U.S. patent application number 11/649663 was filed with the patent office on 2007-08-16 for nucleotide sequences and polypeptides encoded thereby useful for modifying plant characteristics.
This patent application is currently assigned to CERES, INC.. Invention is credited to Nickolai Alexandrov, Vyacheslav Brover.
Application Number | 20070192907 11/649663 |
Document ID | / |
Family ID | 36613359 |
Filed Date | 2007-08-16 |
United States Patent
Application |
20070192907 |
Kind Code |
A1 |
Alexandrov; Nickolai ; et
al. |
August 16, 2007 |
Nucleotide sequences and polypeptides encoded thereby useful for
modifying plant characteristics
Abstract
Isolated polynucleotides and polypeptides encoded thereby are
described, together with the use of those products for making
transgenic plants.
Inventors: |
Alexandrov; Nickolai;
(Thousand Oaks, CA) ; Brover; Vyacheslav; (Simi
Valley, CA) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
CERES, INC.
Thousand Oaks
CA
|
Family ID: |
36613359 |
Appl. No.: |
11/649663 |
Filed: |
January 3, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11174307 |
Jun 30, 2005 |
|
|
|
11649663 |
Jan 3, 2007 |
|
|
|
60583671 |
Jun 30, 2004 |
|
|
|
60583781 |
Jun 30, 2004 |
|
|
|
60583651 |
Jun 30, 2004 |
|
|
|
Current U.S.
Class: |
800/288 ;
435/419; 435/468; 435/6.15; 536/23.6 |
Current CPC
Class: |
C07K 14/415
20130101 |
Class at
Publication: |
800/288 ;
435/006; 435/419; 435/468; 536/023.6 |
International
Class: |
A01H 1/00 20060101
A01H001/00; C12Q 1/68 20060101 C12Q001/68; C07H 21/04 20060101
C07H021/04; C12N 15/82 20060101 C12N015/82; C12N 5/04 20060101
C12N005/04 |
Claims
1. An isolated nucleic acid molecule comprising: a) a nucleic acid
having a nucleotide sequence which encodes an amino acid sequence
exhibiting at least 85% sequence identity to an amino acid sequence
in Sequence Listing; b) a nucleic acid which is a complement of a
nucleotide sequence according to paragraph (a); c) a nucleic acid
which is the reverse of the nucleotide sequence according to
subparagraph (a), such that the reverse nucleotide sequence has a
sequence order which is the reverse of the sequence order of the
nucleotide sequence according to subparagraph (a); or d) a nucleic
acid capable of hybridizing to a nucleic acid according to any one
of paragraphs (a)-(c), under conditions that permit formation of a
nucleic acid duplex at a temperature from about 40.degree. C. and
48.degree. C. below the melting temperature of the nucleic acid
duplex.
2. The isolated nucleic acid molecule according to claim 1, which
has the nucleotide sequence according to any polynucleotide
sequence in the Sequence Listing.
3. The isolated nucleic acid molecule according to claim 1, wherein
said amino acid sequence comprises any polypeptide sequence in the
Sequence Listing.
4. A vector construct comprising: a) a first nucleic acid having a
regulatory sequence capable of causing transcription and/or
translation in a plant; and b) a second nucleic acid having the
sequence of the isolated nucleic acid molecule according to claim
1; wherein said first and second nucleic acids are operably linked
and wherein said second nucleic acid is heterologous to any element
in said vector construct.
5. The vector construct according to claim 4, wherein said first
nucleic acid is native to said second nucleic acid.
6. The vector construct according to claim 4, wherein said first
nucleic acid is heterologous to said second nucleic acid.
7. A host cell comprising an isolated nucleic acid molecule
according to claim 1 wherein said nucleic acid molecule is flanked
by exogenous sequence.
8. A host cell comprising a vector construct according to claim
4.
9. An isolated polypeptide comprising an amino acid sequence
exhibiting at least 85% sequence identity of an amino acid sequence
of the Sequence Listing.
10. A method of introducing an isolated nucleic acid into a host
cell comprising: a) providing an isolated nucleic acid molecule
according to claim 1; and b) contacting said isolated nucleic with
said host cell under conditions that permit insertion of said
nucleic acid into said host cell.
11. A method of transforming a host cell which comprises contacting
a host cell with a vector construct according to claim 4.
12. A method for detecting a nucleic acid in a sample which
comprises: a) providing an isolated nucleic acid molecule according
to claim 1; b) contacting said isolated nucleic acid molecule with
a sample under conditions which permit a comparison of the sequence
of said isolated nucleic acid molecule with the sequence of DNA in
said sample; and c) analyzing the result of said comparison.
13. A host cell or organism which comprises a nucleic acid molecule
according to claim 1 which is exogenous or heterologous to said
plant or plant cell.
14. A host cell or organism which comprises a vector construct
according to claim 4.
15. A host cell or organism according to claim 13, which is a
plant, plant cell, plant material or seed of a plant.
16. A host cell or organism according to claim 14, which is a
plant, plant cell, plant material or seed of a plant.
17. A plant which has been regenerated from a plant cell or seed
according to claims 15.
18. A plant which has been regenerated from a plant cell or seed
according to claims 16.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a Continuation of co-pending application
Ser. No. 11/174,307 filed on Jun. 30, 2005, and for which priority
is claimed under 35 U.S.C. .sctn. 120; and this application claims
priority under 35 U.S.C. .sctn. 119 on U.S. Provisional Application
No(s). 60/583,671; 60/583,781 and 60/583,651 filed on Jun. 30,
2004; the entire contents of which are hereby incorporated by
reference.
[0002] This application contains two (2) CDRs (Copy 1 and Copy 2)
in place of the paper copy of the Sequence Listing, the entire
contents of which are hereby incorporated by reference. The CDRs
contain the following File:
[0003] File Name: 2005-11-21.sub.--2750-1601PUS2-ST25.txt
[0004] Date of Creation: Jan. 3, 2007
[0005] File Size: 30,409 KB
FIELD OF THE INVENTION
[0006] The present invention relates to isolated polynucleotides,
polypeptides encoded thereby, and the use of those products for
making transgenic plants or organisms, such as transgenic
plants.
BACKGROUND OF THE INVENTION
[0007] There are more than 300,000 species of plants. They show a
wide diversity of forms, ranging from delicate liverworts, adapted
for life in a damp habitat, to cacti, capable of surviving in the
desert. The plant kingdom includes herbaceous plants, such as corn,
whose life cycle is measured in months, to the giant redwood tree,
which can live for thousands of years. This diversity reflects the
adaptations of plants to survive in a wide range of habitats. This
is seen most clearly in the flowering plants (phylum
Angiospermophyta), which are the most numerous, with over 250,000
species. They are also the most widespread, being found from the
tropics to the arctic.
[0008] The process of plant breeding involving man's intervention
in natural breeding and selection is some 20,000 years old. It has
produced remarkable advances in adapting existing species to serve
new purposes. The world's economics was largely based on the
successes of agriculture for most of these 20,000 years.
[0009] Plant breeding involves choosing parents, making crosses to
allow recombination of gene (alleles) and searching for and
selecting improved forms. Success depends on the genes/alleles
available, the combinations required and the ability to create and
find the correct combinations necessary to give the desired
properties to the plant. Molecular genetics technologies are now
capable of providing new genes, new alleles and the means of
creating and selecting plants with the new, desired
characteristics.
[0010] Plants specifically improved for agriculture, horticulture
and other industries can be obtained using molecular technologies.
As an example, great agronomic value can result from modulating the
size of a plant as a whole or of any of its organs. The green
revolution came about as a result of creating dwarf wheat plants,
which produced a higher seed yield than taller plants because they
could withstand higher levels and inputs of fertilizer and
water.
[0011] Similarly, modulation of the size and stature of an entire
plant, or a particular portion of a plant, allows production of
plants better suited for a particular industry. For example,
reductions in the height of specific ornamentals, crops and tree
species can be beneficial by allowing easier harvesting.
Alternatively, increasing height may be beneficial by providing
more biomass. Other examples of commercially desirable traits
include increasing the length of the floral stems of cut flowers,
increasing or altering leaf size and shape, enhancing the size of
seeds and/or fruits, enhancing yields by specifically stimulating
hormone (e.g. Brassinolide) synthesis and stimulating early
flowering or evoking late flowering by altering levels of
gibberellic acid or other hormones in specific cells. Changes in
organ size and biomass also result in changes in the mass of
constituent molecules such as secondary products. To summarize,
molecular genetic technologies provide the ability to modulate and
manipulate growth, development and biochemistry of the entire plant
as well as at the cell, tissue and organ levels. Thus, plant
morphology, development and biochemistry are altered to maximize or
minimize the desired plant trait.
SUMMARY OF THE INVENTION
[0012] The present invention, therefore, relates to isolated
polynucleotides, polypeptides encoded thereby, and the use of those
products for making transgenic organisms, such as plants, bacteria,
yeast, fungi and mammals, depending upon the desired
characteristics.
[0013] In the field of agriculture and forestry efforts are
constantly being made to produce plants with improved
characteristics, such as increased overall yield or increased yield
of biomass or chemical components, in particular in order to
guarantee the supply of the constantly increasing world population
with food and to guarantee the supply of reproducible raw
materials. Conventionally, people try to obtain plants with an
increased yield by breeding, but this is time-consuming and
labor-intensive. Furthermore, appropriate breeding programs must be
performed for each relevant plant species.
[0014] Recently, progress has been made by the genetic manipulation
of plants. That is, by introducing into and expressing recombinant
nucleic acid molecules in plants. Such approaches have the
advantage of not usually being limited to one plant species, but
being transferable to other plant species as well. EP-A 0 511 979,
for example, discloses that the expression of a prokaryotic
asparagine synthetase in plant cells inter alia leads to an
increase in biomass production. Similarly, WO 96/21737 describes
the production of plants with increased yield from the expression
of deregulated or unregulated fructose-1,6-bisphosphatase due to an
increased rate of the photosynthesis. Nevertheless, there still is
a need for generally applicable processes that improve yield in
plants interesting for agriculture or forestry purposes.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0015] The following terms are utilized throughout this
application:
[0016] Domain: Domains are fingerprints or signatures that can be
used to characterize protein families and/or parts of proteins.
Such fingerprints or signatures can comprise conserved (1) primary
sequence, (2) secondary structure, and/or (3) three-dimensional
conformation. Generally, each domain has been associated with
either a family of proteins or motifs. Typically, these families
and/or motifs have been correlated with specific in-vitro and/or
in-vivo activities. A domain can be any length, including the
entirety of the sequence of a protein. Detailed descriptions of the
domains, associated families and motifs, and correlated activities
of the polypeptides of the instant invention are described below.
Usually, the polypeptides with designated domain(s) can exhibit at
least one activity that is exhibited by any polypeptide that
comprises the same domain(s). Domains also define areas of
non-coding sequences such as promoters and miRNAs.
Endogenous: The term "endogenous," within the context of the
current invention refers to any polynucleotide, polypeptide or
protein sequence which is a natural part of a cell or organisms
regenerated from said cell.
[0017] Exogenous: "Exogenous," as referred to within, is any
polynucleotide, polypeptide or protein sequence, whether chimeric
or not, that is initially or subsequently introduced into the
genome of an individual host cell or the organism regenerated from
said host cell by any means other than by a sexual cross. Examples
of means by which this can be accomplished are described below, and
include Agrobacterium-mediated transformation (of dicots--e.g.
Salomon et al. EMBO J. 3:141 (1984); Herrera-Estrella et al. EMBO
J. 2:987 (1983); of monocots, representative papers are those by
Escudero et al., Plant J. 10:355 (1996), Ishida et al., Nature
Biotechnology 14:745 (1996), May et al., Bio/Technology 13:486
(1995)), biolistic methods (Armaleo et al., Current Genetics 17:97
1990)), electroporation, in planta techniques, and the like. Such a
plant containing the exogenous nucleic acid is referred to here as
a T.sub.0 for the primary transgenic plant and T.sub.1 for the
first generation. The term "exogenous" as used herein is also
intended to encompass inserting a naturally found element into a
non-naturally found location.
[0018] Gene: The term "gene," as used in the context of the current
invention, encompasses all regulatory and coding sequence
contiguously associated with a single hereditary unit with a
genetic function. Genes can include non-coding sequences that
modulate the genetic function that include, but are not limited to,
those that specify polyadenylation, transcriptional regulation, DNA
conformation, chromatin conformation, extent and position of base
methylation and binding sites of proteins that control all of
these. Genes comprised of "exons" (coding sequences), which may be
interrupted by "introns" (non-coding sequences), encode proteins. A
gene's genetic function may require only RNA expression or protein
production, or may only require binding of proteins and/or nucleic
acids without associated expression. In certain cases, genes
adjacent to one another may share sequence in such a way that one
gene will overlap the other. A gene can be found within the genome
of an organism, artificial chromosome, plasmid, vector, etc., or as
a separate isolated entity.
[0019] Heterologous sequences: "Heterologous sequences" are those
that are not operatively linked or are not contiguous to each other
in nature. For example, a promoter from corn is considered
heterologous to an Arabidopsis coding region sequence. Also, a
promoter from a gene encoding a growth factor from corn is
considered heterologous to a sequence encoding the corn receptor
for the growth factor. Regulatory element sequences, such as UTRs
or 3' end termination sequences that do not originate in nature
from the same gene as the coding sequence originates from, are
considered heterologous to said coding sequence. Elements
operatively linked in nature and contiguous to each other are not
heterologous to each other. On the other hand, these same elements
remain operatively linked but become heterologous if other filler
sequence is placed between them. Thus, the promoter and coding
sequences of a corn gene expressing an amino acid transporter are
not heterologous to each other, but the promoter and coding
sequence of a corn gene operatively linked in a novel manner are
heterologous.
[0020] Homologous gene: In the current invention, "homologous gene"
refers to a gene that shares sequence similarity with the gene of
interest. This similarity may be in only a fragment of the sequence
and often represents a functional domain such as, examples
including without limitation a DNA binding domain, a domain with
tyrosine kinase activity, or the like. The functional activities of
homologous genes are not necessarily the same.
[0021] Misexpression: The term "misexpression" refers to an
increase or a decrease in the transcription of a coding region into
a complementary RNA sequence as compared to the parental wild-type.
This term also encompasses expression of a gene or coding region
for a different time period as compared to the wild-type and/or
from a non-natural location within the plant genome.
[0022] Percentage of sequence identity: "Percentage of sequence
identity," as used herein, is determined by comparing two optimally
aligned sequences over a comparison window, where the fragment of
the polynucleotide or amino acid sequence in the comparison window
may comprise additions or deletions (e.g., gaps or overhangs) 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. Optimal alignment
of sequences for comparison may be conducted by the local homology
algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by
the homology alignment algorithm of Needleman and Wunsch J. Mol.
Biol. 48:443 (1970), by the search for similarity method of Pearson
and Lipman Proc. Natl. Acad. Sci. (USA) 85:2444 (1988), by
computerized implementations of these algorithms (GAP, BESTFIT,
BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group (GCG), 575 Science Dr., Madison,
Wis.), or by inspection. Given that two sequences have been
identified for comparison, GAP and BESTFIT are preferably employed
to determine their optimal alignment. Typically, the default values
of 5.00 for gap weight and 0.30 for gap weight length are used. The
term "substantial sequence identity" between polynucleotide or
polypeptide sequences refers to polynucleotide or polypeptide
comprising a sequence that has at least 80% sequence identity,
preferably at least 85%, more preferably at least 90% and most
preferably at least 95%, even more preferably, at least 96%, 97%,
98% or 99% sequence identity compared to a reference sequence using
the programs.
[0023] Regulatory Sequence: The term "regulatory sequence," as used
in the current invention, refers to any nucleotide sequence that
influences transcription or translation initiation and rate, and
stability and/or mobility of the transcript or polypeptide product.
Regulatory sequences include, but are not limited to, promoters,
promoter control elements, protein binding sequences, 5' and 3'
UTRs, transcriptional start site, termination sequence,
polyadenylation sequence, introns, certain sequences within a
coding sequence, etc.
[0024] Stringency: "Stringency" as used herein is a function of
probe length, probe composition (G+C content), and salt
concentration, organic solvent concentration, and temperature of
hybridization or wash conditions. Stringency is typically compared
by the parameter T.sub.m, which is the temperature at which 50% of
the complementary molecules in the hybridization are hybridized, in
terms of a temperature differential from T.sub.m. High stringency
conditions are those providing a condition of T.sub.m-5.degree. C.
to T.sub.m-10.degree. C. Medium or moderate stringency conditions
are those providing T.sub.m-20.degree. C. to T.sub.m-29.degree. C.
Low stringency conditions are those providing a condition of
T.sub.m-40.degree. C. to T.sub.m-48.degree. C. The relationship of
hybridization conditions to T.sub.m(in 0.degree. C.) is expressed
in the mathematical equation
T.sub.m=81.5-16.6(log.sub.10[Na.sup.+])+0.41(% G+C)-(600/N) (1)
where N is the length of the probe. This equation works well for
probes 14 to 70 nucleotides in length that are identical to the
target sequence. The equation below for T.sub.m of DNA-DNA hybrids
is useful for probes in the range of 50 to greater than 500
nucleotides, and for conditions that include an organic solvent
(formamide). T.sub.m=81.5+16.6 log
{[Na.sup.+]/(1+0.7[Na.sup.+])}+0.41(% G+C)-500/L 0.63(% formamide)
(2) where L is the length of the probe in the hybrid. (P. Tijessen,
"Hybridization with Nucleic Acid Probes" in Laboratory Techniques
in Biochemistry and Molecular Biology, P. C. vand der Vliet, ed.,
c. 1993 by Elsevier, Amsterdam.) The T.sub.m of equation (2) is
affected by the nature of the hybrid; for DNA-RNA hybrids T.sub.m
is 10-15.degree. C. higher than calculated, for RNA-RNA hybrids
T.sub.m is 20-25.degree. C. higher. Because the T.sub.m decreases
about 1.degree. C. for each 1% decrease in homology when a long
probe is used (Bonner et al., J. Mol. Biol. 81:123 (1973)),
stringency conditions in polynucleotide hybridization reactions can
be adjusted to favor hybridization of polynucleotides from
identical genes or related family members.
[0025] Equation (2) is derived assuming equilibrium and therefore,
hybridizations according to the present invention are most
preferably performed under conditions of probe excess and for
sufficient time to achieve equilibrium. The time required to reach
equilibrium can be shortened by inclusion of a hybridization
accelerator such as dextran sulfate or another high volume polymer
in the hybridization buffer.
[0026] Stringency conditions can be selected during the
hybridization reaction or after hybridization has occurred by
altering the salt and temperature conditions of the wash solutions
used. The formulas shown above are equally valid when used to
compute the stringency of a wash solution. Preferred wash solution
stringencies lie within the ranges stated above; high stringency is
5-8.degree. C. below T.sub.m, medium or moderate stringency is
26-29.degree. C. below T.sub.m and low stringency is 45-48.degree.
C. below T.sub.m.
[0027] Substantially free of: A composition containing A is
"substantially free of" B when at least 85% by weight of the total
A+B in the composition is A. Preferably, A comprises at least about
90% by weight of the total of A+B in the composition, more
preferably at least about 95% or even 99% by weight. For example, a
plant gene or DNA sequence can be considered substantially free of
other plant genes or DNA sequences.
Translational start site: In the context of the current invention,
a "translational start site" is usually an ATG in the cDNA
transcript, more usually the first ATG. A single cDNA, however, may
have multiple translational start sites.
[0028] Transcription start site: "Transcription start site" is used
in the current invention to describe the point at which
transcription is initiated. This point is typically located about
25 nucleotides downstream from a TFIID binding site, such as a TATA
box. Transcription can initiate at one or more sites within the
gene, and a single gene may have multiple transcriptional start
sites, some of which may be specific for transcription in a
particular cell-type or tissue.
[0029] Untranslated region (UTR): A "UTR" is any contiguous series
of nucleotide bases that is transcribed, but is not translated.
These untranslated regions may be associated with particular
functions such as increasing mRNA message stability. Examples of
UTRs include, but are not limited to polyadenylation signals,
terminations sequences, sequences located between the
transcriptional start site and the first exon (5' UTR) and
sequences located between the last exon and the end of the mRNA (3'
UTR).
[0030] Variant: The term "variant" is used herein to denote a
polypeptide or protein or polynucleotide molecule that differs from
others of its kind in some way. For example, polypeptide and
protein variants can consist of changes in amino acid sequence
and/or charge and/or post-translational modifications (such as
glycosylation, etc).
2. Important Characteristics of the Polynucleotides of the
Invention
[0031] The genes and polynucleotides of the present invention are
of interest because when they are misexpressed (i.e. when expressed
at a non-natural location or in an increased amount) they produce
plants with important modified characteristics as discussed below.
These traits can be used to exploit or maximize plant products or
to minimize undesirable characteristics. For example, an increase
in plant height is beneficial in species grown or harvested for
their main stem or trunk, such as ornamental cut flowers, fiber
crops (e.g. flax, kenaf, hesperaloe, hemp) and wood producing
trees. Increase in inflorescence thickness is also desirable for
some ornamentals, while increases in the number, shape and size of
leaves can lead to increased production/harvest from leaf crops
such as lettuce, spinach, cabbage and tobacco. Likewise, a decrease
in plant height is beneficial in species that are particularly
susceptible to lodging or uprooting due to wind stress.
[0032] The polynucleotides and polypeptides of the invention were
isolated from Arabidopsis thaliana, corn, soybean, wheat, Brassica
and others as noted in the Sequence Listing. The polynucleotides
and polypeptides are useful to confer on transgenic plants the
properties identified for each sequence in the relevant portion
(miscellaneous feature section) of the Sequence Listing. The
miscellaneous feature section of the sequence listing contains, for
each sequence, a description of the domain or other characteristic
from which the sequence has the function known in the art for other
sequences. Some identified domains are indicated with "PFam Name",
signifying that the pfam name and description can be found in the
pfam database at http://pfam.wustl.edu. Other domains are indicated
by reference to a "GI Number" from the public sequence database
maintained by GenBank under the NCBI, including the non-redundant
(NR) database.
[0033] The sequences of the invention can be applied to substrates
for use in array applications such as, but not limited to, assays
of global gene expression, under varying conditions of development,
and growth conditions. The arrays are also used in diagnostic or
forensic methods
[0034] The polynucleotides, or fragments thereof, can also be used
as probes and primers. Probe length varies depending on the
application. For use as primers, probes are 12-40 nucleotides,
preferably 18-30 nucleotides long. For use in mapping, probes are
preferably 50 to 500 nucleotides, preferably 100-250 nucleotides
long. For Southern hybridizations, probes as long as several
kilobases are used.
[0035] The probes and/or primers are produced by synthetic
procedures such as the triester method of Matteucci et al. J. Am.
Chem. Soc. 103:3185(1981) or according to Urdea et al. Proc. Natl.
Acad. 80:7461 (1981) or using commercially available automated
oligonucleotide synthesizers.
[0036] The polynucleotides of the invention can be utilized in a
number of methods known to those skilled in the art as probes
and/or primers to isolate and detect polynucleotides including,
without limitation: Southerns, Northerns, Branched DNA
hybridization assays, polymerase chain reaction microarray assays
and variations thereof. Specific methods given by way of examples,
and discussed below include:
[0037] Hybridization
[0038] Methods of Mapping
[0039] Southern Blotting
[0040] Isolating cDNA from Related Organisms
[0041] Isolating and/or Identifying Homologous and Orthologous
Genes.
Also, the nucleic acid molecules of the invention can be used in
other methods, such as high density oligonucleotide hybridizing
assays, described, for example, in U.S. Pat. Nos. 6,004,753 and
5,945,306.
[0042] The polynucleotides or fragments thereof of the present
invention can be used as probes and/or primers for detection and/or
isolation of related polynucleotide sequences through
hybridization. Hybridization of one nucleic acid to another
constitutes a physical property that defines the polynucleotide of
the invention and the identified related sequences. Also, such
hybridization imposes structural limitations on the pair. A good
general discussion of the factors for determining hybridization
conditions is provided by Sambrook et al. ("Molecular Cloning, a
Laboratory Manual, 2nd ed., c. 1989 by Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; see esp., chapters 11
and 12). Additional considerations and details of the physical
chemistry of hybridization are provided by G. H. Keller and M. M.
Manak "DNA Probes", 2.sup.nd Ed. pp. 1-25, c. 1993 by Stockton
Press, New York, N.Y.
[0043] When using the polynucleotides to identify orthologous genes
in other species, the practitioner will preferably adjust the
amount of target DNA of each species so that, as nearly as is
practical, the same number of genome equivalents are present for
each species examined. This prevents faint signals from species
having large genomes, and thus small numbers of genome equivalents
per mass of DNA, from erroneously being interpreted as absence of
the corresponding gene in the genome.
[0044] The probes and/or primers of the instant invention can also
be used to detect or isolate nucleotides that are "identical" to
the probes or primers. Two nucleic acid sequences or polypeptides
are said to be "identical" if the sequence of nucleotides or amino
acid residues, respectively, in the two sequences is the same when
aligned for maximum correspondence as described below.
[0045] Isolated polynucleotides within the scope of the invention
also include allelic variants of the specific sequences presented
in the Sequence Listing. The probes and/or primers of the invention
are also used to detect and/or isolate polynucleotides exhibiting
at least 80% sequence identity with the sequences of the Sequence
Listing or fragments thereof. Related polynucleotide sequences can
also be identified according to the methods described in U.S.
Patent Publication 20040137466A1, dated Jul. 15, 2004 to Jofuku et
al.
[0046] With respect to nucleotide sequences, degeneracy of the
genetic code provides the possibility to substitute at least one
nucleotide of the nucleotide sequence of a gene with a different
nucleotide without changing the amino acid sequence of the
polypeptide. Hence, the DNA of the present invention also has any
base sequence that has been changed from a sequence in the Sequence
Listing by substitution in accordance with degeneracy of genetic
code. References describing codon usage include: Carels et al., J.
Mol. Evol. 46: 45 (1998) and Fennoy et al., Nucl. Acids Res.
21(23): 5294 (1993).
[0047] The polynucleotides of the invention are also used to create
various types of genetic and physical maps of the genome of corn,
Arabidopsis, soybean, rice, wheat, or other plants. Some are
absolutely associated with particular phenotypic traits, allowing
construction of gross genetic maps. Creation of such maps is based
on differences or variants, generally referred to as polymorphisms,
between different parents used in crosses. Common methods of
detecting polymorphisms that can be used are restriction fragment
length polymorphisms(RFLPs, single nucleotide polymorphisms(SNPs)
or simple sequence repeats (SSRs).
[0048] The use of RFLPs and of recombinant inbred lines for such
genetic mapping is described for Arabidopsis by Alonso-Blanco et
al. (Methods in Molecular Biology, vol. 82, "Arabidopsis
Protocols", pp. 137-146, J. M. Martinez-Zapater and J. Salinas,
eds., c. 1998 by Humana Press, Totowa, N.J.) and for corn by Burr
("Mapping Genes with Recombinant Inbreds", pp. 249-254. In
Freeling, M. and V. Walbot (Ed.), The Maize Handbook, c. 1994 by
Springer-Verlag New York, Inc.: New York, N.Y., USA; Berlin
Germany; Burr et al. Genetics (1998) 118: 519; Gardiner, J. et al.,
(1993) Genetics 134: 917). This procedure, however, is not limited
to plants and is used for other organisms (such as yeast) or for
individual cells.
[0049] The polynucleotides of the present invention are also used
for simple sequence repeat (SSR) mapping. Rice SSR mapping is
described by Morgante et al. (The Plant Journal (1993) 3: 165),
Panaud et al. (Genome (1995) 38: 1170); Senior et al. (Crop Science
(1996) 36: 1676), Taramino et al. (Genome (1996) 39: 277) and Ahn
et al. (Molecular and General Genetics (1993) 241: 483-90). SSR
mapping is achieved using various methods. In one instance,
polymorphisms are identified when sequence specific probes
contained within a polynucleotide flanking an SSR are made and used
in polymerase chain reaction (PCR) assays with template DNA from
two or more individuals of interest. Here, a change in the number
of tandem repeats between the SSR-flanking sequences produces
differently sized fragments (U.S. Pat. No. 5,766,847).
Alternatively, polymorphisms are identified by using the PCR
fragment produced from the SSR-flanking sequence specific primer
reaction as a probe against Southern blots representing different
individuals (U. H. Refseth et al., (1997) Electrophoresis 18:
1519).
[0050] The polynucleotides of the invention can further be used to
identify certain genes or genetic traits using, for example, known
AFLP technologies, such as in EP0534858 and U.S. Pat. No.
5,878,215.
[0051] The polynucleotides of the present invention are also used
for single nucleotide polymorphism (SNP) mapping.
[0052] Genetic and physical maps of crop species have many uses.
For example, these maps are used to devise positional cloning
strategies for isolating novel genes from the mapped crop species.
In addition, because the genomes of closely related species are
largely syntenic (i.e. they display the same ordering of genes
within the genome), these maps are used to isolate novel alleles
from relatives of crop species by positional cloning
strategies.
[0053] The various types of maps discussed above are used with the
polynucleotides of the invention to identify Quantitative Trait
Loci (QTLs). Many important crop traits, such as the solids content
of tomatoes, are quantitative traits and result from the combined
interactions of several genes. These genes reside at different loci
in the genome, often times on different chromosomes, and generally
exhibit multiple alleles at each locus. The polynucleotides of the
invention are used to identify QTLs and isolate specific alleles as
described by de Vicente and Tanksley (Genetics 134:585(1993)). Once
a desired allele combination is identified, crop improvement is
accomplished either through biotechnological means or by directed
conventional breeding programs (for review see Tanksley and
McCouch, Science 277:1063 (1997)). In addition to isolating QTL
alleles in present crop species, the polynucleotides of the
invention are also used to isolate alleles from the corresponding
QTL of wild relatives.
[0054] In another embodiment, the polynucleotides are used to help
create physical maps of the genome of corn, Arabidopsis and related
species. Where polynucleotides are ordered on a genetic map, as
described above, they are used as probes to discover which clones
in large libraries of plant DNA fragments in YACs, BACs, etc.
contain the same polynucleotide or similar sequences, thereby
facilitating the assignment of the large DNA fragments to
chromosomal positions. Subsequently, the large BACs, YACs, etc. are
ordered unambiguously by more detailed studies of their sequence
composition (e.g. Marra et al. (1997) Genomic Research 7:1072-1084)
and by using their end or other sequences to find the identical
sequences in other cloned DNA fragments. The overlapping of DNA
sequences in this way allows building large contigs of plant
sequences to be built that, when sufficiently extended, provide a
complete physical map of a chromosome. Sometimes the
polynucleotides themselves provide the means of joining cloned
sequences into a contig. All scientific and patent publications
cited in this paragraph are hereby incorporated by reference.
[0055] U.S. Pat. Nos. 6,287,778 and 6,500,614, both hereby
incorporated by reference, describe scanning multiple alleles of a
plurality of loci using hybridization to arrays of
oligonucleotides. These techniques are useful for each of the types
of mapping discussed above.
[0056] Following the procedures described above and using a
plurality of the polynucleotides of the present invention, any
individual is genotyped. These individual genotypes are used for
the identification of particular cultivars, varieties, lines,
ecotypes and genetically modified plants or can serve as tools for
subsequent genetic studies involving multiple phenotypic
traits.
[0057] Identification and isolation of orthologous genes from
closely related species and alleles within a species is
particularly desirable because of their potential for crop
improvement. Many important crop traits, result from the combined
interactions of the products of several genes residing at different
loci in the genome. Generally, alleles at each of these loci make
quantitative differences to the trait. Once a more favorable allele
combination is identified, crop improvement is accomplished either
through biotechnological means or by directed conventional breeding
programs (Tanksley et al. Science 277:1063(1997)).
4. Use of the Genes to Make Transgenic Plants
[0058] To use the sequences of the present invention or a
combination of them or parts and/or mutants and/or fusions and/or
variants of them, recombinant DNA constructs are prepared which
comprise the polynucleotide sequences of the invention inserted
into a vector, and which are suitable for transformation of plant
cells. The construct is made using standard recombinant DNA
techniques (Sambrook et al. 1989) and is introduced to the species
of interest by Agrobacterium-mediated transformation or by other
means of transformation as referenced below.
[0059] The vector backbone is any of those typical in the art such
as plasmids (such as Ti plasmids), viruses, artificial chromosomes,
BACs, YACs and PACs and vectors of the sort described by
(a) BAC: Shizuya et al., Proc. Natl. Acad. Sci. USA 89: 8794-8797
(1992); Hamilton et al., Proc. Natl. Acad. Sci. USA 93: 9975-9979
(1996);
(b) YAC: Burke et al., Science 236:806-812 (1987);
(c) PAC: Sternberg N. et al., Proc Natl Acad Sci USA. January
;87(1):103-7 (1990);
(d) Bacteria-Yeast Shuttle Vectors: Bradshaw et al., Nucl Acids Res
23: 4850-4856 (1995);
[0060] (e) Lambda Phage Vectors: Replacement Vector, e.g.,
Frischauf et al., J. Mol Biol 170: 827-842 (1983); or Insertion
vector, e.g., Huynh et al., In: Glover NM (ed) DNA Cloning: A
practical Approach, Vol. 1 Oxford: IRL Press (1985); T-DNA gene
fusion vectors: Walden et al., Mol Cell Biol 1: 175-194 (1990);
and
(g) Plasmid vectors: Sambrook et al., infra.
[0061] Typically, the construct comprises a vector containing a
sequence of the present invention with any desired transcriptional
and/or translational regulatory sequences, such as promoters, UTRs,
and 3' end termination sequences. Vectors can also include origins
of replication, scaffold attachment regions (SARs), markers,
homologous sequences, introns, etc. The vector may also comprise a
marker gene that confers a selectable phenotype on plant cells. The
marker may encode biocide resistance, particularly antibiotic
resistance, such as resistance to kanamycin, G418, bleomycin,
hygromycin, or herbicide resistance, such as resistance to
chlorosulfuron, glyphosate or phosphinotricin.
[0062] A plant promoter fragment is used that directs transcription
of the gene in all tissues of a regenerated plant and/or is a
constitutive promoter. Alternatively, the plant promoter directs
transcription of a sequence of the invention in a specific tissue
(tissue-specific promoter) or is otherwise under more precise
environmental control (inducible promoter).
[0063] If proper polypeptide production is desired, a
polyadenylation region at the 3'-end of the coding region is
typically included. The polyadenylation region is derived from the
natural gene, from a variety of other plant genes, or from T-DNA,
synthesized in the laboratory.
Transformation
[0064] Techniques for transforming a wide variety of higher plant
species are well known and described in the technical and
scientific literature. See, e.g. Weising et al., Ann. Rev. Genet.
22:421 (1988); and Christou, Euphytica, v. 85, n.1-3:13-27,
(1995).
[0065] The person skilled in the art knows processes for the
transformation of monocotyledonous and dicotyledonous plants. A
variety of techniques are available for introducing DNA into a
plant host cell. These techniques comprise transformation of plant
cells by DNA injection, DNA electroporation, use of bolistics
methods, protoplast fusion and via T-DNA using Agrobacterium
tumefaciens or Agrobacterium rhizogenes, as well as further
possibilities, or other bacterial hosts for Ti plasmid vectors. See
for example, Broothaerts et al., Gene Transfer to Plants by Diverse
Species of Bacteria, Nature, Vol. 433, pp. 629-633, 10 Feb.
2005.
[0066] DNA constructs of the invention are introduced into the cell
or the genome of the desired plant host by a variety of
conventional techniques. For example, the DNA construct is
introduced using techniques such as electroporation, microinjection
and polyethylene glycol precipitation of plant cell protoplasts or
protoplast fusion. Electroporation techniques are described in
Fromm et al. Proc. Natl Acad. Sci. USA 82:5824 (1985).
Microinjection techniques are known in the art and well described
in the scientific and patent literature. The plasmids do not have
to fulfill specific requirements for use in DNA electroporation or
DNA injection into plant cells. Simple plasmids such as pUC
derivatives can be used.
[0067] The introduction of DNA constructs using polyethylene glycol
precipitation is described in Paszkowski et al. EMBO J. 3:2717
(1984). Introduction of foreign DNA using protoplast fusion is
described by Willmitzer (Willmitzer, L., 1993 Transgenic plants.
In: Biotechnology, A Multi-Volume Comprehensive Treatise (H. J.
Rehm, G. Reed, A. Puhler, P. Stadler, eds.), Vol. 2, 627-659, VCH
Weinheim-New York-Basel-Cambridge).
[0068] Alternatively, the DNA constructs of the invention are
introduced directly into plant tissue using ballistic methods, such
as DNA particle bombardment. Ballistic transformation techniques
are described in Klein et al. Nature 327:773 (1987). Introduction
of foreign DNA using ballistics is described by Willmitzer
(Willmitzer, L., 1993 Transgenic plants. In: Biotechnology, A
Multi-Volume Comprehensive Treatise (H. J. Rehm, G. Reed, A.
Puhler, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim-New
York-Basel-Cambridge).
[0069] DNA constructs are also introduced with the help of
Agrobacteria. The use of Agrobacteria for plant cell transformation
is extensively examined and sufficiently disclosed in the
specification of EP-A 120 516, and in Hoekema (In: The Binary Plant
Vector System Offsetdrukkerij Kanters B. V., Alblasserdam (1985),
Chapter V), Fraley et al. (Crit. Rev. Plant. Sci. 4, 1-46) and
DePicker et al. (EMBO J. 4 (1985), 277-287). Using this technique,
the DNA constructs of the invention are combined with suitable
T-DNA flanking regions and introduced into a conventional
Agrobacterium tumefaciens host vector. The virulence functions of
the Agrobacterium tumefaciens host direct the insertion of the
construct and adjacent marker(s) into the plant cell DNA when the
cell is infected by the bacteria (McCormac et al., 1997, Mol.
Biotechnol. 8:199; Hamilton, 1997, Gene 200:107; Salomon et al.,
1984 EMBO J. 3:141; Herrera-Estrella et al., 1983 EMBO J. 2:987).
Agrobacterium tumefaciens-mediated transformation techniques,
including disarming and use of binary or co-integrate vectors, are
well described in the scientific literature. See, for example
Hamilton, C M., Gene 200:107 (1997); Muller et al. Mol. Gen. Genet.
207:171 (1987); Komari et al. Plant J. 10:165 (1996); Venkateswarlu
et al. Biotechnology 9:1103 (1991) and Gleave, A P., Plant Mol.
Biol. 20:1203 (1992); Graves and Goldman, Plant Mol. Biol. 7:34
(1986) and Gould et al., Plant Physiology 95:426 (1991).
[0070] For plant cell T-DNA transfer of DNA, plant organs, e.g.
infloresences, plant explants, plant cells that have been cultured
in suspension or protoplasts are co-cultivated with Agrobacterium
tumefaciens or Agrobacterium rhizogenes or other suitable T-DNA
hosts. Whole plants are regenerated from the infected plant
material or seeds generated from infected plant material using a
suitable medium that contains antibiotics or biocides for the
selection of transformed cells or by spraying the biocide on plants
to select the transformed plants. Plants obtained in this way are
then examined for the presence of the DNA introduced. The
transformation of dicotyledonous plants via Ti-plasmid-vector
systems and Agrobacterium tumefaciens is well established.
[0071] Monocotyledonous plants are also transformed by means of
Agrobacterium based vectors (See Chan et al., Plant Mol. Biol. 22
(1993), 491-506; Hiei et al., Plant J. 6 (1994), 271-282; Deng et
al., Science in China 33 (1990), 28-34; Wilmink et al., Plant Cell
Reports 11 (1992), 76-80; May et al., Bio/Technology 13 (1995),
486-492; Conner and Domisse; Int. J. Plant Sci. 153 (1992),
550-555; Ritchie et al., Transgenic Res. 2 (1993), 252-265). Maize
transformation in particular is described in the literature (see,
for example, WO95/06128, EP 0 513 849; EP 0 465 875; Fromm et al.,
Biotechnology 8 (1990), 833-844; Gordon-Kamm et al., Plant Cell 2
(1990), 603-618; Koziel et al., Biotechnology 11 (1993), 194-200).
In EP 292 435 and in Shillito et al. (1989, Bio/Technology 7, 581)
fertile plants are obtained from a mucus-free, soft (friable) maize
callus. Prioli and Sondahl (1989, Bio/Technology 7, 589) also
report regenerating fertile plants from maize protoplasts of the
maize Cateto inbred line, Cat 100-1.
[0072] Other cereal species have also been successfully
transformed, such as barley (Wan and Lemaux, see above; Ritala et
al., see above) and wheat (Nehra et al., 1994, Plant J. 5,
285-297).
[0073] Alternatives to Agrobacterium transformation for plants are
ballistics, protoplast fusion, electroporation of partially
permeabilized cells and use of glass fibers (See Wan and Lemaux,
Plant Physiol. 104 (1994), 37-48; Vasil et al., Bio/Technology 11
(1993), 1553-1558; Ritala et al., Plant Mol. Biol. 24 (1994),
317-325; Spencer et al., Theor. Appl. Genet. 79 (1990),
625-631)).
[0074] Introduced DNA is usually stable after integration into the
plant genome and is transmitted to the progeny of the transformed
cell or plant. Generally the transformed plant cell contains a
selectable marker that makes the transformed cells resistant to a
biocide or an antibiotic such as kanamycin, G 418, bleomycin,
hygromycin, phosphinotricin or others. Therefore, the individually
chosen marker should allow the selection of transformed cells from
cells lacking the introduced DNA.
[0075] The transformed cells grow within the plant in the usual way
(McCormick et al., 1986, Plant Cell Reports 5, 81-84) and the
resulting plants are cultured normally. Transformed plant cells
obtained by any of the above transformation techniques are cultured
to regenerate a whole plant that possesses the transformed genotype
and thus the desired phenotype. Such regeneration techniques rely
on manipulation of certain phytohormones in a tissue culture growth
medium, typically relying on a biocide and/or herbicide marker that
has been introduced together with the desired nucleotide
sequences.
[0076] Plant regeneration from cultured protoplasts is described in
Evans et al., Protoplasts Isolation and Culture in "Handbook of
Plant Cell Culture," pp. 124-176, MacMillan Publishing Company, New
York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts,
pp. 21-73, CRC Press, Boca Raton, 1988. Regeneration also occurs
from plant callus, explants, organs, or parts thereof. Such
regeneration techniques are described generally in Klee et al. Ann.
Rev. of Plant Phys. 38:467 (1987). Regeneration of monocots (rice)
is described by Hosoyama et al. (Biosci. Biotechnol. Biochem.
58:1500 (1994)) and by Ghosh et al. (J. Biotechnol 32:1 (1994)).
Useful and relevant procedures for transient expression are also
described in U.S. Application Ser. No. 60/537,070 filed on Jan. 16,
2004 and PCT Application No. PCT/US2005/001153 filed on Jan. 14,
2005.
[0077] After transformation, seeds are obtained from the plants and
used for testing stability and inheritance. Generally, two or more
generations are cultivated to ensure that the phenotypic feature is
stably maintained and transmitted.
[0078] One of skill will recognize that after the expression
cassette is stably incorporated in transgenic plants and confirmed
to be operable, it can be introduced into other plants by sexual
crossing. Any of a number of standard breeding techniques can be
used, depending upon the species to be crossed.
[0079] The nucleotide sequences according to the invention
generally encode an appropriate protein from any organism, in
particular from plants, fungi, bacteria or animals. The sequences
preferably encode proteins from plants or fungi. Preferably, the
plants are higher plants, in particular starch or oil storing
useful plants, such as potato or cereals such as rice, maize,
wheat, barley, rye, triticale, oat, millet, etc., as well as
spinach, tobacco, sugar beet, soya, cotton etc.
[0080] In principle, the process according to the invention can be
applied to any plant. Therefore, monocotyledonous as well as
dicotyledonous plant species are particularly suitable. The process
is preferably used with plants that are interesting for
agriculture, horticulture and/or forestry. Examples are vegetable
plants such as cucumber, melon, pumpkin, eggplant, zucchini,
tomato, spinach, cabbage species, peas, beans, etc., as well as
fruits such as pears, apples, etc.
[0081] Thus, the invention has use over a broad range of plants,
preferably higher plants, pertaining to the classes of Angiospennae
and Gymnospermae. Plants of the subclasses of the Dicotylodenae and
the Monocotyledonae are particularly suitable. Dicotyledonous
plants belong to the orders of the Magniolales, Illiciales,
Laurales, Piperales Aristochiales, Nymphaeales, Ranunculales,
Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales,
Eucomiales, Leitneriales, Myricales, Fagales, Casuarinales,
Caryophyllales, Batales, Polygonales, Plumbaginales, Dilleniales,
Theales, Malvales, Urticales, Lecythidales, Violales, Salicales,
Capparales, Ericales, Diapensales, Ebenales, Primulales, Rosales,
Fabales, Podostemales, Haloragales, Myrtales, Cornales, Proteales,
Santales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales,
Sapindales, Juglandales, Geraniales, Polygalales, Umbellales,
Gentianales, Polemoniales, Lamiales, Plantaginales,
Scrophulariales, Campanulales, Rubiales, Dipsacales, and Asterales.
Monocotyledonous plants belong to the orders of the Alismatales,
Hydrocharitales, Najadales, Triuridales, Commelinales,
Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales,
Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales,
Arales, Lilliales, and Orchidales. Plants belonging to the class of
the Gymnospermae are Pinales, Ginkgoales, Cycadales and
Gnetales.
[0082] The method of the invention is preferably used with plants
that are interesting for agriculture, horticulture, biomass for
bioconversion and/or forestry. Examples are tobacco, oilseed rape,
sugar beet, potato, tomato, cucumber, pepper, bean, pea, citrus
fruit, apple, pear, berries, plum, melon, eggplant, cotton,
soybean, sunflower, rose, poinsettia, petunia, guayule, cabbage,
spinach, alfalfa, artichoke, corn, wheat, rye, barley, grasses such
as switch grass or turf grass, millet, hemp, banana, poplar,
eucalyptus trees, conifers.
[0083] The invention being thus described, it will be apparent to
one of ordinary skill in the art that various modifications of the
materials and methods for practicing the invention can be made.
Such modifications are to be considered within the scope of the
invention as defined by the following claims.
[0084] Each of the references from the patent and periodical
literature cited herein is hereby expressly incorporated in its
entirety by such citation.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070192907A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070192907A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References