U.S. patent application number 16/819972 was filed with the patent office on 2020-07-16 for methods and systems for predicting the risk of transgene silencing.
This patent application is currently assigned to Dow AgroSciences LLC. The applicant listed for this patent is Dow AgroSciences LLC. Invention is credited to Lakshmi Sastry-Dent, Shreedharan Sriram, PoHao Wang.
Application Number | 20200224269 16/819972 |
Document ID | / |
Family ID | 58663279 |
Filed Date | 2020-07-16 |
![](/patent/app/20200224269/US20200224269A1-20200716-D00001.png)
United States Patent
Application |
20200224269 |
Kind Code |
A1 |
Wang; PoHao ; et
al. |
July 16, 2020 |
METHODS AND SYSTEMS FOR PREDICTING THE RISK OF TRANSGENE
SILENCING
Abstract
The present invention is based on the use of the H3K9me2
methylation levels in plant genomes to predict transgene silencing,
transgene stability, and/or transgene expression level. Provided
are methods and/or systems for generating whole-genome H3K9me2 maps
and its use with an assigned threshold value for predicting gene
silencing. The methods and/or systems provided herein can be used
in high-throughput setting for screening large number of
transformed event in a relatively short period of time as compared
to existing technologies.
Inventors: |
Wang; PoHao; (Carmel,
IN) ; Sriram; Shreedharan; (Indianapolis, IN)
; Sastry-Dent; Lakshmi; (Avon, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dow AgroSciences LLC |
Indianapolis |
IN |
US |
|
|
Assignee: |
Dow AgroSciences LLC
Indianapolis
IN
|
Family ID: |
58663279 |
Appl. No.: |
16/819972 |
Filed: |
March 16, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15333574 |
Oct 25, 2016 |
10633703 |
|
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16819972 |
|
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62253213 |
Nov 10, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6876 20130101;
C12Q 1/6895 20130101; G16B 20/00 20190201; C12Q 2600/158 20130101;
C12Q 2600/154 20130101; C12N 15/82 20130101; G16B 25/00
20190201 |
International
Class: |
C12Q 1/6876 20060101
C12Q001/6876; G16B 25/00 20060101 G16B025/00; C12Q 1/6895 20060101
C12Q001/6895; C12N 15/82 20060101 C12N015/82; G16B 20/00 20060101
G16B020/00 |
Claims
1.-15. (canceled)
16. A computerized system for predicting the risk of transgene
silencing, comprising, (a) a histone methylation/acetylation
database showing peak numbers of the histone
methylation/acetylation profiles in a plant's genome; (b) a first
computer module for the input of data where a user inputs sequences
of at least one transgene insertion site from a transgenic event;
and (c) a second computer module for the output of data where data
predicting the risk of transgene silencing is generated to select a
transgenic event with a lower probability of transgene silencing,
wherein the data predicting the risk of transgene silencing is
based on comparison of the inputted sequences of Step (b) and the
histone methylation/acetylation profiles of Step (a).
17. The system of claim 16, wherein the histone
methylation/acetylation profile data using a chromatin
immunoprecipitation sequencing (ChIP-seq) assay.
18. The system of claim 16, wherein the histone
methylation/acetylation profile data is associated with histone
methylation/acetylation selected from the group consisting of
H3K4me2, H3K4me3, H3K9/14ac, H3K9me2, H3K9me3, H3K27me1, H3K27me3
and H4K20me3, and combinations thereof.
19. The system of claim 16, wherein the histone
methylation/acetylation profile data is associated with histone
methylation/acetylation selected from the group consisting of
H3K9me2, H3K9me3, H3K27me1, H3K27me3 and H4K20me3, and combinations
thereof.
20. The system of claim 16, wherein the histone
methylation/acetylation profile data is associated with H3K9me2
methylation.
21. The system of claim 16, wherein the plant is selected from
soybean, maize, canola, cotton, wheat, and rice.
22. A process for use in a computerized system for predicting the
risk of transgene silencing, comprising, (a) inputting sequences of
at least one transgene insertion site into the system of claim 16
by a user; and (b) receiving output from the system of claim 16 for
prediction of the risk of transgene silencing, wherein the
prediction is based on comparison of the inputted sequences of the
transgene insertion site and the histone methylation/acetylation
profiles within the system.
23. A transgenic plant, plant part, or plant cell comprising a
transgene inserted within a genomic locus, wherein the transgene
insertion is calculated to fall into peak zero or peak one of the
histone methylation/acetylation profile as determined by the method
comprising, a. generating histone methylation/acetylation profile
data of a plant's genome from a physical assay; b. assembling the
histone methylation/acetylation profile data of step (a) into a
histone methylation/acetylation database showing peak numbers of
the histone methylation/acetylation profiles; c. analyzing
sequences of at least one transgene insertion site from a
transgenic event; d. comparing the sequences of the transgene
insertion site of step (c) with the histone methylation/acetylation
database of step (b); wherein i. if the transgene insertion site
falls into peak zero or peak one of the histone
methylation/acetylation profiles, there is no risk of transgene
silencing, or ii. if the transgene insertion site falls into peak
two or more of the histone methylation/acetylation profiles, there
is significant risk of transgene silencing; and, e. selecting the
transgenic event with a lower probability of transgene
silencing.
24. The transgenic plant of claim 23, wherein the histone
methylation/acetylation profile data is generated using a chromatin
immunoprecipitation sequencing (ChIP-seq) assay.
25. The transgenic plant of claim 23, wherein the histone
methylation/acetylation profile data is associated with histone
methylation/acetylation selected from the group consisting of
H3K4me2, H3K4me3, H3K9/14ac, H3K9me2, H3K9me3, H3K27me1, H3K27me3
and H4K20me3, and combinations thereof.
26. The transgenic plant of claim 23, wherein the histone
methylation/acetylation profile data is associated with histone
methylation/acetylation selected from the group consisting of
H3K9me2, H3K9me3, H3K27me1, H3K27me3 and H4K20me3, and combinations
thereof.
27. The transgenic plant of claim 23, wherein the histone
methylation/acetylation profile data is associated with H3K9me2
methylation.
28. The transgenic plant of claim 23, wherein the transgenic plant
is selected from soybean, maize, canola, cotton, wheat, and
rice.
29. The transgenic plant of claim 23, wherein the sequences of the
transgene insertion site are obtained by sequencing.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn. 119
of U.S. Provisional Patent Application Ser. No. 62/253,213, filed
Nov. 10, 2015, which is hereby incorporated by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0002] The genome of plants, for example soybean or maize plants,
was successfully transformed with transgenes in the 1990's. Over
the last twenty years, numerous methodologies have been developed
for transforming the genome of plants, wherein a transgene is
stably integrated into the genome of plants. The evolution of plant
transformation methodologies has resulted in the capability to
successfully introduce a transgene comprising an agronomic trait
within the genome of plants. The introduction of insect resistance
and herbicide tolerant traits within plants provided producers with
a new and convenient technological innovation for controlling
insects and a wide spectrum of weeds, which was unparalleled in
cultivation farming methods.
[0003] Current transformation methodologies rely upon the random
insertion of transgenes within the genome of plants. Because
transgenic events may randomly integrate within gene
transcriptional sequences, such events may interrupt the expression
of endogenous traits and alter the growth and development of the
plant. In addition, the transgenic events may indiscriminately
integrate into locations of the genome that are susceptible to gene
silencing, culminating in the reduced or complete inhibition of
transgene expression either in the first or subsequent generations
of transgenic plants. Finally, the random integration of transgenes
within the plant genome requires considerable effort and cost in
identifying the location of the transgenic event and selecting
transgenic events that perform as designed without agronomic impact
to the plant.
[0004] Therefore, there is the need for inventions that are useful
to evaluate the effectiveness of the integration sites of transgene
for gene expression, and/or to predict the risk for transgene
silencing.
SUMMARY OF THE INVENTION
[0005] This invention is related to methods and systems for
predicting the risk of transgene silencing, and/or predicting
expression levels of transgenes, and/or predicting stability and/or
effectiveness of transgene integration sites. In one aspect,
provided is a method for predicting the risk of transgene
silencing. The method comprises: [0006] (a) generating histone
methylation/acetylation profile data in a plant's genome; [0007]
(b) assemble the histone methylation/acetylation profile data of
step (a) into a histone methylation/acetylation database showing
peak numbers of the histone methylation/acetylation profiles;
[0008] (c) analyzing sequences of at least one transgene insertion
site; and [0009] (d) comparing the sequences of the transgene
insertion site of step (c) with the histone methylation/acetylation
database of step (b); wherein (i) if the transgene insertion site
falls into peak zero or peak one of the methylation/acetylation
profiles, there is no risk of transgene silencing, or [0010] (ii)
if the transgene insertion site falls into peak two or more of the
histone methylation/acetylation profiles, there is significant risk
of transgene silencing.
[0011] In one embodiment, the histone methylation/acetylation
profile data is generated using a chromatin immunoprecipitation
sequencing (ChIP-seq) assay. In another embodiment, the histone
methylation/acetylation profile data is associated with histone
methylation/acetylation selected from the group consisting of
H3K4me2, H3K4me3, H3K9/14ac, H3K9me2, H3K9me3, H3K27me1, H3K27me3
and H4K20me3, and combinations thereof. In another embodiment, the
histone methylation/acetylation profile data is associated with
histone methylation/acetylation selected from the group consisting
of H3K9me2, H3K9me3, H3K27me1, H3K27me3 and H4K20me3, and
combinations thereof. In another embodiment, the histone
methylation/acetylation profile data is associated with H3K9me2
methylation. In another embodiment, the plant is selected from
soybean, maize, canola, cotton, wheat, and rice. In another
embodiment, the sequences of the transgene insertion site are
obtained by direct sequencing.
[0012] In another aspect, provided is computerized system for
predicting the risk of transgene silencing. The system comprises:
[0013] (a) a histone methylation/acetylation database showing peak
numbers of the histone methylation/acetylation profiles in a
plant's genome; [0014] (b) an input module where an user inputs
sequences of at least one transgene insertion site; and [0015] (c)
an output module shows prediction of the risk of transgene
silencing, wherein the prediction is based on comparison of the
inputted sequences of Step (b) and the histone
methylation/acetylation profiles of Step (a).
[0016] In one embodiment, the histone methylation/acetylation
profile data is generated using a chromatin immunoprecipitation
sequencing (ChIP-seq) assay. In another embodiment, the histone
methylation/acetylation profile data is associated with histone
methylation/acetylation selected from the group consisting of
H3K4me2, H3K4me3, H3K9/14ac, H3K9me2, H3K9me3, H3K27me1, H3K27me3
and H4K20me3, and combinations thereof. In another embodiment, the
histone methylation/acetylation profile data is associated with
histone methylation/acetylation selected from the group consisting
of H3K9me2, H3K9me3, H3K27me1, H3K27me3 and H4K20me3, and
combinations thereof. In another embodiment, the histone
methylation/acetylation profile data is associated with H3K9me2
methylation. In another embodiment, the plant is selected from
soybean, maize, canola, cotton, wheat, and rice. In another
embodiment, the sequences of the transgene insertion site are
obtained by direct sequencing.
[0017] In another aspect, provided is a process for use in a
computerized system for predicting the risk of transgene silencing.
The process comprises: [0018] (a) inputting sequences of at least
one transgene insertion site into the system provided herein by an
user; and [0019] (b) receiving output from the system provided
herein for prediction of the risk of transgene silencing, wherein
the prediction is based on comparison of the inputted sequences of
the transgene insertion site and the histone
methylation/acetylation profiles within the system.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 shows H3K9me2 abundance within Soybean gene features
(gene body, 2 kb upstream, 1 kb downstream) as a function of
different expression categories (High, Medium High, Medium, Medium
low, Low).
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention is based on the use of the H3K9me2
methylation levels in plant genomes to predict transgene silencing,
transgene stability, and/or transgene expression level. Provided
are methods and/or systems for generating whole-genome H3K9me2 maps
and its use with an assigned threshold value for predicting gene
silencing. The methods and/or systems provided herein can be used
in high-throughput setting for screening large number of
transformed event in a relatively short period of time as compared
to existing technologies.
[0022] Transgene performance and heritable transgene stability are
important for the success of trait product concepts. Several
approaches have been used in the past to assess transgene
expression and performance including quantitative expression
analyses techniques including for example RT-PCR, DNA methylation,
and transgenerational stability testing. These approaches take time
and resources during the trait development process and require
multiple generations of tracking transgenic events and could
potentially lead to picking bad transgenic events to promote for
downstream product development.
[0023] Epigenetic marks refer to enzyme-mediated chemical
modifications of DNA and of its associated chromatin proteins.
Typically epigenetic marks do not alter the primary sequence of
DNA, but they are important in regulating genome function. DNA
modifications (including cytosine methylation, posttranslational
modifications of histone tails and the histone core, and the
positioning of nucleosomes), may influence the transcriptional
state and other functional aspects of chromatin. Histone
modifications are a cellular system involving transcriptional
regulatory mechanisms. Both activating type (for example H3K4me3
and H3K9/14ac) and repressive type (for example H3K27me3, H3K9me3,
H3K9me2, and H4K20me3) histone marks have been previously
identified. In particular di-methylation of a specific lysine (K9)
in the histone H3, commonly known as H3K9me2, has been
characterized as an epigenetic mark indicating closed chromatin or
transcriptional repression at a genome scale. Provided are methods
and systems for the use of H3K9me2 as a diagnostic tool to
understand the chromatin state of the genomic location around an
integrated transgene and predict the potential risk of gene
silencing in transgenic events. The applicability of the methods
and systems provided include for example event sorting or selection
in the trait development process. Specifically, data for
genome-wide H3K9me2 profiling in Soybean roots and shoots are
provided as an example. In some embodiment, H3K9me2 methylation
level at the locus of a transgene insertion site can be used to
identify/predict events with a higher probability of stable
expression.
[0024] In some embodiment, whole-genome H3K9me2 maps are generated
for each crop plant. In some embodiment, a database is generated
according to such whole-genome H3K9me2 maps. In some embodiment,
such database can be used to predict the chromatin structure within
the locus of our interest according to the methods and/or systems
provided.
[0025] Unless otherwise indicated, all technical and scientific
terms used herein have the same meaning as they would to one
skilled in the art of the present invention. Practitioners are
particularly directed to Sambrook et al. Molecular Cloning: A
Laboratory Manual (Second Edition), Cold Spring Harbor Press,
Plainview, N.Y., 1989, and Ausubel F M et al. Current Protocols in
Molecular Biology, John Wiley & Sons, New York, N.Y., 1993, for
definitions and terms of the art. It is to be understood that this
invention is not limited to the particular methodology, protocols,
and reagents described, as these may vary.
[0026] As used herein, the phrase "about" refers to greater or
lesser than the value or range of values stated by 10 percent, but
is not intended to designate any value or range of values to only
this broader definition. Each value or range of values preceded by
the term "about" is also intended to encompass the embodiment of
the stated absolute value or range of values
[0027] As used herein, the phrase "vector" refers to a piece of
DNA, typically double-stranded, which can have inserted into it a
piece of foreign DNA. The vector can be for example, of plasmid or
viral origin, which typically encodes a selectable or screenable
marker or transgenes. The vector is used to transport the foreign
or heterologous DNA into a suitable host cell. Once in the host
cell, the vector can replicate independently of or coincidental
with the host chromosomal DNA. Alternatively, the vector can target
insertion of the foreign or heterologous DNA into a host
chromosome.
[0028] As used herein, the phrase "transgene vector" refers to a
vector that contains an inserted segment of DNA, the "transgene"
that is transcribed into mRNA or replicated as a RNA within a host
cell. The phrase "transgene" refers not only to that portion of
inserted DNA that is converted into RNA, but also those portions of
the vector that are necessary for the transcription or replication
of the RNA. A transgene typically comprises a gene-of-interest but
needs not necessarily comprise a polynucleotide sequence that
contains an open reading frame capable of producing a protein.
[0029] As used herein, the phrase "transformed" or "transformation"
refers to the introduction of DNA into a cell. The phrases
"transformant" or "transgenic" refers to plant cells, plants, and
the like that have been transformed or have undergone a
transformation procedure. The introduced DNA is usually in the form
of a vector containing an inserted piece of DNA.
[0030] As used herein, the phrase "transgenic plant" refers to a
plant whose genome has been altered by the stable integration of
recombinant DNA. A transgenic plant includes a plant regenerated
from an originally-transformed plant cell and progeny transgenic
plants from later generations or crosses of a transformed
plant.
[0031] As used herein, the phrase "recombinant DNA" refers to DNA
which has been genetically engineered and constructed outside of a
cell including DNA containing naturally occurring DNA or cDNA or
synthetic DNA.
[0032] As used herein, the phrase "selectable marker" or
"selectable marker gene" refers to a gene that is optionally used
in plant transformation to, for example, protect the plant cells
from a selective agent or provide resistance/tolerance to a
selective agent. Only those cells or plants that receive a
functional selectable marker are capable of dividing or growing
under conditions having a selective agent. Examples of selective
agents can include, for example, antibiotics, including
spectinomycin, neomycin, kanamycin, paromomycin, gentamicin, and
hygromycin. These selectable markers include gene for neomycin
phosphotransferase (npt II), which expresses an enzyme conferring
resistance to the antibiotic kanamycin, and genes for the related
antibiotics neomycin, paromomycin, gentamicin, and G418, or the
gene for hygromycin phosphotransferase (hpt), which expresses an
enzyme conferring resistance to hygromycin. Other selectable marker
genes can include genes encoding herbicide resistance including Bar
(resistance against BASTA.RTM. (glufosinate ammonium), or
phosphinothricin (PPT)), acetolactate synthase (ALS, resistance
against inhibitors such as sulfonylureas (SUs), imidazolinones
(IMIs), triazolopyrimidines (TPs), pyrimidinyl oxybenzoates (POBs),
and sulfonyl amino carbonyl triazolinones that prevent the first
step in the synthesis of the branched-chain amino acids),
glyphosate, 2,4-D, and metal resistance or sensitivity. The phrase
"marker-positive" refers to plants that have been transformed to
include the selectable marker gene.
[0033] Various selectable or detectable markers can be incorporated
into the chosen expression vector to allow identification and
selection of transformed plants, or transformants. Many methods are
available to confirm the expression of selection markers in
transformed plants, including for example DNA sequencing and PCR
(polymerase chain reaction), Southern blotting, RNA blotting,
immunological methods for detection of a protein expressed from the
vector, e g., precipitated protein that mediates phosphinothricin
resistance, or other proteins such as reporter genes
.beta.-glucuronidase (GUS), luciferase, green fluorescent protein
(GFP), DsRed, .beta.-galactosidase, chloramphenicol
acetyltransferase (CAT), alkaline phosphatase, and the like (See
Sambrook, et al., Molecular Cloning: A Laboratory Manual, Third
Edition, Cold Spring Harbor Press, N.Y., 2001, the content of which
is incorporated herein by reference in its entirety).
[0034] Selectable marker genes are utilized for the selection of
transformed cells or tissues. Selectable 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.
Herbicide resistance genes generally code for a modified target
protein insensitive to the herbicide or for an enzyme that degrades
or detoxifies the herbicide in the plant before it can act. For
example, resistance to glyphosate or has been obtained by using
genes coding for the mutant target enzymes,
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Genes and
mutants for EPSPS have been disclosed in U.S. Pat. Nos. 4,940,835,
5,188,642, 5,310,667, 5,633,435, 5,633,448, and 6,566,587, the
contents of which are incorporated by reference in their
entireties. Resistance to glufosinate ammonium, bromoxynil, and
2,4-dichlorophenoxyacetate (2,4-D) have been obtained by using
bacterial genes encoding phosphinothricin acetyltransferase, a
nitrilase, or a 2,4-dichlorophenoxyacetate monooxygenase, which
detoxify the respective herbicides. Enzymes/genes for glufosinate
resistance/tolerance have been disclosed in U.S. Pat. Nos.
5,273,894, 5,276,268, 5,550,318, and 5,561,236, the contents of
which are incorporated by reference in their entireties.
Enzymes/genes for 2,4-D resistance have been previously disclosed
in U.S. Pat. Nos. 6,100,446 and 6,153,401, as well as patent
applications US 2009/0093366 (AAD-1) and WO 2007/053482 (AAD-12),
the contents of which are hereby incorporated by reference in their
entireties. Enzymes/genes for nitrilase have been previously
disclosed in U.S. Pat. No. 4,810,648, the content of which is
incorporated by reference in its entirety.
[0035] Other herbicides can inhibit the growing point or meristem,
including imidazolinone or sulfonylurea, and genes for
resistance/tolerance of acetohydroxy acid synthase (AHAS) and
acetolactate synthase (ALS) for these herbicides have been
described. Genes and mutants for AHAS and mutants have been
disclosed in U.S. Pat. Nos. 4,761,373, 5,304,732, 5,331,107,
5,853,973, and 5,928,937, the contents of which are incorporated by
reference in their entireties. Genes and mutants for ALS have been
disclosed in U.S. Pat. Nos. 5,013,659 and 5,141,870, the contents
of which are incorporated by reference in their entireties.
[0036] Glyphosate resistance genes include mutant
5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) genes (via the
introduction of recombinant nucleic acids and/or various forms of
in vivo mutagenesis of native EPSPs genes), aroA genes and
glyphosate acetyl transferase (GAT) genes, respectively).
Resistance genes for other phosphono compounds include glufosinate
(phosphinothricin acetyl transferase (PAT) genes from Streptomyces
species, including Streptomyces hygroscopicus and Streptomyces
viridichromogenes), and pyridinoxy or phenoxy proprionic acids and
cyclohexones (ACCase inhibitor-encoding genes). Herbicide
resistance/tolerance genes of acetyl coenzyme A carboxylase
(ACCase) have been described in U.S. Pat. Nos. 5,162,602 and
5,498,544, the contents of which are incorporated by reference in
their entireties.
[0037] A DNA molecule encoding a mutant aroA gene can be obtained
under ATCC accession number 39256, and the nucleotide sequence of
the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai,
European patent application No. 0 333 033 to Kumada et al., and
U.S. Pat. No. 4,975,374 to Goodman et al., disclosing nucleotide
sequences of glutamine synthetase genes which confer resistance to
herbicides such as L-phosphinothricin. The nucleotide sequence of a
PAT gene is provided in European application No. 0 242 246 to
Leemans et al. Also DeGreef et al., Bio/Technology 7:61 (1989),
describes the production of transgenic plants that express chimeric
bar genes coding for PAT activity. Exemplary of genes conferring
resistance to phenoxy proprionic acids and cyclohexones, including
sethoxydim and haloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3
genes described by Marshall et al., Theon. Appl. Genet. 83:435
(1992). GAT genes capable of conferring glyphosate resistance are
described in WO 2005012515 to Castle et al. Genes conferring
resistance to 2,4-D, fop and pyridyloxy auxin herbicides are
described in WO 2005107437 and U.S. patent application Ser. No.
11/587,893.
[0038] Other herbicides can inhibit photosynthesis, including
triazine (psbA and 1s+ genes) or benzonitrile (nitrilase gene).
Przibila et al., Plant Cell 3:169 (1991), describes the
transformation of Chlamydomonas with plasmids encoding mutant psbA
genes. Nucleotide sequences for nitrilase genes are disclosed in
U.S. Pat. No. 4,810,648 to Stalker, and DNA molecules containing
these genes are available under ATCC Accession Nos. 53435, 67441,
and 67442. Cloning and expression of DNA coding for a glutathione
S-transferase is described by Hayes et al., Biochem. J. 285:173
(1992).
[0039] For purposes of the present invention, selectable marker
genes include, but are not limited to genes encoding: neomycin
phosphotransferase II (Fraley et al. (1986) CRC Critical Reviews in
Plant Science, 4:1-25); cyanamide hydratase (Maier-Greiner et al.
(1991) Proc. Natl. Acad. Sci. USA, 88:4250-4264); aspartate kinase;
dihydrodipicolinate synthase (Perl et al. (1993) Bio/Technology,
11:715-718); tryptophan decarboxylase (Goddijn et al. (1993) Plant
Mol. Bio., 22:907-912); dihydrodipicolinate synthase and
desensitized aspartade kinase (Perl et al. (1993) Bio/Technology,
11:715-718); bar gene (Toki et al. (1992) Plant Physiol.,
100:1503-1507 and Meagher et al. (1996) and Crop Sci., 36:1367);
tryptophan decarboxylase (Goddijn et al. (1993) Plant Mol. Biol.,
22:907-912); neomycin phosphotransferase (NEO) (Southern et al.
(1982) J. Mol. Appl. Gen., 1:327; hygromycin phosphotransferase
(HPT or HYG) (Shimizu et al. (1986) Mol. Cell Biol., 6:1074);
dihydrofolate reductase (DHFR) (Kwok et al. (1986) PNAS USA 4552);
phosphinothricin acetyltransferase (DeBlock et al. (1987) EMBO J.,
6:2513); 2,2-dichloropropionic acid dehalogenase
(Buchanan-Wollatron et al. (1989) J. Cell. Biochem. 13D:330);
acetohydroxyacid synthase (Anderson et al., U.S. Pat. No.
4,761,373; Haughn et al. (1988) Mol. Gen. Genet. 221:266);
5-enolpyruvyl-shikimate-phosphate synthase (aroA) (Comai et al.
(1985) Nature 317:741); haloarylnitrilase (Stalker et al.,
published PCT application WO87/04181); acetyl-coenzyme A
carboxylase (Parker et al. (1990) Plant Physiol. 92:1220);
dihydropteroate synthase (sul I) (Guerineau et al. (1990) Plant
Mol. Biol. 15:127); and 32 kD photosystem II polypeptide (psbA)
(Hirschberg et al. (1983) Science, 222:1346).
[0040] Also included are genes encoding resistance to:
chloramphenicol (Herrera-Estrella et al. (1983) EMBO J.,
2:987-992); methotrexate (Herrera-Estrella et al. (1983) Nature,
303:209-213; Meijer et al. (1991) Plant Mol Bio., 16:807-820
(1991); hygromycin (Waldron et al. (1985) Plant Mol. Biol.,
5:103-108; Zhijian et al. (1995) Plant Science, 108:219-227 and
Meijer et al. (1991) Plant Mol. Bio. 16:807-820); streptomycin
(Jones et al. (1987) Mol. Gen. Genet., 210:86-91); spectinomycin
(Bretagne-Sagnard et al. (1996) Transgenic Res., 5:131-137);
bleomycin (Hille et al. (1986) Plant Mol. Biol., 7:171-176);
sulfonamide (Guerineau et al. (1990) Plant Mol. Bio., 15:127-136);
bromoxynil (Stalker et al. (1988) Science, 242:419-423); 2,4-D
(Streber et al. (1989) Bio/Technology, 7:811-816); glyphosate (Shaw
et al. (1986) Science, 233:478-481); and phosphinothricin (DeBlock
et al. (1987) EMBO J., 6:2513-2518). All references recited in the
disclosure are hereby incorporated by reference in their entireties
unless stated otherwise.
[0041] The above list of selectable marker and reporter genes are
not meant to be limiting. Any reporter or selectable marker gene
are encompassed by the present invention. If necessary, such genes
can be sequenced by methods known in the art.
[0042] The reporter and selectable marker genes are synthesized for
optimal expression in the plant. That is, the coding sequence of
the gene has been modified to enhance expression in plants. The
synthetic marker gene is designed to be expressed in plants at a
higher level resulting in higher transformation efficiency. Methods
for synthetic optimization of genes are available in the art. In
fact, several genes have been optimized to increase expression of
the gene product in plants.
[0043] The marker gene sequence can be optimized for expression in
a particular plant species or alternatively can be modified for
optimal expression in plant families. The plant preferred codons
may be determined from the codons of highest frequency in the
proteins expressed in the largest amount in the particular plant
species of interest. See, for example, EPA 0359472; EPA 0385962; WO
91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA,
88:3324-3328; and Murray et al. (1989) Nucleic Acids Research, 17:
477-498; U.S. Pat. Nos. 5,380,831; and 5,436,391, herein
incorporated by reference. In this manner, the nucleotide sequences
can be optimized for expression in any plant. It is recognized that
all or any part of the gene sequence may be optimized or synthetic.
That is, fully optimized or partially optimized sequences may also
be used.
[0044] In addition, several transformation strategies utilizing the
Agrobacterium-mediated transformation system have been developed.
For example, the binary vector strategy is based on a two-plasmid
system where T-DNA is in a different plasmid from the rest of the
Ti plasmid. In a co-integration strategy, a small portion of the
T-DNA is placed in the same vector as the foreign gene, which
vector subsequently recombines with the Ti plasmid.
[0045] As used herein, the phrase "plant" includes dicotyledons
plants and monocotyledons plants. Examples of dicotyledons plants
include tobacco, Arabidopsis, soybean, tomato, Papaya, canola,
sunflower, cotton, alfalfa, potato, grapevine, pigeon pea, pea,
Brassica, chickpea, sugar beet, rapeseed, watermelon, melon,
pepper, peanut, pumpkin, radish, spinach, squash, broccoli,
cabbage, carrot, cauliflower, celery, Chinese cabbage, cucumber,
eggplant, and lettuce. Examples of monocotyledons plants include
corn, rice, wheat, sugarcane, barley, rye, sorghum, orchids,
bamboo, banana, cattails, lilies, oat, onion, millet, and
triticale.
[0046] As used herein, the term "plant" also includes a whole plant
and any descendant, cell, tissue, or part of a plant. The term
"plant parts" include any part(s) of a plant, including, for
example and without limitation: seed (including mature seed and
immature seed); a plant cutting; a plant cell; a plant cell
culture; a plant organ (e.g., pollen, embryos, flowers, fruits,
shoots, leaves, roots, stems, and explants). A plant tissue or
plant organ may be a seed, callus, or any other group of plant
cells that is organized into a structural or functional unit. A
plant cell or tissue culture may be capable of regenerating a plant
having the physiological and morphological characteristics of the
plant from which the cell or tissue was obtained, and of
regenerating a plant having substantially the same genotype as the
plant. In contrast, some plant cells are not capable of being
regenerated to produce plants. Regenerable cells in a plant cell or
tissue culture may be embryos, protoplasts, meristematic cells,
callus, pollen, leaves, anthers, roots, root tips, silk, flowers,
kernels, ears, cobs, husks, or stalks.
[0047] Plant parts include harvestable parts and parts useful for
propagation of progeny plants. Plant parts useful for propagation
include, for example and without limitation: seed; fruit; a
cutting; a seedling; a tuber; and a rootstock. A harvestable part
of a plant may be any useful part of a plant, including, for
example and without limitation: flower; pollen; seedling; tuber;
leaf; stem; fruit; seed; and root.
[0048] As used herein, the phrase "plant cell described" or
"transformed plant cell" refers to a plant cell that is transformed
with stably-integrated, non-natural, recombinant DNA, e.g., by
Agrobacterium-mediated transformation or by bombardment using
microparticles coated with recombinant DNA or other means. A plant
cell of this invention can be an originally-transformed plant cell
that exists as a microorganism or as a progeny plant cell that is
regenerated into differentiated tissue, e.g., into a transgenic
plant with stably-integrated, non-natural recombinant DNA, or seed
or pollen derived from a progeny transgenic plant.
[0049] As used herein, the phrase "consensus sequence" refers to an
artificial sequence of amino acids in a conserved region of an
alignment of amino acid sequences of homologous proteins, e.g., as
determined by a CLUSTALW alignment of amino acid sequence of
(functional) homolog proteins.
[0050] As used herein, the phrase "homolog" refers to a nucleic
acid or a protein in a group of proteins that perform the same
biological function, e.g., proteins that belong to the same protein
family or similar nucleic acids that provide a common enhanced
trait in transgenic plants of this invention. Homologs are
expressed by homologous genes. With reference to homologous genes,
homologs include orthologs, i.e., genes expressed in different
species that evolved from common ancestral genes by speciation and
encode proteins that retain the same function, but do not include
paralogs, i.e., genes that are related by duplication but have
evolved to encode proteins with different functions. Homologous
genes include naturally occurring alleles and artificially-created
variants. Degeneracy of the genetic code provides the possibility
to substitute at least one base of the protein encoding sequence of
a gene with a different base without causing the amino acid
sequence of the polypeptide produced from the gene to be changed.
When optimally aligned, homolog genes have at least 50%, 60%, 70%,
80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity
over the full length of the gene identified as being associated
with imparting an enhanced trait when expressed in plant cells. In
one aspect of the invention homolog genes have a nucleic acid or
amino acid sequence similarity that has at least 85%, 90%, 95%,
96%, 97%, 98% or 99% identity to a consensus sequence of proteins,
nucleotides and homologs disclosed herein.
[0051] Homologs can be identified by comparison of amino acid
sequence, e.g., manually or by use of a computer-based tool using
known homology-based search algorithms such as those commonly known
and referred to as BLAST, FASTA, and Smith-Waterman. A local
sequence alignment program, (e.g., Basic Local Alignment Search
Tool (BLAST)) can be used to search a database of sequences to find
similar sequences, and the summary Expectation value (E-value) used
to measure the sequence base similarity. Because a protein hit with
the best E-value for a particular organism may not necessarily be
an ortholog, i.e., have the same function, or be the only ortholog,
a reciprocal query is used to filter hit sequences with significant
E-values for ortholog identification. The reciprocal query entails
search of the significant hits against a database of amino acid
sequences from the base organism that are similar to the sequence
of the query protein. A hit can be identified as an ortholog, when
the reciprocal query's best bit is the query protein itself or a
protein encoded by a duplicated gene after speciation. A further
aspect of the homologs encoded by DNA useful in the transgenic
plants of the invention are those proteins that differ from a
disclosed protein as the result of deletion or insertion of one or
more amino acids in a native sequence.
[0052] As used herein, the phrase "percent identity" or "%
identity" refers to the extent to which the sequences of DNA or
protein segments are invariant throughout a window of alignment of
sequences, for example nucleotide sequences or amino acid
sequences. An "identity fraction" for a sequence aligned with a
reference sequence is the number of identical components which are
shared by the sequences, divided by the length of the alignment not
including gaps introduced by the alignment algorithm. "Percent
identity" is the identity fraction times 100. Percent identity is
calculated over the aligned length preferably using a local
alignment algorithm, for example BLASTp.
[0053] As used herein, the phrase "functional activity" or
"functionally active" refers to the proteins/enzymes for use
according to the subject invention which have the ability to
provide for stress tolerance which can result in an increased
yield. Transfer of the functional activity to plant or bacterial
systems can involve a nucleic acid sequence, encoding the amino
acid sequence for a protein of the subject invention, integrated
into a protein expression vector appropriate to the host in which
the vector will reside. One way to obtain a nucleic acid sequence
encoding a protein with functional activity is to isolate the
native genetic material from the bacterial species which produces
the protein of interest, using information deduced from the
protein's amino acid sequence, as disclosed herein. The native
sequences can be optimized for expression in plants. Optimized
polynucleotides can also be designed based on the protein
sequence.
[0054] As used herein, the phrases "control sequences" and
"regulatory sequences" are interchangeable and refer to nucleic
acid sequences useful for transcription/gene expression in plants.
"Control sequences" or "regulatory sequences" may include, but not
limited to, promoters, operators, enhancers, origins of
replication, ribosome binding sites, termination and
polyadenylation signals.
[0055] As used herein, the phrase "promoter" refers to regulatory
DNA for initializing transcription. A "plant promoter" is a
promoter capable of initiating transcription in plant cells whether
or not its origin is a plant cell, e.g., is it well known that
Agrobacterium T-DNA promoters are functional in plant cells. Thus,
plant promoters include promoter DNA obtained from plants, plant
viruses and bacteria such as Agrobacterium and Bradyrhizobium
bacteria. Examples of promoters under developmental control include
promoters that preferentially initiate transcription in certain
tissues, including leaves, roots, or seeds. Such promoters are
referred to as "tissue preferred." Promoters that initiate
transcription only in certain tissues are referred to as "tissue
specific." A "cell type" specific promoter primarily drives
expression in certain cell types in one or more organs, for
example, vascular cells in roots or leaves. An "inducible" or
"repressible" promoter is a promoter which is under environmental
or chemical control. Examples of environmental conditions that may
affect transcription by inducible promoters include anaerobic
conditions, or certain chemicals, or the presence of light. Tissue
specific, tissue preferred, cell type specific, and inducible
promoters belong to the class of "non-constitutive" promoters. A
"constitutive" promoter refers to a promoter which is active under
most conditions and in most tissues.
[0056] Numerous promoters that are active in plant cells have been
previously described. These include promoters present in plant
genomes as well as promoters from other sources, including nopaline
synthase (NOS) promoter and octopine synthase (OCS) promoters
carried on tumor-inducing plasmids of Agrobacterium tumefaciens and
the CaMV35S promoters from the cauliflower mosaic virus as
disclosed in U.S. Pat. Nos. 5,164,316 and 5,322,938. Useful
promoters derived from plant genes are found in U.S. Pat. No.
5,641,876 disclosing a rice actin promoter; U.S. Pat. No. 7,151,204
disclosing a maize chloroplast aldolase promoter and a maize
aldolase (FDA) promoter; and U.S. Patent Application Publication
2003/0131377 disclosing a maize nicotianamine synthase promoter.
These and numerous other promoters that function in plant cells are
known to those skilled in the art and available for use in
recombinant polynucleotides described herein to provide for
expression of desired genes in transgenic plant cells.
[0057] Furthermore, the promoters may be altered to contain
multiple "enhancer sequences" to assist in elevating gene
expression. Such enhancers are known in the art. By including an
enhancer sequence with such constructs, the expression of the
selected protein may be enhanced. These enhancers often are found
5' to the start of transcription in a promoter that functions in
eukaryotic cells, but can often be inserted upstream (5') or
downstream (3') to the coding sequence. In some instances, these 5'
enhancing elements are introns. Particularly useful as enhancers
are the 5' introns of the rice actin 1 (see for example U.S. Pat.
No. 5,641,876) and rice actin 2 genes, the maize alcohol
dehydrogenase gene intron(s), the maize heat shock protein 70 gene
intron (see for example U.S. Pat. No. 5,593,874) and the maize
shrunken 1 gene intron. See also U.S. Patent Application
Publication 2002/0192813A1 disclosing 5', 3' and intron elements
useful in the design of effective plant expression vectors.
[0058] In some embodiments, sufficient expression in plant seed
tissues is desired to affect improvements in seed composition.
Exemplary promoters for use for seed composition modification
include promoters from seed genes such as napin as disclosed in
U.S. Pat. No. 5,420,034, maize L3 oleosin as disclosed in U.S. Pat.
No. 6,433,252), zein Z27 as disclosed by Russell et al. (1997)
Transgenic Res. 6(2): 157-166), globulin 1 as disclosed by Belanger
et al (1991) Genetics 129:863-872), glutelin 1 as disclosed by
Russell (1997, supra), and peroxiredoxin antioxidant (Perl) as
disclosed by Stacy et al. (1996) Plant Mol Biol.
31(6):1205-1216.
[0059] As used herein, the phrase "operably linked" refers to the
association of two or more DNA fragments in a recombinant DNA
construct so that the function of one, e.g., protein-encoding DNA,
is controlled by the other, e.g., a promoter.
[0060] Recombinant DNA constructs are assembled using methods well
known to persons of ordinary skill in the art and typically
comprise a promoter operably linked to DNA, the expression of which
provides the enhanced agronomic trait. Other construct components
may include additional regulatory elements, including 5' leaders
and introns for enhancing transcription, 3' untranslated regions
(for example, polyadenylation signals and sites), and DNA for
transit or signal peptides.
[0061] Recombinant DNA constructs described herein also generally
include a 3' element that typically contains a polyadenylation
signal and site. Well-known 3' elements include those from
Agrobacterium tumefaciens genes such as nos 3', tml 3', tmr 3', tms
3', ocs 3', tr73', for example disclosed in U.S. Pat. No.
6,090,627; 3' elements from plant genes such as wheat (Triticum
aesevitum) heat shock protein 17 (Hspl 73), a wheat ubiquitin gene,
a wheat fructose-1,6-biphosphatase gene, a rice glutelin gene, a
rice lactate dehydrogenase gene and a rice beta-tubulin gene, all
of which are disclosed in U.S. Patent Application Publication
2002/0192813; and the pea (Pisum sativum) ribulose biphosphate
carboxylase gene (rbs 3'), and 3' elements from the genes within
the host plant.
[0062] Constructs and vectors may also include a transit peptide
for targeting of a gene to a plant organelle, particularly to a
chloroplast, leucoplast or other plastid organelle. For
descriptions of the use of chloroplast transit peptides see for
example U.S. Pat. Nos. 5,188,642 and 5,728,925.
[0063] As used herein, the phrase "expressed" refers to a protein
which is expressed or produced in a plant cell when its cognate DNA
is transcribed to mRNA that is translated to the protein. As used
herein, the phrase "suppressed" refers to decreased expression or
activity of a protein. Typically a protein is suppressed in a plant
cell when there is a decrease in the amount and/or activity of the
protein in the plant cell. The presence or activity of the protein
can be decreased by any amount up to and including a total loss of
protein expression and/or activity.
[0064] As used herein, the phrase "trait" refers to a
physiological, morphological, biochemical, or physical
characteristic of a plant or particular plant material or cell. In
some instances, this characteristic is visible to the human eye,
including seed or plant size, or can be measured by biochemical
techniques, including detecting the protein, starch, or oil content
of seed or leaves, or by observation of a metabolic or
physiological process (e.g., by measuring uptake of carbon
dioxide), or by the observation of the expression level of a gene
or genes (e.g., by employing Northern analysis, RT-PCR, microarray
gene expression assays), or reporter gene expression systems, or by
agricultural observations including stress tolerance, yield, or
pathogen tolerance.
[0065] As used herein, the phrase "overexpression" refers to a
greater expression level of a gene in a plant, plant cell or plant
tissue, compared to expression in a wild-type plant, cell or
tissue, at any developmental or temporal stage for the gene.
Overexpression can occur when, for example, the genes are under the
control of a strong expression signal, including one of the
promoters described herein or the cauliflower mosaic virus 35S
transcription initiation region known in the art. Overexpression
may occur throughout a plant or in specific tissues of the plant,
depending on the promoter used.
[0066] As used herein, a "nongenic sequence" or "nongenic genomic
sequence" is a native DNA sequence found in the nuclear genome of a
plant, having a length of at least 1 Kb, and devoid of any open
reading frames, gene sequences, or gene regulatory sequences.
Furthermore, the nongenic sequence does not comprise any intron
sequence (i.e., introns are excluded from the definition of
nongenic). The nongenic sequence cannot be transcribed or
translated into protein. Many plant genomes contain nongenic
regions, where as much as 95% of the genome can be nongenic, and
these regions may be comprised of mainly repetitive DNA.
[0067] As used herein, a "genic region" is defined as a
polynucleotide sequence that comprises an open reading frame
encoding an RNA and/or polypeptide. The genic region may also
encompass any identifiable adjacent 5' and 3' non-coding nucleotide
sequences involved in the regulation of expression of the open
reading frame up to about 2 Kb upstream of the coding region and 1
Kb downstream of the coding region, but possibly further upstream
or downstream. A genic region further includes any introns that may
be present in the genic region. Further, the genic region may
comprise a single gene sequence, or multiple gene sequences
interspersed with short spans (less than 1 Kb) of nongenic
sequences.
[0068] As used herein, the term "hypomethylation" or
"hypomethylated," in reference to a DNA sequence, defines a reduced
state of methylated DNA nucleotide residues in a given sequence of
DNA. Typically, the decreased methylation relates to the number of
methylated adenine or cytosine residues, relative to the average
level of methylation found in nongenic sequences present in the
genome of a plant for example soybean or maize.
[0069] The term "in proximal location to a genic region" when used
in reference to a nongenic sequence defines the relative location
of the nongenic sequence to a genic region. Specifically, the
number of genic regions within a 40 Kb neighborhood (i.e., within
40 Kb on either end of the selected optimal soybean genomic loci
sequence) is analyzed. The number of genic regions may range from a
minimum of 1 gene to a maximum of 18 genes within the 40 Kb
neighborhood.
[0070] The term "evidence of recombination" as used herein relates
to the meiotic recombination frequencies between any pair of
genomic markers across a chromosome region comprising the selected
sequence. The recombination frequencies were calculated based on
the ratio of the genetic distance between markers (in centimorgan
(cM)) to the physical distance between the markers (in megabases
(Mb)). For a selected sequence to have evidence of recombination,
the selected sequence must contain at least one recombination event
between two markers flanking the selected sequence as detected
using a high resolution marker dataset generated from multiple
mapping populations.
[0071] As used herein the term "relative location value" is a
calculated value defining the distance of a genomic locus from its
corresponding chromosomal centromere. For each selected sequence,
the genomic distance from the native location of the selected
sequence to the centromere of the chromosome that it is located on,
is measured (in Bp). The relative location of selected sequence
within the chromosome is represented as the ratio of its genomic
distance to the centromere relative to the length of the specific
chromosomal arm (measured in Bp) that it lies on. These relative
location values for the optimal nongenic genomic loci can be
generated for different plants, the relative location values for
the dataset may range from a minimum of 0 to a maximum of 0.99682
ratio of genomic distance.
[0072] The term ""exogenous DNA sequence" as used herein is any
nucleic acid sequence that has been removed from its native
location and inserted into a new location altering the sequences
that flank the nucleic acid sequence that has been moved. For
example, an exogenous DNA sequence may comprise a sequence from
another species.
[0073] "Recombination" refers to a process of exchange of genetic
information between two polynucleotides, including but not limited
to, donor capture by non-homologous end joining (NHEJ) and
homologous recombination. As used herein, "homologous recombination
(HR)" refers to the specialized form of such exchange that takes
place, for example, during repair of double-strand breaks in cells
via homology-directed repair mechanisms. This process requires
nucleotide sequence homology, uses a "donor" molecule to template
repair of a "target" molecule (i.e., the nucleotide sequence that
experienced the double-strand break), and is variously known as
"non-crossover gene conversion" or ""short tract gene conversion,"
because it leads to the transfer of genetic information from the
donor to the target. Without wishing to be bound by any particular
theory, such transfer can involve mismatch correction of
heteroduplex DNA that forms between the broken target and the
donor, and/or "synthesis-dependent strand annealing," in which the
donor is used to resynthesize genetic information that will become
part of the target, and/or related processes. Such specialized HR
often results in an alteration of the sequence of the target
molecule such that part or all of the sequence of the donor
polynucleotide is incorporated into the target polynucleotide. For
HR-directed integration, the donor molecule contains at least 2
regions of homology to the genome ("homology arms") of least 50-100
base pairs in length. See, e.g., U.S. Patent Publication No.
20110281361, the content of which is hereby incorporated by
reference.
[0074] As used herein, the phrase "gene expression" refers to the
conversion of the information, contained in a gene, into a gene
product. A gene product can be the direct transcriptional product
of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, interfering RNA,
ribozyme, structural RNA or any other type of RNA) or a protein
produced by translation of a mRNA. Gene products also include RNAs
which are modified, by processes such as capping, polyadenylation,
methylation, and editing, and proteins modified by, for example,
methylation, acetylation, phosphorylation, ubiquitination,
ADP-ribosylation, myristilation, and glycosylation.
[0075] As used herein, the term "substantially homologous" or
""substantial homology," with regard to a contiguous nucleic acid
sequence, refers to contiguous nucleotide sequences that hybridize
under stringent conditions to the reference nucleic acid sequence.
For example, nucleic acid sequences that are substantially
homologous to a reference nucleic acid sequence are those nucleic
acid sequences that hybridize under stringent conditions (e.g., the
Moderate Stringency conditions set forth, supra) to the reference
nucleic acid sequence. Substantially homologous sequences may have
at least 80% sequence identity. For example, substantially
homologous sequences may have from about 80% to 100% sequence
identity, such as about 81%; about 82%; about 83%; about 84%; about
85%; about 86%; about 87%; about 88%; about 89%; about 90%; about
91%; about 92%; about 93%; about 94% about 95%; about 96%; about
97%; about 98%; about 98.5%; about 99%; about 99.5%; and about
100%. The property of substantial homology is closely related to
specific hybridization. For example, a nucleic acid molecule is
specifically hybridizable when there is a sufficient degree of
complementarity to avoid non-specific binding of the nucleic acid
to non-target sequences under conditions where specific binding is
desired, for example, under stringent hybridization conditions.
[0076] In some instances "homologous" may be used to refer to the
relationship of a first gene to a second gene by descent from a
common ancestral DNA sequence. In such instances, the term,
homolog, indicates a relationship between genes separated by the
event of speciation (see ortholog) or to the relationship between
genes separated by the event of genetic duplication (see paralog).
In other instances "homologous" may be used to refer to the level
of sequence identity between one or more polynucleotide sequences,
in such instances the one or more polynucleotide sequences do not
necessarily descend from a common ancestral DNA sequence. Those
with skill in the art are aware of the interchangeably of the term
"homologous" and appreciate the proper application of the term.
[0077] While the invention has been described with reference to
specific methods and embodiments, it will be appreciated that
various modifications and changes may be made without departing
from the invention. All publications cited herein are expressly
incorporated herein by reference for the purpose of describing and
disclosing compositions and methodologies that might be used in
connection with the invention. All cited patents, patent
applications, and sequence information in referenced websites and
public databases are also incorporated by reference.
EXAMPLES
Example 1
[0078] According to the methods and/or systems provided herein,
database and/or map data can be used for assessment or prediction
of transgene performance and/or stability by profiling the local
chromatin state, in particular the abundance of H3K9me2, in the
vicinity of the transgene-inserted locus. As provided here, a
threshold value can be assigned to predict the potential risk of
transgene silencing for transgenic integration occurring with
highly H3K9me2 enriched genomic regions. To assess the local
chromatin structure, a genome-wide histone3 lysine (K) 9
di-methylation (H3K9me2) map in soybean (Glycine max) cv Maverick
is constructed. H3K9me2 are often observed within the silenced
heterochromatic regions including centromere and telomere-carrying
repetitive sequences. This genome-wide H3K9me2 map allows us to
predict the chromatin structure (opened or packed) in any locus
within the soybean genome and provides an assessment on potential
for silencing.
[0079] Shoot and root tissues of soybean (Glycine max) cv Maverick
are harvested at v1 (.about.12 days after planting) stage, and then
cross-linked using 1% formaldehyde. The cross-linked tissues is
used for the chromatin immunoprecipitation (ChIP) assay using
H3K9me2 antibody (Millipore 17-648) where normal Rabbit IgG
(Millipore Cat. 12-370) is used as a negative control. Three
independent ChIP assays from each tissue are performed and the
ChIPed DNA is prepared for sequencing using HiSeq. The sequencing
reads are listed in Table 1.
TABLE-US-00001 TABLE 1 ChIP-seq reads from HiSeq Sample Total Read
Mapped Mapping Name Label Species Barcode # Read # Ratio x486_7
Shoot-1 (1) Soybean Index 5 76,305,548 57,296,302 75.09% (Gmax_109)
x486_8 Shoot-2 (2) Soybean Index 6 98,536,615 78,863,536 80.03%
(Gmax_109) x486_9 Shoot-2 (3) Soybean Index 12 90,263,804
74,632,245 82.68% (Gmax_109) x486_10 Root-3(1) Soybean Index 4
91,350,018 68,443,285 74.92% (Gmax_109) x486_11 Root-3(2) Soybean
Index 12 99,169,687 74,526,487 75.15% (Gmax_109) x486_12 Root-4(3)
Soybean Index 6 92,090,396 69,694,991 75.68% (Gmax_109)
[0080] ChIP-Seq data analysis--Raw sequencing data from the H3K9me2
ChIP-seq experiments are aligned using Bowtie to the Glycine max
Williams82 genome. The MACS (model-based analysis of ChIP-seq) peak
finding algorithm can be used to identify regions of H3K9me2
enrichment over background, where a "peak" represents a genomic
stretch that is predicted to be enriched for H3K9me2 as compared to
its genomic neighborhood. On the other hand, a "summit" represents
the genomic location that has the greatest enrichment within a
predicted "peak."
[0081] According to this example, ChIP-seq data is consistent among
technical replicates. To reduce the variation and noise resulted
from the ChIP-seq data, three technical replicates of ChIP-seq are
performed individually. To examine if the ChIP pull-downs from each
round are consistent and reflect the genuine H3K9me2 modification
in the soybean genome, the pull-down from each technical rep can be
compared. Among the three technical replicates, the ChIP-seq data
is consistent to each other, indicating that the pull-downs are
consistent from each round of ChIP experiment. Therefore, that the
CHIP-seq protocols used in this example are highly replicable
across root and shoot technical replicate samples.
Example 2
[0082] H3K9me2 enrichment is associated with heterochromatic
regions and gene silencing--H3K9me2 is associated with
heterochromatin (tightly packed chromatin structure) and one of the
highly conserved histone marks accounting for transcriptional
repression in eukaryotes. To determine whether H3K9me2 is
associated with heterochromatic region and gene silencing in
soybean, the enrichment of H3K9me2 within the genic region
(measured as the number of H3K9me2 "peaks" predicted within a genic
region--higher peak number represents greater extent of H3K9H3K9me2
enrichment) is investigated and compared with the corresponding
gene expression level (measured using RNAseq technology, in units
of normalized read counts). Herein, we observed that the genic
regions with zero or one peak of H3K9me2 show a significant range
of gene expression. On the other hand, the genic regions with more
than two peaks of H3K9me2 are associated relatively lower levels of
gene expression. This indicates that higher enrichment of H3K9me2
is highly associated with the lower levels of gene expression in
soybean.
[0083] In addition to understanding H3K9me2 content in genic
regions, we further identify higher levels of H3K9me2 in
transposable elements which are often associated with highly
repetitive and transcriptionally repressed regions within the
genome. We compare the distribution of H3K9me2 enrichment between
transposable elements and genes in soybean genome. On average, the
H3K9me2 enrichment of the majority of transposon elements is much
higher than regular genes. This data further demonstrates that the
enrichment of H3K9me2 is highly associated with the repressive
status of soybean genome.
[0084] To build on our understanding of how H3K9me2 abundance is
associated with gene expression, we categorize genes based on their
expression levels into five different expression categories--High,
Medium High, Medium, Medium Low, and Low--and then selected a
representative number of 500 genes from each category (see FIG. 1).
For each gene within each category, the H3K9me2 abundance across
the genic region was calculated. The genic region is then divided
into three sections--gene body, 2 kb upstream, and 1 kb downstream.
An average value for H3K9me2 abundance within each section is
plotted for each expression category in FIG. 1. High and Medium
high expression categories can be distinguished from Low and Medium
Low expression categories based on their H3K9me2 abundance within
all three genic sections. This provides a unique view and a
statistical model for how H3K9me2 data can be used to predict the
expression level for a particular transgene of interest. Together,
the data above indicated that H3K9me2 is a conserved repressive
histone mark in soybean as well and the abundance of H3K9me2 is
associated with heterochromatic region and transcriptional
repression in soybean. We conclude a threshold is link to
acceptable expression levels where the genic region has one peak of
H3K9me2 profiling data mapped to soybean genomes. According to this
example, a databased for the soybean genome is established based on
the H3K9me2 profiling data.
Example 3
[0085] Assessment of the H3K9me2 enrichment using the soybean lead
events to validate the prediction of expression levels based on the
H3K9me2 profiling data. To evaluate the practical use of applying
H3K9me2 map as a tool to assess the potential risk of transgene
silencing, nine soybean lead events from Enlist and Insect
Resistance constructs are selected and listed in Table 2. Because
the insertion sites of these lead events are already known, the
H3K9me2 enrichment profiles in the vicinity (5 kb) of
transgene-inserted site are evaluated to predict the risk of gene
silencing. Because all the lead events are selected based on stable
transgene expression and stability, it is expect that the transgene
is inserted in a locus with low H3K9me2 enrichment (peak no.
.ltoreq.1). In the 5 kb window of the transgene-inserted site among
nine distinct events, six events showed zero peak and three events
show one peak of H3K9me2. The low H3K9me2 enrichment is
corresponding to the stable transgene expression. This indicates
that H3K9me2 profiling provides an effective tool to predict the
risk of gene silencing.
TABLE-US-00002 TABLE 2 Enrichment of H3K9me2 in the 5kb vicinity of
transgene- inserted site of the commercial soybean events Soybean
events Chromosome Start Stop pDAB4468.04.16.1 Gm 04 46004353
46004354 pDAB4464.11.04.1 Gm 15 876318 876319 pDAB4472.16.06.1 Gm
08 8345349 8345350 pDAB9582.812.09.1 Gm 05 2810964 2810965
pDAB9582.814.19.1 Gm 02 10027415 10027416 pDAB9582.146.06.1 Gm 17
7737798 7737799 pDAB9582.140.11.1 Gm 18 57058706 57058707
pDAB9582.816.15.1 Gm 03 46300211 46300212 pDAB9582.816.12.1 Gm 04
48406612 48406613
* * * * *