaattt atatagagta tatattgtcc 6660ggcctgttgg
tgataaagat agaatgcatg cacgctaact gccaacatgc atgggtgctg
6720catcgaattt ttggtatggt gcatgcatac cgtgcattgg tgctctgcta
gtactaggac 6780caatctccat ggctccatta gatctcttgt ttactcgtct
ccatgtgcct ctcaaagtgt 6840gtactagcta gttgcggcac acaagttggc
agttgtttgt tgtttcagcg gggaagaagg 6900aggtcaccgt tgtcatcgtc
aggggcgaag ctaggatcag aagacagagg gggcaggctt 6960agcctccaag
cgaccaaacc agtaagaaca caatataaaa atggcaagag aaccaaccat
7020aatatatata ttgatatata atcttcatta aaaaacagta taatgaaaca
acatctattt 7080tgtcaaacaa aataaaatta aatctcagtt attttgaatt
tagctctacg tgtattagct 7140agatcatagg tgaaagtcgc ctagaggggg
ggtgaatagg gcgaaactga aatttacaaa 7200tataaacaca actacaagcc
gggttagcgt tataag 723647306DNAZea mays 4tagaatatat aatctagagc
aaactagtta gtccaaatat ttgtgttggg aattcaacca 60ccaaaattat ttataggaaa
aggttaaacc ctatttccct ttcactaatt aattggaaga 120acttgaggtg
tagtcttctt cgtcgtcgtg ccgttaatgg ggtcctagca cagtacttgc
180tctaccgagg ttgggtacca aggttctttt gttttgcttt tgttagacac
cccatgtggg 240gaggggtact atgtttatca aactgtagaa acctaacagg
cgactttgac ctctggagaa 300tctttgtaaa tgctacatag tgaaaccttg
ttgactcacc ataggagtgt ttaagggttt 360gatcgactta tggcaaaaag
ggggtcacgg ctcgtgagta aagtgtaaga cctttgcata 420gggttagaaa
ctgatatatc agtcatgctc acaattaaga acggccttgg gagctccttt
480gattagagat actgtagata cattcatgat gatggtttga tgatggtgcc
tctaattatg 540atttctagta ttttctctac gaggaggtac tatttgggat
aataagctag gttttaagat 600aaaatttggc ttatattaat gattaaaacc
tgataaagta aaagcaacct gctatcagct 660taactccaca taaagctagt
ccattttagc caaacaagat atttgctgag tacgttgatg 720tgtgcaaaat
ggagaacttt tatcttaaaa caccaggttg tccacactgc aaccactgct
780caagcgagga tgaaggcaac atgaagaact ttcaggagtt tctagacttc
aaggagtttt 840aaactagatt agtggtaaac cccagtcagc tgcctgtgaa
ggccttatct ttactacgtt 900ccgcgttagc actttgttta cttgttaagt
tgatggatac atcatgttgt aataagttaa 960tacctcttta tatcattatt
tgaacactgt gcaatgatgt tcatttatgt aatcgctgtg 1020tatgtcagtt
ctaattctag cacatacatg gttcacatcc agtttgtctt ctaaaaaacg
1080aatgtgacat aatgtcatat gtatgtgata atgctttttg ttggggtcct
tcgtctttca 1140aaggtcctca aaaacacatt taaccattgg ttgttagcac
atccttaagt gttgcaggag 1200ctttggtatt gaataccttc ggagcaggac
atggaggaag acgaagatgt tagcttcgtc 1260ataacaacac aaggaaacga
aggcagaagt ggaacaaggc cgggatatgg tgttttcaag 1320actctgtaac
caaagcaaaa aagacataaa gacgatactg tccttacata atttgtaaac
1380tatatgaaca agttttatgg acatgtttgt aactttacac gaaactgtac
caccacacta 1440tagatagata aatagtgccc tgcatgaggc gcctcttggg
aacaatgagg aacaactgtg 1500tgtaatcctt tttcttctaa gtaccttcgg
gttttctcct catcaaaaag cggaaggtac 1560tattgtaaat ttgtttcata
taaagaaaga aatcccaagt tgtttgagat aagtaatctt 1620atctagcttc
gttatagccc tgtgtgtaat ctttatcttt atcctctgac aatcctatat
1680attatatata ataaccttcg tactttactt ggatgtctcg aaggacaaac
tctttaagta 1740cgaaggataa catctttttt aataatatgt tgccttgttt
ttttattgtg tacaacaatt 1800aaaaacgagt gaccaacatt ttcatgtcgg
ggtatggaga cccattggag actcctaaat 1860gaggatgggt atatgatgaa
tcctatacct atgatgagta taagtatgag aatcaggatg 1920agtataactt
catcagaaaa ggtacggggg cgtccttgtg ggcgtgccta cagtgcgatc
1980gcacaaggcc tccaaaacca tagagcccca aaatttataa caatctttat
atacaatata 2040aataaaaaaa tattatttta tataaaatat ttaccaacat
atagcataga atcgtaaaag 2100cgttgaaatc gatatgttct tattgttatt
caaactattt acctccagca tattgtagtc 2160attagataaa aaagattgag
atcttattgt cactatctta agacgacaca gttaaaagag 2220gtagacaata
tgatttgatg caagtaacca attcgtggcg ttgagtttcc tttaagattt
2280tttaaaaaaa aattgctatg ttgacattct aaattttata aagcagagga
gcaaaactga 2340gtaaaatcgt aattaatgat aaaaatgcgg aaagtgacaa
aactaagaat acaattttaa 2400atagtccaat atctttttac tatcttttgc
acagggcctc tcaacttggg aggacgcttc 2460tgggtgtggg tttacaaatt
cgatgaaaaa ttccccattg acaaacgata ggaggatatt 2520tttctcccag
cacaaaatag catagccata aggcaacaag gcatggcaaa ggatcgtatc
2580atcgtcatcc gagacccatt gctttctctc tctctcctcg tgctttcatt
actggggtgg 2640gggtggagtg gaccagtgga gtggagaaat gacaaatcca
ggcccgcagg cagccccacc 2700caccaaatcg gccgagcagg gtgcccaaat
caggaaggat tttaaggtta accggctgcc 2760accgcccacc gccggtgacc
ccagtctctc ttctatctat atattacccg cctccttttc 2820tcctctctct
ccgccccacc ctccttcctc agctccgttg cgcaccgcca ccgccggccg
2880gccagccgcc ggagcaccga aagacccccg ttctttcctg taaaaaaaaa
cccgccgcct 2940ttagctagct aaccggtcgt cctcttcacc ccctagcttt
gctagctcta gctaggaacg 3000aaagaaatta aaggataact gagattgctg
attggtggtc cgggtacggt gttcttgagt 3060cgtgaagcga cagtacagtg
gctagggtcg tgccgcccct gcagtctccg gggttgcgtg 3120caggatggtc
gtcagggatc aggagtgagg aggcatcagc tctcgcggtc gtggagccta
3180aatgtaccgc aacaacgact cggcactctc ctgcttctac ctcttcctcc
tctggttctt 3240cttcttgaag tagacaccac cagttcgcca ggtagttagc
agcccagttg cgactgggga 3300tcggtggcgg gctgccgctt gcgagttgta
agcttggagg ggaggggagc aggagcagga 3360gatgcagctg gatctgaacg
tggccgaggc gccgccgccg gtggagatgg aggcgagcga 3420ctcggggtcg
tcggtgctga acgcgtcgga agcggcgtcg gcgggcggcg cgcccgcgcc
3480ggcggaggag gggtccagct caacgccggc cgcgctggag ttcagcatcc
tcatccggag 3540cgacagcgac gcggccggcg cggacgagga cgaggacgcc
acgccgtcgc ctcctcctcg 3600ccaccgccac cagcaccagc agcagctcgt
gacccgcgag ctgttcccgg ctggcgccgg 3660cccgccggcc ccggcgccgc
ggcattgggc cgagctcggc ttcttccgcg ccgacctgca 3720gcagcaacag
gcgccgggcc ccaggatcgt gccgcaccca cacgccgcgc cgccgccggc
3780caagaagagc cgccgcggcc cgcgctcccg cagctcgcag taccgcggcg
tcaccttcta 3840ccgccgcaca ggccgctggg agtcccacat ctggtcagta
ctaccactgt ctacaactag 3900ccacaccaca ccgattgctt ccgactctca
ttaatttctg acacaaactc tccgtcttcc 3960tcctcttctc ccgcgacgca
gggattgcgg caagcaggtg tacctaggtg agcaagagca 4020gatctctttt
gcgttcccaa agattttccc cttttagctc ctcatcccat ctcgaatggc
4080ctagctaacc gattcactgg tggtccggct gctggccgat atacgcaggt
ggattcgaca 4140ccgctcacgc cgctgcaagg cacgcactgg actggacgcc
cagaattctt cgtcatgtga 4200gtctctgacc ggattggttg attgattgat
taacgagtct ctggctcctg gaactcgcag 4260ggcgtacgac cgggcggcga
tcaagttccg cggcgtggac gccgacatca acttcaacct 4320cagcgactac
gaggacgaca tgaagcagat ggggagcctg tccaaggagg agttcgtgca
4380cgtcctgcgc cgccagagca ccggcttctc gagaggcagc tccaggtaca
gaggcgtcac 4440cctgcacaag tgcggccgct gggaggcgcg catggggcag
ttcctcggca agaagtaaga 4500accaaccaac gcttcttttt ttttatagca
tgcagatgat gattcacact tagttgtgcc 4560tctcctccta atcctatgta
ggatttagta ttggttgact acatatctat tgttatatat 4620gtattgttca
gtaaaagtat acataggtta gctgcatgtt tatgtatgta gctggttgtt
4680tcaatcagaa gataaaaaga aagggaagta gtggctaggg aattcctcca
atcctcaccg 4740gtgggaacgc cgtgcttggg tgcaggtaca tataccttgg
gctattcgac agcgaagtag 4800aggctgcaag gttcttcatc ttggattctg
ccgttcatat atgcataacc atgtcttttc 4860atttccaaag ggttgagtac
cgactcgatt cctctttttt tttctttctt tcttcgaaat 4920ccagagctta
cgacaaggcc gccatcaaat gcaatggcag agaggccgtg acgaacttcg
4980agccgagcac gtatcacggg gagctgccga ctgaaggtac gtatttcttt
ctgcatatat 5040ataatcttca ggtattattg gctattaaac tgcttggatt
ttactgcttc ttctgcagtt 5100gctgatgtcg atctgaacct gagcatatct
cagccgagcc cccaaagaga caagaacagc 5160tgcctaggtc tgcagctcca
ccacggacca ttcgagggct ccgaactgaa gaaaaccaag 5220gcaagcgcta
acgatagata taccttgaca agctagtatc aaacaaaacc agtatttttt
5280ttactttctt gtcgaatttc attgcctacc tgatgtacgt acttgtgctt
ctgcacagaa 5340taacgaaatc cttttgccct ctgatgatga tgcagatcga
cgatgctccc tctgacctcc 5400cgggccgccc tcgtcggctg tctcctctcg
tggctgagca tccgccggcc tggcctgcgc 5460agccgcctca ccccttcttc
gtcttcacaa accatgaggt taggtgacag ctactgatcg 5520agatgcagca
gcagttcaaa acttagatcc ggtcccttgt cctgtctgtt ccaaggacct
5580ttaggccgga ttaccaaatc atcggtcaac tgtcctgtct gttatatatt
tatgtgttta 5640taatacaagt gtgactattt ttcaaacctt ccttcaaaat
gcatgaaaag agtttttttt 5700ttaacgaaag gcgaaaagaa aatatgatac
tttgggacag gagcaagctt ggatcatcag 5760aaagtattat taattaggat
cactgagctg ttcattttgt tcttgaatca atcctaatcg 5820tactatgtca
gtgaatgaac ttgtgttgca ccaatgcaga tgagtgcatc aggagatctc
5880cacaggaggc ctgcaggggc tgttcccagc tgggcatggc aggtggcagc
agcagctcct 5940cctcctgccg ccctgccgtc gtccgctgca gcatcatcag
gattctccaa caccgccacg 6000acagctgcca ccgccgcccc atcggcctcc
tccctccggt actgcccgcc gccgccgccg 6060ccgccgtcga gccatcacca
tcgccgctga gagaatcaag aagccgcact gtaaatctgc 6120cgggaatgaa
gctagcattt tccccccggc cctcccctct ccgggcgttg cgactttttc
6180agttttgcgc cgccagccgg ggtggtggtt tcttgtaacc gatcggttgg
attcctcgta 6240ttactgctta cactcccaat taagtgggaa aaaacgctcc
tctactcttt acactagaca 6300cactgttagc tgatcgattg gacgtacttg
ctagctgctg ttgctgctgc tagctagaga 6360ttgactaact gaagcacttg
gattgatcta tatctatatg actatataga cacattgtgt 6420acgtgtagat
aatatttctt tatttcctga ccgccataaa actgtttact ctagccattt
6480tgaactaaag gctagctaca aatgagtgtc cgtctcggcc ttctacatgt
tctggtcatg 6540gacatcgaga gatcaaactt ctctgtcctg cttactagat
acgtactaga tttacttagc 6600ctagatagat ttcgttccaa actcgaggcc
aggcgcatcg agatccgagc acttcatcca 6660ctcgtcgttc atcgtgctgc
atgcatgatg gtctcaactc tgaggcatgc aaacgcagtg 6720agaacgaact
gggaggaatt tatatagagt atatattgtc cggcctgttg gtgataaaga
6780tagaatgcat gcacgctaac tgccaacatg catgggtgct gcatcgaatt
tttggtatgg 6840tgcggtgcat gcataccgtg cattggtggg gagaatccat
gaaatagcac ttgtttgaga 6900cggcgttcac gaaatagcat ccgatttcaa
gtaattcata gaatagcact tgttttacca 6960aattaattca gaaaataaca
ctctatctat attttgcatt cttttattgc ttacctcaca 7020tacaaatgga
ccaaattacc cctatttatc acaaccttct ttttttatct cttatgtcca
7080taagaacaac ataaataaat aaaatatatg acctatattt ttatagtgtt
aaacatacaa 7140atagacacat gttttatgaa cgaagaagtc tatatttaac
tattaaattt agaactaggt 7200atcttcggtt gtcgaacttt taaaattaga
catatttggt ccattaaaag gtcttagatt 7260gtacttccta tgttcttttt
tatttgatac ggttgttttt tttcaa 730654230DNAZea mays 5ctccacctct
ctcgtacgct ttgattggcc gctcgcacgc atttatcgcc aagatcgggc 60ttgcccatca
ctgggatcat cctggctggc tggctggccg gccacatatc aaacatggat
120ttgctatatt ttaaatgcct gaaatatgtt atactatgtt ttttagataa
cgatctttac 180agtcgattta tgaatgtgta gaattaagat tgagcttcct
gatgaaaaaa tcaggcaaga 240ggctcttttg gttggagacc tggcatggca
cttgcctgcg agtcaagcaa accctatgcc 300actgtttgga ccaggccact
ttcctagaga gccgaatcag acatcactat cggaattcga 360atgagtgctc
agcactttac cgagtgcaat taatcggaca ctcggcaaag cattctttgt
420cgagtgccac tctcggcgaa ctaataaggc tctcgggaca gatctcgtat
gtcgagagcg 480gaacactcga catagaaaaa cactcggcaa aagaggtttt
gtcgaatgac aagctctcgg 540caaaatgtga cactcgataa gggtcgtcaa
gagtcgtcta ttgttgacgg ccattaactt 600tcccgagtgt caaacgttga
cacttgggaa ttatcttctt tttgtcgatt gtaacctggc 660aaaccctcgg
caaaagtata ctttgcggag tgtcttccct caacactcga taaagaatat
720ttgtttcttt ttcttttttc tataccaaac tctttgtgac gttttttctg
cagtatatag 780acatacatat tcaattttac acaattatca aagtgtttgc
tataattaat agatttagtt 840tgtttaattg aatttctgag aaccaatcaa
atagacccga attagacata tctagacatt 900taaaaagtaa gatactaaaa
aataatagtg tttaccttca accggtacta aatgtcattc 960ctgtagtaga
caggataacg aaagctagtc tatttagatc atcagttcca gttcgagatt
1020ttaaatgcta gtccctccat ttcaatttac aatttattta atttttttag
tgtgataccg 1080tttagcgtat gtagctttga tttttttata tatttttgca
aaattttgaa taagacaagt 1140gtgtcaaatt tggtgtaaaa attaaacgaa
attataaatt ggagcggagg gagtagaaat 1200ctacgatttt tctagctgag
gcagactgtg cgcattccca tccatcgagt ccacttgcac 1260ctctcctcga
catgaatacg aatgtacgat ccgatgaatt ccccacaaag aagcaattaa
1320cgtcaaatcc atcatcgtca taaaacgacg ataccgagct agcgtttaat
tagtttgtta 1380gacgaaccag aaactatatt atatcgcttt gtgtccaaaa
attacttata ttatatatag 1440ttctcgtgca tctacgtaca cgtaacatcg
ctcttaaatt tgtccttact tggtgtaaaa 1500agattaatct acaaagacta
tattgttaaa gcaaaattga ggaagctgta tttaacctcg 1560tgaccaccta
aactagtggg aatgccccat ccctcaatag aactagtttt atttggcaaa
1620catcatggaa agaatatatc agatacacta ctctcgcaca gagagagatg
tcaagtggtt 1680tgggcatcaa aaccataagc ggacgggttt cgagtttgga
cctttgaatc tggttgatgt 1740tggagcaaga agaagcaaaa atgacacatg
gcatcattgt gaagcttgcg tcgagacgaa 1800gcaagacaaa ggcaaagtag
cgaaggtaca ctgttaccgc cggcaataac tacgtacgta 1860catgcatgta
agacatgcat tgtaccgtga catacatgta tgtcatataa atctatgtag
1920atcactccta cgaccggcgg ctaatttcac tacacacatg atgcatggat
ggaatggatg 1980tgaagtgaga ctgtgagagc actatcggaa tcactttctt
cgccgagtgt cgctttccat 2040gaataaacat gtgaacctag gcctaggcac
cttccacttc agggcttgtt cggttagctc 2100tcaatccatg tggattgagc
gggattggat gggtttgaat cccaaacaag tcaaacttct 2160tcacaatttt
ttccaatccc atccaatcca tgtgtattgg gaataaccga acaagccctc
2220aggcatcatc ttctacaaat gtagaatgtg tgaaggtagg caaacgtaac
ggaaaggcag 2280gaagctcatc gccaacgcat ctcctctcct gctccttttg
acgacctttc atacctgcac 2340ccgctttttc tggaaagggc atcaagattt
atatatatat atgttattca ccagtaaatt 2400tcattattat tagctttgtt
tacaacaagt attattatta ttatccgggc tgtgagcaga 2460gggagctggt
acaacttgtc ccttgagacg gccaagaggc aacagtgttg tgggcttgcg
2520gccatgacgc gacgttgcta gctgccgtgt ccaacaggaa gagaagacgg
cgacgtggtc 2580gcactgtacg tttttcccgc cacacaaacg gggcgggggg
cggtggtata ctggtatggt 2640ggccactggc cagccgccgt gccggtgcag
gcagcagccc acaggaccaa cgccgccgcc 2700aatggatcgg acggcctctg
ctactgctag aaatggaaag cacgcaggta cgtggggccc 2760cctccctttc
ccgcgcaagt gcagtgccag tgcggcagtg cgtgtgtcat tattctgtcc
2820ggaccggtag gtagtagtat cagatgtact accagtcaaa cgacagtgcc
ttccgcggcg 2880gccaaaggta cagtgacact ttgccaaaaa caaaaaaaaa
acagcaaata aagaaaggaa 2940cgcgcgcggg aatatatcga tctcatcttt
ttttttcttt tttgttgttg ttgtctacag 3000agatggtaag gaataaataa
ataaaggtgc taaataaaga ccggattctt tatttctttc 3060caaatccaga
aaaggaatta tcttccccgg aatctatttt cgagcaaata ataataataa
3120tatatgattt tgttattttt cattggttct ctggttaatc attttggacg
tcatctacct 3180aataacatag gtcgtccact atgtggagcg cacggcctta
gcttaagaca caatttgttg 3240acttccagga ttatataatc caccttatag
attatataat catataatga tatctagtta 3300tcaagattat ataataatcc
acctaataat ttgtgttgtt tgtttgcctc ttgatatagt 3360aggactatgt
agcctactga catgatcaat ttacttctct aatcaccggc aaaatgaaaa
3420atccattgtt taccttgcct tgagttgtat taacggtttc caccaaagtc
gttcaagact 3480ctaagtagct cacatgtatt ccatccagtc ttcaaatact
ttaaggggtc ttcactttgg 3540atgtaaatac tttatgttta agcatttgaa
gctcatttta gactaggata cccttgatct 3600aggtggtcca catattctcc
taggcatgac aaattactca gagacacgct tcctcttccc 3660actacatgtg
tgcatgcatt gaaaccgtga aaaaacctac gagtgcaatc gatcaggtcg
3720ggaaaaaaag gcacacctag aggctagagc tcagtcagtc ccgaacgatt
gcaaaaaaaa 3780aagacactag agctcgacct caaaacccag tgtgtcaatt
tttacagcca gcgttgcaac 3840catctaagct aacatcttgt atttaaatat
tatagaaaca acaaccctat ttaatagttc 3900cttggtatat atacaagatc
atgtccaaaa catcaagcat ttgaagacta gatgtagtac 3960atggaccaac
ctgcaacctt actttatcag agaaatcgtc agtccacaca tatttgtcat
4020taattaccaa ggcactactg agaggttaga tgcccacacc gtttcaccaa
atgattggtt 4080aaaaagcacg gactcctgtg gagctgctac tctgcagagg
agcgggagtc agaaccattt 4140ttagaagagc caaagtccta ccaaacatac
tcttactctc tctttttaaa ggtactacct 4200cttttgaatt taaatgtatt
actccctctt 423064236DNAZea mays 6ctccacctct ctcgtacgct ttgattggcc
gctcgcacgc atttatcgcc aagatcgggc 60ttgcccatca ctgggatcat cctggctggc
tggctggccg gccacatatc aaacatggat 120ttgctatatt ttaaatgcct
gaaatatgtt atactatgtt ttttagataa cgatctttac 180agtcgattta
tgaatgtgta gaattaagat tgagcttcct gatgaaaaaa tcaggcaaga
240ggctcttttg gttggagacc tggcatggca cttgcctgcg agtcaagcaa
accctatgcc 300actgtttgga ccaggccact ttcctagaga gccgaatcag
acatcactat cggaattcga 360atgagtgctc agcactttac cgagtgcaat
taatcggaca ctcggcaaag cattctttgt 420cgagtgccac tctcggcgaa
ctaataaggc tctcgggaca gatctcgtat gtcgagagcg 480gaacactcga
catagaaaaa cactcggcaa aagaggtttt gccgaatgcc aagctctcgg
540caaaatgcga cactcggtaa gggtcgtcaa gagtcgtcta ttgttgacgg
ccattaactt 600tcccgagtgt caaacgttga cacttgggaa ttatcttctt
tttgtcgagt gtaacctggc 660aaaccctcgg caaaaatata ctttgcggag
tgtcttccct cgacactcga taaagaatat 720ttgtttcttt ttcttttttc
tataccaaac tctttgtgac gttttttctg cagtatatag 780acatacatat
tcaattttac acaattatca aagtgtttgc tataattaat agatttagtt
840tgtttaattg aatttctgag aaccaaccaa atagacccga attagacata
tctagacatt 900taaaaagtaa gatactaaaa aataatagtg tttaccttca
accggtacta aatatcattc 960ctgtagtaga caggataacg aaagctagtc
tatttagatc atcagttcca gttcgagatt 1020ttaaatgcta gtccctccat
ttcaatttac aatttattta atttttttag tgtgataccg 1080tttagcgtat
gtagctttga ttttttttat atatttttgc aaaattttga ataagacaag
1140tgtgtcaaat ttggtgtaaa aattaaacga aattataaat tggagcggag
ggagtagaaa 1200tctacgattt ttctagctga ggcagactgt gcgcattccc
atccatcgag tccacttgca 1260cctctcctcg acatgaatac gaatgtacga
tccgatgaat tccccacaaa gaagcaatta 1320acgtcaaatc catcatcgtc
ataaaacgac gataccgagc tagcgtttaa ttagtttgtt 1380agacgaacca
gaaactatat tatatcgctt tgtgtccaaa aattacttat attatatata
1440gttctcgtgc atctacgtac acgtaacatc gctcttaaat ttgtccttac
ttggtgtaaa 1500aagattaatc tacaaagact atattgttaa agcaaaattg
aggaagctgt atttaacctc 1560gtgaccacct aaactagtgg gaatgcccca
tccctcaata gaactagttt tatttggcaa 1620acatcatgga aagaatatat
cagatacact actctcgcac agagagagat gtcaagtggt 1680ttgggcatca
aaaccataag cggacgggtt tcgagtttgg acctttgaat ctggttgatg
1740ttggagcaag aagaagcaaa aatgacacat ggcatcattg tgaagcttgc
gtcgagacga 1800agcaagacaa aggcaaagta gcgaaggtac actgttaccg
ccggcaataa ctacgtacgt 1860acatgcatgt aagacatgca ttgtaccgtg
acatacatgt atgtcatata aatctatgta 1920gatcactcct acgaccggcg
gctaatttca ctacacacat gatgcatgga tggaatggat 1980gtgaagtgag
actgtgagag cactatcgga atcactttct tcgccgagtg tcgctttcca
2040tgaataaaca tgtgaaccta ggcctaggca ccttccactt cagggcttgt
tcggttagct 2100ctcaatccat gtggattgag cgggattgga tgggtttgaa
tcccaaacaa gtcaaacttc 2160ttcacaattt tttccaatcc catccaatcc
atgtgtattg ggaataaccg aacaagccct 2220caggcatctt ctacaaatgt
agaatgtgtg aaggtaggca aacgtaacgg aaaggcagga 2280agctcatcgc
caacgcatct cctctcctgc tccttttgac gacctttcat acctgcaccc
2340gctttttctg gaaagggcat caagatttat atatatatat atatgttatt
caccagtaaa 2400tttcattatt attagctttg tttacaacaa gtattattat
tattatccgg gctgtgagca 2460gagggagctg gtacaacttg tcccttgaga
cggccaagag gcaacagtgt tgtgggcttg 2520cggccatgac gcgacgttgc
tagctgccgt gtccaacagg aagagaagac ggcgacgtgg 2580tcgcactgta
cgtttttccc gccacacaaa cggggcgggg ggcgggggta tactggtatg
2640gtggccactg gccagccgcc gtgccggtgc aggcagcagc ccacaggacc
aacgccgccg 2700ccaatggatc ggacggcctc tgctactgct agaaatggaa
agcacgcagg tacgtggggc 2760cccctccctt tcccgcgcaa gtgcagtgcc
agtgcggcag tgcgtgtgtc attattctgt 2820ccggaccggt aggtagtagt
atcagatgta ctaccagtca aacgacagtg ccttccgcgg 2880cggccaaagg
tacagtgaca ctttgccaaa aacaaaaaaa aaacagcaaa taaagaaagg
2940aacgcgcgcg ggaatatatc gatctcatct ttttttttct tttttgttgt
tgttgtctac 3000agagatggta aggaataaat aaataaaggt gctaaataaa
gaccggattc tttatttctt 3060tccaaatcca gaaaaggaat tatcttcccc
ggaatctatt ttcgagcaaa taataataat 3120aataatatat gattttgtta
tttttcattg gttctctggt taatcatttt ggacgtcatc 3180tacctaataa
cataggtcgt ccactatgtg gagcgcacgg ccttagctta agacacaatt
3240tgttgacttc caggattata taatccacct tatagattat ataatcatat
aatgatatct 3300agttatcaag attatataat aatccaccta ataatttgtg
ttgtttgttt gcctcttgat 3360atagtaggac tatgtagcct actgacatga
tcaatttact tctctaatca ccggcaaaat 3420gaaaaatcca ttgtttacct
tgccttgagt tgtattaacg gtttccacca aagtcgttca 3480agactctaag
tagctcacat gtattccatc cagtcttcaa atactttaag gggtcttcac
3540tttggatgta aatactttat gtttaagcat ttgaagctca ttttagacta
ggataccctt 3600gatctaggtg gtccacatat tctcctaggc atgacaaatt
actcagagac acgcttcctc 3660ttcccactac atgtgtgcat gcattgaaac
cgtgaaaaaa cctacgagtg caatcgatca 3720ggtcgggaaa aaaaggcaca
cctagaggct agagctcagt cagtcccgaa cgattgcaaa 3780aaaaaaaaga
cactagagct cgacctcaaa acccagtgtg tcaattttta cagccagcgt
3840tgcaaccatc taagctaaca tcttgtattt aaatattata gaaacaacaa
ccctatttaa 3900tagttccttg gtatatatac aagatcatgt ccaaaacatc
aagcatttga agactagatg 3960tagtacatgg accaacctgc aaccttactt
tatcagagaa atcgtcagtc cacacatatt 4020tgtcattaat taccaaggca
ctactgagag gttagatgcc cacaccgttt caccaaatga 4080ttggttaaaa
agcacggact cctgtggagc tgctactctg cagaggagcg ggagtcagaa
4140ccatttttag aagagccaaa gtcctaccaa acatactctt actctctctt
tttaaaggta 4200ctacctcttt tgaatttaaa tgtattactc cctctt
423674313DNAZea mays 7ataaatttat tttgaagata taaatatttt aacggtattt
tttattaata aaatgattta 60tatggattat acagatagag tctctctcta ctctctagtc
ctgtgtacgt gcaggtttgg 120caaggtatga acaggacgtc aaacgcgtac
gtgtactcca cacacactcc acctctctcg 180tacgctttga ttggccgctc
gcacgcattt atcgccaaga tcgggcttgc ccatcactgg 240gatcatcctg
gctggctggc tggccggcca catatcaaac atggatttgc tcatttgcta
300tattttaaat gcctgaaata tgttatacta tgttttttag ataacgatct
ttacagtcga 360tttatgaatg tgtagaatta agattgagct tcctgatgaa
aaaatcaggc aagaggctct 420tttggttgga gacctggcat ggcacttgcc
tgcgagtcaa gcaaacccta tgccactgtt 480tggaccaagc cactttccta
gagagccgaa tcagacatca ctatcggaat tcgaatgagt 540gctcagcact
ttaccgagtg taattaatcg gacactcggc aaagcattct ttgtcgagtg
600tcactctcgg cgaactaata aggctctcgg gacagatctc gtatgtcgag
agcggaacac 660tcgacataga aaaacactcg gcaaaagagg ttttaccgaa
tgccaagctc tcggcaaaat 720gcgacactcg gtaagggtcg tcaagagtcg
tctattgttg acggccatta actttcccga 780gtgtcaaacg ttgacacttg
ggaattatct tctttttgtc gattgtaacc tggcaaaccc 840tcggcaaaag
tatactttac ggagtgtctt ccctcaacac tcgataaaga atatttgttt
900ctttttcttt tttctatacc aaactctttg tgacgttttt tctgcagtat
atagacatac 960atattcaatt ttacacaatt atcaaagtgt ttgctataat
taatagattt agtttgttta 1020attgaatttc tgagaaccaa ccaaatagac
ccgaattaga catatctaga catttaaaaa 1080gtaagatact aaaaaataat
agtgtttacc ttcaaccggt actaaatatc attcctgtag 1140tagacaggat
aacgaaagct agtctattta gatcatcagt tccagttcga gattttaaat
1200gctagtccct ccatttcaat ttacaattta tttaattttt ttagtgtgat
accgtttagc 1260atataatata ctttaagtgt agctttgatt tttttatata
tttttgcaaa attttgaata 1320agacaagtat gtcaaatttg gtgtaaaaat
taaacgaatt tataaattgg agcggaggga 1380gtagaaatct acgatttttc
tagctgaggc agactgtgcg cattcccatc catcgagtcc 1440acttgcacct
ctcctcgaca tgaatacgaa tgtacgatcc gatgaattcc ccacaaagaa
1500gcaattaacg tcaaatccat catcgtcata aaacgacgat accgagctag
cgtttaatta 1560gtttgttaga cgaaccagaa actatattat atcgctttgt
gtccaaaaat tacttatatt 1620atatatagtt ctcgtgcatc tacgtacacg
taacatcgct cttaaatttg tccttacttg 1680gtgtaaaaag attaatctac
aaagactata ttgttaaagc aaaattgagg aagctgtatt 1740taacctcgtg
accacctaaa ctagtgggaa ttccccatcc ctcaatagaa ctagttttat
1800ttggcaaaca tcatggaaag aatatatcag atacactact ctcgcacaga
aagagacgtc 1860aagtggtttg ggcatcaaaa ccataagcgg acgggtttcg
agtttggacc tttgaatctg 1920gttgatgttg gagcaagaag aagcagaaat
gacacatggc atcattgtga agcttgcgtc 1980gagacgaagc aagacaaagg
caaagtagca aaggtacact gttaccgccg gcaataacta 2040cgtacgtacg
tgcatgtaag acatgcattg taccgtgaca tacatgtatg tcatataaat
2100ctatgtagat cactcctacg accggcggct tatcgtcact acaacgcatg
atacatggat 2160ggaatggatg tgaagtgaga ctgtgagagc actatcggaa
tcgcgttctt tgtcaaatgt 2220cgctttccat aaataaacat gtgaacctag
gcctaggcac cttccacttc aggcatcttc 2280tacaaatgta gaatgtgtga
aggtaggcaa acgtaacggg aaaggcagga agctcatcgc 2340caacgcatct
cctctcctgc tccttttgac gacctttcat acctgcaccc gctttttctg
2400gaaagggcat caagatttat atatatatgt tatttttcat tattattagc
tttgtttaca 2460acaagtatta ttattattat ccgggctgtg agcagaggga
gctggtacaa cttgtccctt 2520gagacggcca agaggcaaca gtgttgtggg
cttgcggcca tgacgacgtt gctagctgcc 2580gtgtccaaca ggaagagaag
acggcgacgt ggtcgcactg tacgtttttc ccgccacaca 2640aacggggcgg
ggggcggtgg tatactggta tggtggccac tggccagccg ccgtgccggt
2700gcaggcagca gcccacagga ccaacgccgc cgccaatgga
tcggacggcc tctgctactg 2760ctagaaatgg aaagcacgca ggtacgtggg
gccccctccc tttcccgcgc aagtgcagtg 2820ccagtgcggc agtgcgtgtg
tcattattct gtccggaccg gtaggtagta gtatcagatg 2880tactaccagt
caaacgacag tgccttccgc ggcggccaaa ggtacagtga cactttgcca
2940aaaacaaaaa aaaaacagca aataaagaaa ggaacgcgcg cgggaatata
tcgatctcat 3000cttttttttt ctttgttgtt gttgttgtct acagagatgg
taaggaaata aataaataaa 3060ggtgctacat aaagaccgga ttctttattt
ctttccaaat ccagaaaagg aattatcttc 3120cccggaatct attttcgaca
aataataata ataatatatg attttgttat ttttcattgg 3180ttctctggtt
aatcattttg gacgtcatct acctaataac ataggtcgtc cactatgtgg
3240agcgcacggc cttagcttaa gacacaattt gttgacttcc aggattatat
aatccacctt 3300atagattata taatcatata atgatatcta gttatcaaga
ttatataata atccacctaa 3360taatttgtgt tgtttgtttg cctcttgata
tagtaggact atgtagccta ctgacatgat 3420caatttactt ctctaatcac
cggcaaaatg aaaaatccat tgtttacctt gccttgagtt 3480gtattaacgg
tttccaccaa agtcgttcaa gactctaagt agctcacatg tattccatcc
3540agtcttcaaa tactttaagg ggtcttcact ttggatgtaa atactttatg
tttaagcatt 3600tgaagctcat tttagactag gatacccttg atctaggtgg
tccacatatt ctcctaggca 3660tgacaaatta ctcagagaca cgcttcctct
tcccactaca tgtgtgcatg cattgaaacc 3720gtgaaaaaac ctacgagtgc
aatcgatcag gtcgggaaaa aaaggcacac ctagaggcta 3780gagctcagtc
agtcccgaac gattgcaaaa aaaaaaagac actagagctc gacctcaaaa
3840cccagtgtgt caatttttac agccagcgtt gcaaccatct aagctaacat
cttgtattta 3900aatattatag aaacaacaac cctatttaat agttccttgg
tatatataca agatcatgtc 3960caaaacatca agcatttgaa gactagatgt
agtacatgga ccaacctgca accttacttt 4020atcagagaaa tcgtcagtcc
acacatattt gtcattaatt accaaggcac tactgagagg 4080ttagatgccc
acaccgtttc accaaatgat tggttaaaaa gcacggactc ctgtggagct
4140gctactctgc agaggagcgg gagtcagaac catttttaga agagccaaag
tcctaccaaa 4200catactctta ctctctcttt ttaaaggtac tacctctttt
gaatttaaat gtattactcc 4260ctctttaaag agataataga gaagacgaac
aagaaaggga gagggagaaa cga 431384232DNAZea mays 8ctccacctct
ctcgtacgct ttgattggcc gctcgcacgc atttatcgcc aagatcgggc 60ttgcccatca
ctgggatcat cctggctggc tggctggccg gccacatatc aaacatggat
120ttgctatatt ttaaatgcct gaaatatgtt atactatgtt ttttagataa
cgatctttac 180agtcgattta tgaatgtgta gaattaagat tgagcttcct
gatgaaaaaa tcaggcaaga 240ggctcttttg gttggagacc tggcatggca
cttgcctgcg agtcaagcaa accctatgcc 300actgtttgga ccaggccact
ttcctagaga gccgaatcag acatcactat cggaattcga 360atgagtgctc
agcactttac cgagtgcaat taatcggaca ctcggcaaag cattctttgt
420cgagtgccac tctcggcgaa ctaataaggc tctcgggaca gatctcgtat
gtcgagagcg 480gaacactcga catagaaaaa cactcggcaa aagaggtttt
gccgaatgcc aagctctcgg 540caaaatgcga cactcggtaa gggtcgtcaa
gagtcgtcta ttgttgacgg ccattaactt 600tcccgagtgt caaacgttga
cacttgggaa ttatcttctt tttgtcgatt gtaacctggc 660aaaccctcgg
caaaagtata ctttgcggag tgtcttccct caacactcga taaagaatat
720ttgtttcttt ttcttttttc tataccaaac tctttgtgac gttttttctg
cagtatatag 780acatacatat tcaattttac acaattatca aagtgtttgc
tataattaat agatttagtt 840tgtttaattg aatttctgag aaccaaccaa
atagacccga attagacata tctagacatt 900taaaaagtaa gatactaaaa
aataatagtg tttaccttca accggtacta aatatcattc 960ctgtagtaga
caggataacg aaagctagtc tatttagatc atcagttcca gttcgagatt
1020ttaaatgcta gtccctccat ttcaatttac aatttattta atttttttag
tgtgataccg 1080tttagcgtat gtagctttga tttttttata tatttttgca
aaattttgaa taagacaagt 1140gtgtcaaatt tggtgtaaaa attaaacgaa
attataaatt ggagcggagg gagtagaaat 1200ctacgatttt tctagctgag
gcagactgtg cgcattccca tccatcgagt ccacttgcac 1260ctctcctcga
catgaatacg aatgtacgat ccgatgaatt ccccacaaag aagcaattaa
1320cgtcaaatcc atcatcgtca taaaacgacg ataccgagct agcgtttaat
tagtttgtta 1380gacgaaccag aaactatatt atatcgcttt gtgtccaaaa
attacttata ttatatatag 1440ttctcgtgca tctacgtaca cgtaacatcg
ctcttaaatt tgtccttact tggtgtaaaa 1500agattaatct acaaagacta
tattgttaaa gcaaaattga ggaagctgta tttaacctcg 1560tgaccaccta
aactagtggg aatgccccat ccctcaatag aactagtttt atttggcaaa
1620catcatggaa agaatatatc agatacacta ctctcgcaca gagagagatg
tcaagtggtt 1680tgggcatcaa aaccataagc ggacgggttt cgagtttgga
cctttgaatc tggttgatgt 1740tggagcaaga agaagcaaaa atgacacatg
gcatcattgt gaagcttgcg tcgagacgaa 1800gcaagacaaa ggcaaagtag
cgaaggtaca ctgttaccgc cggcaataac tacgtacgta 1860catgcatgta
agacatgcat tgtaccgtga catacatgta tgtcatataa atctatgtag
1920atcactccta cgaccggcgg ctaatttcac tacacacatg atgcatggat
ggaatggatg 1980tgaagtgaga ctgtgagagc actatcggaa tcactttctt
cgccgagtgt cgctttccat 2040gaataaacat gtgaacctag gcctaggcac
cttccacttc agggcttgtt cggttagctc 2100tcaatccatg tggattgagc
gggattggat gggtttgaat cccaaacaag tcaaacttct 2160tcacaatttt
ttccaatccc atccaatcca tgtgtattgg gaataaccga acaagccctc
2220aggcatcttc tacaaatgta gaatgtgtga aggtaggcaa acgtaacgga
aaggcaggaa 2280gctcatcgcc aacgcatctc ctctcctgct ccttttgacg
acctttcata cctgcacccg 2340ctttttctgg aaagggcatc aagatttata
tatatatata tatgttattc accagtaaat 2400ttcattatta ttagctttgt
ttacaacaag tattattatt attatccggg ctgtgagcag 2460agggagctgg
tacaacttgt cccttgagac ggccaagagg caacagtgtt gtgggcttgc
2520ggccatgacg cgacgttgct agctgccgtg tccaacagga agagaagacg
gcgacgtggt 2580cgcactgtac gtttttcccg ccacacaaac ggggcggggg
gcggtggtat actggtatgg 2640tggccactgg ccagccgccg tgccggtgca
ggcagcagcc cacaggacca acgccgccgc 2700caatggatcg gacggcctct
gctactgcta gaaatggaaa gcacgcaggt acgtggggcc 2760ccctcccttt
cccgcgcaag tgcagtgcca gtgcggcagt gcgtgtgtca ttattctgtc
2820cggaccggta ggtagtagta tcagatgtac taccagtcaa acgacagtgc
cttccgcggc 2880ggccaaaggt acagtgacac tttgccaaaa acaaaaaaaa
aacagcaaat aaagaaagga 2940acgcgcgcgg gaatatatcg atctcatctt
tttttttctt ttttgttgtt gttgtctaca 3000gagatggtaa ggaataaata
aataaaggtg ctaaataaag accggattct ttatttcttt 3060ccaaatccag
aaaaggaatt atcttccccg gaatctattt tcgagcaaat aataataata
3120atatatgatt ttgttatttt tcattggttc tctggttaat cattttggac
gtcatctacc 3180taataacata ggtcgtccac tatgtggagc gcacggcctt
agcttaagac acaatttgtt 3240gacttccagg attatataat ccaccttata
gattatataa tcatataatg atatctagtt 3300atcaagatta tataataatc
cacctaataa tttgtgttgt ttgtttgcct cttgatatag 3360taggactatg
tagcctactg acatgatcaa tttacttctc taatcaccgg caaaatgaaa
3420aatccattgt ttaccttgcc ttgagttgta ttaacggttt ccaccaaagt
cgttcaagac 3480tctaagtagc tcacatgtat tccatccagt cttcaaatac
tttaaggggt cttcactttg 3540gatgtaaata ctttatgttt aagcatttga
agctcatttt agactaggat acccttgatc 3600taggtggtcc acatattctc
ctaggcatga caaattactc agagacacgc ttcctcttcc 3660cactacatgt
gtgcatgcat tgaaaccgtg aaaaaaccta cgagtgcaat cgatcaggtc
3720gggaaaaaaa ggcacaccta gaggctagag ctcagtcagt cccgaacgat
tgcaaaaaaa 3780aaaagacact agagctcgac ctcaaaaccc agtgtgtcaa
tttttacagc cagcgttgca 3840accatctaag ctaacatctt gtatttaaat
attatagaaa caacaaccct atttaatagt 3900tccttggtat atatacaaga
tcatgtccaa aacatcaagc atttgaagac tagatgtagt 3960acatggacca
acctgcaacc ttactttatc agagaaatcg tcagtccaca catatttgtc
4020attaattacc aaggcactac tgagaggtta gatgcccaca ccgtttcacc
aaatgattgg 4080ttaaaaagca cggactcctg tggagctgct actctgcaga
ggagcgggag tcagaaccat 4140ttttagaaga gccaaagtcc taccaaacat
actcttactc tctcttttta aaggtactac 4200ctcttttgaa tttaaatgta
ttactccctc tt 4232949DNAartificial sequenceprimer 9ggggacaagt
ttgtacaaaa aagcaggcta tgcagttgga tctgaacgt 491047DNAartificial
sequenceprimer 10ggggaccact ttgtacaaga aagctgggtt cagcggggat
ggtgatg 471124DNAartificial sequenceprimer 11ggatccgatc tgaacgtggc
cgag 241231DNAartificial sequenceprimer 12gaattcctag gcagctgttc
ttgtctcttt g 311326DNAartificial sequenceprimer 13gcggccgcga
tctgaacgtg gccgag 261430DNAartificial sequenceprimer 14gaattctgtg
ggactcccag cggcctgtgc 30
* * * * *
References