U.S. patent application number 12/767490 was filed with the patent office on 2010-10-14 for corn event pv-zmir13 (mon863) plants and compositions and methods for detection thereof.
Invention is credited to Tracey A. Cavato, Timothy R. Coombe, Scott C. Johnson.
Application Number | 20100260729 12/767490 |
Document ID | / |
Family ID | 31188566 |
Filed Date | 2010-10-14 |
United States Patent
Application |
20100260729 |
Kind Code |
A1 |
Cavato; Tracey A. ; et
al. |
October 14, 2010 |
Corn Event PV-ZMIR13 (MON863) Plants and Compositions and Methods
for Detection Thereof
Abstract
The present invention provides compositions and methods for
detecting the presence of the corn event MON863 DNA inserted into
the corn genome from the transformation of the recombinant
construct containing a Cry3Bb gene and of genomic sequences
flanking the insertion site. The present invention also provides
the corn event MON863 plants, progeny and seeds thereof that
contain the corn event MON863 DNA.
Inventors: |
Cavato; Tracey A.; (St.
Charles, MO) ; Coombe; Timothy R.; (Ellisville,
MO) ; Johnson; Scott C.; (Wildwood, MO) |
Correspondence
Address: |
HOWREY LLP
C/O IP DOCKETING DEPARTMENT, 2941 FAIRVIEW PARK DRIVE SUITE 200
FALLS CHURCH
VA
22042
US
|
Family ID: |
31188566 |
Appl. No.: |
12/767490 |
Filed: |
April 26, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10523290 |
Oct 19, 2005 |
7705216 |
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PCT/US03/22860 |
Jul 23, 2003 |
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12767490 |
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60399279 |
Jul 29, 2002 |
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Current U.S.
Class: |
424/93.7 ;
426/601; 426/622; 435/412; 435/6.15; 536/24.3; 536/24.33; 800/265;
800/279; 800/302; 800/320.1 |
Current CPC
Class: |
Y02A 40/162 20180101;
C12N 15/8286 20130101; Y02A 40/146 20180101; C12Q 1/6895 20130101;
C07K 14/415 20130101; A01H 5/10 20130101 |
Class at
Publication: |
424/93.7 ;
800/302; 800/320.1; 800/265; 800/279; 536/24.3; 536/24.33; 435/6;
435/412; 426/622; 426/601 |
International
Class: |
A61K 36/899 20060101
A61K036/899; A01H 5/00 20060101 A01H005/00; A01H 1/02 20060101
A01H001/02; C12N 15/82 20060101 C12N015/82; C07H 21/04 20060101
C07H021/04; C12Q 1/68 20060101 C12Q001/68; C12N 5/10 20060101
C12N005/10; A23L 1/212 20060101 A23L001/212; A23D 9/00 20060101
A23D009/00 |
Claims
1-42. (canceled)
43. At least one DNA molecule of sufficient length of contiguous
nucleotides homologous or complementary to SEQ ID NO:3 or SEQ ID
NO:4 to function as a DNA primer or probe specific for corn event
MON863 or progeny thereof.
44. The at least one DNA molecule of claim 43, comprising a
nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, or the complement
thereof.
45. The at least one DNA molecule of claim 43, comprising a
nucleotide sequence of SEQ ID NO:3, SEQ ID NO:4, or the complement
thereof.
46. The at least one DNA molecule of claim 43, comprising a pair of
DNA molecules, said pair comprising a first DNA molecule of 11 or
more contiguous nucleotides of any portion of the transgene region
of SEQ ID NO:3 or the complement thereof, and a second DNA molecule
of a similar length of a 5' flanking corn genomic DNA region of SEQ
ID NO:3 or the complement thereof.
47. The at least one DNA molecule of claim 46, wherein said first
DNA molecule comprises 11 or more contiguous nucleotides of the 5'
transgene portion of the DNA sequence of SEQ ID NO:7 or complement
thereof, and said second DNA molecule comprises a similar length of
5' flanking corn DNA sequence of SEQ ID NO:5 or complement
thereof.
48. The at least one DNA molecule of claim 47, wherein said first
DNA molecule comprises a sequence that is homologous or
complementary to SEQ ID NO:7, and said second DNA molecule
comprises a similar length of a sequence that is homologous or
complementary to SEQ ID NO:5.
49. The at least one DNA molecule of claim 46, wherein said first
DNA molecule comprises SEQ ID NO:10 and said second DNA molecule
comprises SEQ ID NO:9.
50. The at least one DNA molecule of claim 43, comprising a pair of
DNA molecules, said pair comprising a first DNA molecule of 11 or
more contiguous nucleotides of any portion of the transgene region
of SEQ ID NO:4 or the complement thereof, and a second DNA molecule
of a similar length of a 3' flanking corn genomic DNA region of SEQ
ID NO:4 or the complement thereof.
51. The at least one DNA molecule of claim 50, wherein said first
DNA molecule comprises 11 or more contiguous nucleotides of the 3'
transgene portion of the DNA sequence of SEQ ID NO:8, or the
complement thereof, and said second DNA molecule comprises a
similar length of 3' flanking corn DNA of SEQ ID NO:6, or the
complement thereof.
52. The at least one DNA molecule of claim 51, wherein said first
DNA molecule comprises a sequence that is homologous or
complementary to SEQ ID NO:8, and said second DNA molecule
comprises a similar length of a sequence that is homologous or
complementary to SEQ ID NO:6.
53. The at least one DNA molecule of claim 50, wherein said first
DNA molecule comprises SEQ ID NO:11 and said second DNA molecule
comprises SEQ ID NO:12.
54. A method of detecting the presence of a DNA molecule selected
from the group consisting of SEQ ID NO:3 and SEQ ID NO:4 in a
biological sample, the method comprising: (a) contacting said
biological sample with at least one DNA molecule according to any
one of claims 46-53; (b) providing a nucleic acid amplification
reaction condition; (c) performing said nucleic acid amplification
reaction, thereby producing a DNA amplicon molecule; and (d)
detecting said DNA amplicon molecule, wherein detection of an
amplicon comprising at least one of SEQ ID NO:1, SEQ ID NO:2 and
the complement thereof is indicative of the presence of a DNA
molecule selected from the group consisting of SEQ ID NO:3 and SEQ
ID NO:4 in said biological sample.
55. The method of claim 54, wherein said biological sample is a DNA
sample extracted from a corn plant and is selected from the group
consisting of corn flour, corn meal, corn syrup, corn oil, corn
starch, and cereals manufactured in whole or in part to contain
corn by-products.
56. A method of detecting the presence of a DNA molecule selected
from the group consisting of SEQ ID NO:3 and SEQ ID NO:4 in a
biological sample, the method comprising: (a) contacting said
biological sample with at least one DNA molecule according to any
one of claims 43-45; (b) subjecting said biological sample and at
least one DNA molecule to stringent hybridization conditions; and
(c) detecting hybridization of said at least one DNA molecule to
said biological sample, wherein detection of hybridization is
indicative of the presence of a DNA molecule selected from the
group consisting of SEQ ID NO:3 and SEQ ID NO:4 in said biological
sample.
57. The method of claim 56, wherein said biological sample is a DNA
sample extracted from a corn plant and is selected from the group
consisting of corn flour, corn meal, corn syrup, corn oil, corn
starch, and cereals manufactured in whole or in part to contain
corn by-products.
58. A DNA detection kit, comprising at least one DNA molecule
according to any one of claims 43 to 53.
59. A method of determining zygosity of DNA of a corn plant
comprising corn event MON863 in a biological sample comprising: (a)
contacting said sample with at least one DNA molecule according to
any one of claims 46 to 53, said at least one DNA molecule is a
primer set comprising SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:12,
or a primer set comprising SEQ ID NO:10, SEQ ID NO:11, and SEQ ID
NO:12, that (1) when used in a nucleic acid amplification reaction
comprising corn event MON863 DNA, produces a first amplicon that is
diagnostic for corn event MON863, and (2) when used in a nucleic
acid amplification reaction comprising corn genomic DNA other than
MON863 DNA, produces a second amplicon that is diagnostic for corn
genomic DNA other than MON863 DNA; (b) performing a nucleic acid
amplification reaction; and (c) detecting the amplicons so
produced, wherein detection of presence of both amplicons indicates
that said sample is heterozygous for corn event MON863 DNA, wherein
detection of only the first amplicon indicates that said sample is
homozygous for corn event MON863 DNA.
60. A transgenic corn plant cell comprising DNA encoding a Cry3Bb
and DNA having nucleotide sequences of SEQ ID NO:1 and SEQ ID
NO:2.
61. An insect resistant corn plant, or parts thereof, comprising
the transgenic corn plant cell of claim 60, wherein said plant, or
parts thereof, comprises said DNA encoding a Cry3Bb and said DNA
having nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:2.
62. Seed of the insect resistant corn plant of claim 61, wherein
said seed comprises said DNA encoding a Cry3Bb and said DNA having
nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:2.
63. Seed of corn plant designated MON863, having representative
sample deposited with the American Type Culture Collection (ATCC)
under accession number PTA-2605.
64. A corn plant MON863 or parts thereof produced by growing the
seed of claim 63.
65. A corn plant, seed, or parts thereof, comprising corn event
MON863.
66. A corn plant, seed, or parts thereof, capable of producing a
MON863 diagnostic amplicon.
67. The corn plant, seed, or parts thereof, of claim 66, wherein
said MON863 diagnostic amplicon comprises SEQ ID NO:1 or SEQ ID
NO:2.
68. A composition derived from the transgenic corn plant, or parts
thereof, as set forth in claim 61, wherein said composition
comprises a detectable amount of said DNA encoding a Cry3Bb and
said DNA having nucleotide sequences of SEQ ID NO:1 and SEQ ID
NO:2, and wherein said composition is a commodity product selected
from the group consisting of corn flour, corn meal, corn syrup,
corn oil, corn starch, popcorn, corn cakes, cereals containing corn
and corn by-products.
69. A method of producing an insect resistant corn plant
comprising: (a) sexually crossing a first insect resistant corn
plant MON863 having representative seed deposited with the American
Type Culture Collection (ATCC) under accession number PTA-2605 and
a second parent corn plant that lacks insect resistance, thereby
producing a plurality of progeny plants; and (b) selecting a
progeny plant that is insect resistant.
70. The method of claim 69, further comprising backcrossing the
progeny plant that is insect resistant to the second parent corn
plant, thereby producing a plant that is insect resistant.
71. A method of producing an insect resistant corn plant,
comprising: (a) crossing the corn plant of any one of claims 61 and
64 to 67 with another corn plant; and (b) selecting a progeny plant
that is insect resistant by analyzing for the presence of at least
one nucleotide sequence of SEQ ID NO:1 and SEQ ID NO:2.
72. The method of claim 71, wherein said selecting step (b)
includes subjecting the progeny plant to a nucleic acid
amplification reaction, wherein progeny plant that produces an
amplicon comprising at least one nucleotide sequence of SEQ ID NO:1
and SEQ ID NO:2 is selected, or subjecting the progeny plant to a
nucleic acid hybridization reaction, wherein progeny hybridizing to
a probe that hybridizes under stringent conditions with one or more
DNA sequence selected from SEQ ID NO:1 and SEQ ID NO:2 is
selected.
73. A method of producing an insect resistant corn plant,
comprising (a) transforming a corn plant cell with DNA encoding a
Cry3Bb and DNA having nucleotide sequences of SEQ ID NO:1 and SEQ
ID NO:2; and (b) regenerating a corn plant from said transformed
cell, wherein said corn plant comprises said DNA encoding a Cry3Bb
and said DNA having nucleotide sequences of SEQ ID NO:1 and SEQ ID
NO:2 and is insect resistant.
74. A method for protecting a corn plant from insect infestation,
comprising providing in the diet of a Coleopteran pest of corn an
insecticidally effective amount of cell(s) or tissue(s) of the corn
plant, or parts thereof, of any one of claims 61 and 64 to 67.
75. The method of claim 74, wherein said Coleopteran pest is
selected from the group consisting of Diabrotica vergifera,
Diabrotica undecimpunctata, and Leptinotarsa decemlineata.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of plant
molecular biology. The invention more specifically relates to a
coleopteran resistant corn plant (Zea mays) PV-ZMIR13, designated
MON863, and to seeds and progeny of the corn plant MON863. The corn
plant MON863 and its progeny are particularly resistant to
Diabrotica vergifera, Diabrotica undecimpunctata, and Leptinotarsa
decemlineata.
[0002] The present invention more specifically also relates to a
DNA construct inserted into the corn plant genome in event MON863
for conferring resistance to insect infestation by a coleopteran
species. The present invention also relates to assays for detecting
the presence of a corn plant MON863 DNA in a sample and
compositions thereof.
BACKGROUND OF THE INVENTION
[0003] Corn is an important crop and is a primary food source in
many areas of the world. The methods of biotechnology have been
applied to corn plants for improvement of the agronomic traits and
the quality of the product. Expression of foreign genes in plants
is known to be influenced by their chromosomal position, perhaps
due to chromatin structures (e.g., heterochromatin) or the
proximity of transcriptional regulation elements (e.g., enhancers)
close to the integration site (Weising et al., Ann. Rev. Genet
22:421-477, 1988). For this reason, it is often necessary to screen
a large number of events in order to identify an event
characterized by optimal expression of an introduced gene of
interest. For example, it has been observed in plants and in other
organisms that there may be a wide variation in levels of
expression of an introduced gene among events. There may also be
differences in spatial or temporal patterns of expression, for
example, differences in the relative expression of a transgene in
various plant tissues, that may not correspond to the patterns
expected from transcriptional regulatory elements present in the
introduced gene construct. For this reason, it is common to produce
hundreds to thousands of different events and screen those events
for a single event that has desired transgene expression levels and
patterns for commercial purposes. An event that has desired levels
or patterns of transgene expression is useful for introgressing the
transgene into other genetic backgrounds by sexual outcrossing
using conventional breeding methods. Progeny of such crosses
maintain the transgene expression characteristics of the original
transformant. This strategy is used to ensure reliable gene
expression in a number of varieties that are well adapted to local
growing conditions.
[0004] It would be advantageous to be able to detect the presence
of a particular event in order to determine whether progeny of a
sexual cross contain a transgene of interest. In addition, a method
for detecting a particular event would be helpful for complying
with regulations requiring the premarket approval and labeling of
food derived from recombinant crop plants, for example. It is
possible to detect the presence of a transgene by any well-known
nucleic acid detection method such as the polymerase chain reaction
(PCR) or DNA hybridization using nucleic acid probes. These
detection methods generally focus on frequently used genetic
elements, such as promoters, terminators, marker genes, etc. As a
result, such methods may not be useful for discriminating between
different events, particularly those produced using the same DNA
construct unless the sequence of chromosomal DNA adjacent to the
inserted DNA ("flanking DNA") is known. An event-specific PCR assay
is discussed, for example, by Windels et al. (Med. Fac. Landbouww,
Univ. Gent 64/5b: 459-462, 1999), who identified glyphosate
tolerant soybean event 40-3-2 by PCR using a primer set spanning
the junction between the insert and flanking DNA, specifically one
primer that included sequence from the insert and a second primer
that included sequence from flanking DNA.
SUMMARY OF THE INVENTION
[0005] According to one preferred embodiment of the present
invention, compositions and methods are provided for detecting the
presence of the transgene/genomic insertion region from a novel
corn plant PV-ZMIR13, designated MON863. DNA sequences are provided
that comprise at least one junction sequence of MON863 selected
from the group consisting of SEQ ID NO:1 (arbitrarily assigned 5'
end insert-to-genome junction) and SEQ ID NO:2 (arbitrarily
assigned 3' end insert-to-genome junction) and complements thereof,
wherein the junction sequence spans the junction between a
heterologous DNA inserted into the corn genome and the DNA from the
corn cell flanking the insertion site and is diagnostic for the
event.
[0006] According to another preferred embodiment of the present
invention, DNA sequences that comprise the novel transgene/genomic
insertion region, SEQ ID NO:3 (sequence containing the arbitrarily
assigned 5'end of the inserted DNA) and SEQ ID NO:4 (sequence
containing the arbitrarily assigned 3'end of the inserted DNA) for
example, are disclosed.
[0007] According to still another preferred embodiment of the
present invention, the DNA sequences that comprise at least from
about 11 to about 50 or more nucleotides of the 5' transgene
portion of the DNA sequence of SEQ ID NO:7 and a similar length of
5' flanking corn DNA sequence of SEQ ID NO:5, or a similar length
of 3' transgene portion of the DNA sequence of SEQ ID NO:8 and a
similar length of 3' flanking corn DNA of SEQ ID NO:6, for use as
DNA primers in DNA amplification methods are also disclosed in the
present invention. Amplicons produced using these primers are
diagnostic for corn event MON863. An amplicon produced by a first
DNA primer homologous or complementary to SEQ ID NO:7 coupled with
a second DNA primer homologous or complementary to SEQ ID NO:5,
when both are present together in a reaction mixture with corn
event MON863 DNA in a sample are an aspect of the present
invention. An amplicon produced by a third DNA primer homologous or
complementary to SEQ ID NO:8 coupled with a fourth DNA primer
homologous or complementary to SEQ ID NO:6, when both are present
together in a reaction mixture with corn event MON863 DNA in a
sample, are another aspect of the present invention. The corn plant
MON863 and progeny derived therefrom that contain these DNA
sequences used in a DNA amplification reaction to provide one or
more diagnostic amplicons are aspects of the invention.
[0008] According to yet another preferred embodiment of the present
invention, methods of detecting the presence of a DNA corresponding
to the corn event MON863 event in a sample are provided. Such
methods comprise the steps of: (a) contacting a biological sample
suspected of containing an event MON863 DNA with a primer pair
that, when used in a nucleic acid amplification reaction with said
DNA, produces an amplicon that is diagnostic for the corn event
MON863; (b) performing a nucleic acid amplification reaction,
thereby producing the amplicon; and (c) detecting the amplicon. The
amplicons specifically exemplified herein correspond to a first
amplicon of about 508 base pairs as set forth in SEQ ID NO:3 and a
second amplicon of about 584 base pairs as set forth in SEQ ID
NO:4, or longer or shorter amplicons, wherein said first amplicon
contains as least a nucleotide sequence corresponding to SEQ ID
NO:1 from about nucleotide 1 through about nucleotide 11 or from
about nucleotide 10 through about nucleotide 20 and said second
amplicon contains at least a nucleotide sequence corresponding to
SEQ ID NO:2 from about nucleotide 1 through about nucleotide 11 or
from about nucleotide 10 through about nucleotide 20.
[0009] According to yet another preferred embodiment of the present
invention, methods of detecting the presence of a DNA corresponding
to the MON863 event in a sample are provided. Such methods comprise
the steps of: (a) contacting a biological sample suspected of
containing an event MON863 DNA with a probe that hybridizes under
stringent hybridization conditions with said DNA and that does not
hybridize under stringent hybridization conditions with DNA from a
control corn plant that does not contain an inserted DNA derived
from pMON25097; (b) subjecting the sample and the probe to
stringent hybridization conditions; and (c) detecting hybridization
of the probe to the genomic DNA, wherein detection of probe binding
to said DNA is diagnostic for the presence of event MON863 DNA in
said sample.
[0010] According to a further preferred embodiment of the present
invention, there is provided a novel corn plant MON863, that
comprises DNA sequences comprising the novel transgene/genomic
insertion regions as set forth in SEQ ID NO:3 and SEQ ID NO:4. The
seeds of the plants of MON863, the progeny of the plants of MON863
and the methods for producing a corn plant by crossing the corn
plant MON863 with itself or with another corn plant are further
embodiments of the present invention.
[0011] The foregoing and other preferred embodiments of the present
invention will become more apparent from the following detailed
descriptions.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES
Description of Drawings
[0012] FIG. 1 illustrates a plant expression vector PV-ZMIR13, also
designated herein as pMON25097, from which the corn rootworm event
MON863 is generated through particle acceleration technology using
a Mlu I restriction fragment from about nucleotide position 149
through about nucleotide position 4840.
[0013] FIG. 2 is a graphical map illustrating the general
organization of the elements comprising the heterologous nucleic
acid sequences inserted into the corn event MON863 genome and
essentially sets forth the positions at which the inserted nucleic
acid sequences are linked to corn genomic DNA sequences designated
herein as corn genomic nucleic acid sequences which flank the ends
of the inserted heterologous DNA sequences; the corn event MON863
being characterized as follows: corn genomic DNA [1] flanking the
arbitrarily assigned 5' end of the full length primary functional
inserted DNA sequence is adjacent to a non-naturally occurring
CaMV35S AS4 promoter sequence [2] (P-CaMV.AS4, SEQ ID NO:17)
operably connected to a wheat chlorophyll A/B binding protein
untranslated leader sequence [3] (L-Ta.hcb1, SEQ ID NO:18) operably
connected to a rice actin intron sequence [4] (I-Os.Act1, SEQ ID
NO:19) operably connected to a non-naturally occurring sequence
encoding Cry3Bb variant protein [5] (SEQ ID NO:20) operably
connected to a wheat heat shock Hsp17 transcription termination and
polyadenylation sequence [6] (T-Ta.Hsp17, SEQ ID NO:21), and the
full-length primary functional inserted DNA sequence being flanked
by the corn genomic DNA at the arbitrarily assigned 3' end [7], in
which the junction between [1] and [2] ([8]) corresponds to SEQ ID
NO:1, and the junction between [6] and [7] ([9]) corresponds to SEQ
ID NO:2.
DESCRIPTION OF SEQUENCES
[0014] SEQ ID NO:1 corresponds to a junction sequence between corn
genome and inserted DNA that is diagnostic for the arbitrarily
assigned 5' end of the full-length primary functional inserted DNA
sequence in the corn event MON863.
[0015] SEQ ID NO:2 corresponds to a junction sequence between corn
genome and inserted DNA that is diagnostic for the arbitrarily
assigned 3' end of the full-length primary functional inserted DNA
sequence in the corn event MON863.
[0016] SEQ ID NO:3 corresponds to the sequences represented
substantially by [1] and [2] of FIG. 2.
[0017] SEQ ID NO:4 corresponds to the sequences represented
substantially by [6] and [7] of FIG. 2.
[0018] SEQ ID NO:5 corresponds to the partial corn genome DNA
sequence that is adjacent to and flanking the 5' end of the
arbitrarily assigned 5' end of the partial Cry3Bb DNA coding
sequence inserted in the corn event MON863.
[0019] SEQ ID NO:6 corresponds to the partial corn genome DNA
sequence that is adjacent to and flanking the 3' end of the
arbitrarily assigned 3' end of the partial Cry3Bb DNA coding
sequence inserted in the corn event MON863.
[0020] SEQ ID NO:7 corresponds to the sequence of the arbitrarily
assigned 5' end of the partial Cry3Bb DNA coding sequence inserted
in the corn event MON863.
[0021] SEQ ID NO:8 corresponds to the sequence of the arbitrarily
assigned 3' end of the partial Cry3Bb DNA coding sequence inserted
in the corn event MON863.
[0022] SEQ ID NO:9 corresponds to a 5' primer sequence (primer A)
complementary to a part of the corn genomic DNA sequence identified
as flanking the arbitrarily assigned 5' end of the full length
primary functional inserted DNA sequence in the corn event MON863,
and when paired with a primer corresponding to the reverse
complement of the sequence set forth in SEQ ID NO:10 and template
DNA of the corn event MON863, produces an amplicon comprising SEQ
ID NO:3 that is diagnostic for the corn event MON863 DNA in a
sample.
[0023] SEQ ID NO:10 corresponds to the reverse complement of a 3'
primer sequence (primer B) complementary to a part of the
arbitrarily assigned 5' end sequence of the full length primary
functional DNA inserted into the corn genome in the corn event
MON863, and when paired with a primer corresponding to the sequence
set forth in SEQ ID NO:9 and template DNA of the corn event MON863,
produces an amplicon comprising SEQ ID NO:3 that is diagnostic for
the corn event MON863 DNA in a sample.
[0024] SEQ ID NO:11 corresponds to a 5' primer sequence (primer C)
complementary to part of the arbitrarily assigned 3' end sequence
of the full length primary functional DNA inserted into the corn
genome in the corn event MON863, and when paired with a primer
corresponding to the reverse complement of the sequence set forth
in SEQ ID NO:12 and template DNA of the corn event MON863, produces
an amplicon having SEQ ID NO:4 that is diagnostic for the corn
event MON863 DNA in a sample.
[0025] SEQ ID NO:12 corresponds to the reverse complement of a 3'
primer sequence (primer D) complementary to a part of the corn
genomic DNA sequence identified as flanking the arbitrarily
assigned 3' end of the full length primary functional inserted DNA
sequence in corn event MON863, and when paired with a primer
corresponding to the sequence set forth in SEQ ID NO:11 and the
template DNA of the corn event MON863, produces an amplicon having
SEQ ID NO:4 that is diagnostic for corn event MON863 DNA in a
sample.
[0026] SEQ ID NO:13 corresponds to a 5' genome walker primer 1.
[0027] SEQ ID NO:14 corresponds to a 5' genome walker primer 2.
[0028] SEQ ID NO:15 corresponds to a 3' genome walker primer 1.
[0029] SEQ ID NO:16 corresponds to a 3' genome walker primer 2.
[0030] SEQ ID NO:17 corresponds to CaMV35S AS4 promoter
sequence.
[0031] SEQ ID NO:18 corresponds to a wheat chlorophyll A/B binding
protein untranslated leader sequence (L-Ta.hcb1).
[0032] SEQ ID NO:19 corresponds to a rice actin intron sequence
(I-Os.Act1).
[0033] SEQ ID NO:20 corresponds to a non-naturally occurring
sequence encoding a Cry3Bb variant protein.
[0034] SEQ ID NO:21 corresponds to wheat heat shock Hsp17
transcription termination and polyadenylation sequence
(T-Ta.Hsp17).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0035] The following definitions and methods are provided to better
define the present invention and to guide those of ordinary skill
in the art in the practice of the present invention. Unless
otherwise noted, terms are to be understood according to
conventional usage by those of ordinary skill in the relevant art.
Definitions of common terms in molecular biology may also be found
in Rieger et al., Glossary of Genetics: Classical and Molecular,
5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V,
Oxford University Press: New York, 1994. The nomenclature for DNA
bases as set forth at 37 CFR .sctn.1.822 is used.
[0036] As used herein, the term "biological sample", or "sample",
is intended to include nucleic acids, polynucleotides, DNA, RNA,
tRNA, cDNA, and the like in a composition or fixed to a substrate
which enables the sample to be subjected to molecular probe
analysis or thermal amplification using oligonucleotide probes
and/or primers.
[0037] As used herein, the term "corn" means Zea mays or maize and
includes all plant varieties that can be bred with corn, including
wild maize species.
[0038] As used herein, the term "comprising" means "including but
not limited to".
[0039] As used herein, the term "diagnostic" refers to the fact
that, for the purposes of identifying nucleic acid sequences as
those contained within or derived from the corn event MON863, any
one or more of the novel DNA sequences set forth herein comprise
the corn genome flanking sequences adjacent to and linked to the
arbitrarily assigned ends of the inserted heterologous DNA
sequences are necessary and sufficient as being descriptive as a
distinguishing characteristic of the corn event MON863 genome, so
long as the sequence comprises at least a part of one of the ends
of the inserted heterologous DNA sequence or the corn genome
sequence flanking or adjacent to one of these ends and includes at
least the two nucleotides, the di-nucleotide, comprising the point
at which the corn genome sequence and the inserted heterologous DNA
sequence are linked together by a phosphodiester bond. It is well
known in the art that a sequence which is diagnostic for a
particular event, such as those disclosed herein for the corn event
MON863, which is not present in a particular sample containing corn
genome nucleic acids, is indicative that the sample does not
contain the diagnostic sequence and therefore the nucleic acids in
the sample are not or were not derived from and have not been
contained within the genome of the corn event MON863. In addition,
additional novel and diagnostic sequences are present within the
corn event MON863 DNA as exemplified herein selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID
NO:4 and complements thereof.
[0040] A transgenic "event" is produced by transformation of plant
cells with a heterologous DNA, i.e., a nucleic acid construct that
includes a transgene of interest, regeneration of a population of
plants resulting from the insertion of the transgene into the
genome of the plant, and selection of a particular plant
characterized by insertion into a particular genome location. The
term "event" refers to the original transformant and progeny of the
transformant that include the heterologous DNA. The term "event"
also refers to progeny produced by a sexual outcross between the
transformant and another variety that include the heterologous DNA.
Even after repeated backcrossing to a recurrent parent, the
inserted DNA and flanking DNA from the transformed parent is
present in the progeny of the cross at the same chromosomal
location. The term "event" also refers to DNA from the original
transformant comprising the inserted DNA and flanking genomic
sequence immediately adjacent to the inserted DNA that would be
expected to be transferred to a progeny that receives inserted DNA
including the transgene of interest as the result of a sexual cross
of one parental line that includes the inserted DNA (e.g., the
original transformant and progeny resulting from selfing) and a
parental line that does not contain the inserted DNA.
[0041] It is also to be understood that two different transgenic
plants can also be mated to produce offspring that contain two or
more independently segregating exogenous genes (exogenous genes
referring nucleotide sequences that are not naturally occurring in
the plant genome, i.e., heterogeneous to the corn plant). Selling
of appropriate progeny can produce plants that are homozygous for
any combination of the exogenous genes. Backcrossing to a parental
plant and out-crossing with a non-transgenic plant are also
contemplated, as is vegetative propagation. Descriptions of other
breeding methods that are commonly used for different traits and
crops can be found in one of several references, e.g., Fehr, in
Breeding Methods for Cultivar Development, Wilcox J. ed., American
Society of Agronomy, Madison Wis. (1987).
[0042] A "probe" is an isolated nucleic acid to which a
conventional detectable label or reporter molecule, e.g., a
radioactive isotope, ligand, chemiluminescent agent, or enzyme may
be linked or attached. Such a probe is complementary to a sequence
within a target nucleic acid, in the case of the present invention,
to a sequence of genomic DNA from the corn event MON863 whether
from a corn plant or from a sample that includes DNA from the
event. Probes according to the present invention include not only
deoxyribonucleic or ribonucleic acids but also polyamides and other
probe materials that bind specifically to a target DNA sequence and
can be used to detect the presence of that target DNA sequence.
[0043] "Primers" are isolated nucleic acid probes that are annealed
to, for any given single primer, a complementary target DNA
sequence by nucleic acid hybridization to form a hybrid between the
primer and the target DNA sequence, and then extended along the
target DNA strand by a polymerase, e.g., a DNA polymerase. Primer
pairs of the present invention refer to two or more different
primer sequences for is in amplification of a nucleic acid sequence
that is between and linked to the target sequences designated as
the reverse complement or substantially the reverse complement of
the primers, e.g., by the polymerase chain reaction (PCR) or other
conventional nucleic-acid amplification methods.
[0044] Probes and primers are generally from about 11 nucleotides
or more in length, preferably from about 18 nucleotides or more in
length, more preferably from about 24 nucleotides or more in
length, and most preferably from about 30 nucleotides or more in
length. Such probes and primers hybridize specifically to a target
sequence under high stringency hybridization conditions.
Preferably, probes and primers according to the present invention
have complete sequence similarity with the target sequence,
although probes differing from the target sequence and that retain
the ability to hybridize to target sequences may be designed by
conventional methods.
[0045] Methods for preparing and using probes and primers are
described, for example, in Molecular Cloning: A Laboratory Manual,
2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989 (hereinafter,
"Sambrook et al., 1989"); Current Protocols in Molecular Biology,
ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New
York, 1992 (with periodic updates) (hereinafter, "Ausubel et al.,
1992"); and Innis et al., PCR Protocols: A Guide to Methods and
Applications, Academic Press: San Diego, 1990. PCR-primer pairs can
be derived from a known sequence, for example, by using computer
programs intended for that purpose such as Primer (Version 0.5,
.COPYRGT.1991, Whitehead Institute for Biomedical Research,
Cambridge, Mass.).
[0046] Primers and probes constructed based on the flanking DNA,
insert sequences, and junction sequences disclosed herein can be
used to confirm the presence of the disclosed sequences in a sample
by conventional methods, e.g., by re-cloning and sequencing such
sequences.
[0047] Any single nucleic acid probe or primer of the present
invention hybridizes under stringent conditions to a specific
target DNA sequence. Any conventional nucleic acid hybridization or
amplification method can be used to identify the presence of DNA
from a transgenic event in a sample. Nucleic acid molecules or
fragments thereof specifically hybridize to other nucleic acid
molecules under certain circumstances. As used herein, two
different nucleic acid molecules each comprising different
sequences, are said to specifically hybridize to one another if the
two molecules form an anti-parallel, double-stranded nucleic acid
structure. A nucleic acid molecule is said to be the "complement"
of another nucleic acid molecule if they exhibit complete
complementarity. As used herein, molecules are said to exhibit
"complete complementarity" when every nucleotide of one of the
molecules is complementary to a nucleotide of the other. Two
molecules are said to be "minimally complementary" if they can
hybridize to one another with sufficient stability to permit them
to remain annealed to one another under at least conventional
"low-stringency" conditions. Similarly, the molecules are said to
be "complementary" if they hybridize to one another with sufficient
stability to permit them to remain annealed to one another under
conventional "high-stringency" conditions. Conventional stringency
conditions are described by Sambrook et al., 1989, and by Haymes et
al. (In: Nucleic Acid Hybridization, A Practical Approach, IRL
Press, Washington, D.C., 1985). Departures from complete
complementarity are therefore permissible, as long as such
departures do not completely preclude the capacity of the molecules
to form a double-stranded structure. In order for a nucleic acid
molecule to serve as a primer or probe it needs only be
sufficiently complementary in sequence to be able to form a stable
double-stranded structure under the particular solvent and salt
concentrations employed.
[0048] The term "specific for (a target sequence)" indicates that a
probe or primer hybridizes under stringent hybridization conditions
only to the target sequence in a sample comprising the target
sequence, and that the hybridization is detectable.
[0049] As used herein, an "isolated" nucleic acid is one that has
been substantially separated or purified away from other nucleic
acid sequences in the cell of the organism in which the nucleic
acid naturally occurs, i.e., other chromosomal and extrachromosomal
DNA and RNA, by conventional nucleic acid-purification methods. The
term also embraces recombinant nucleic acids and chemically
synthesized nucleic acids.
[0050] As used herein, a "substantially homologous" sequence is a
nucleic acid sequence that specifically hybridizes to the
complement of the nucleic acid sequence to which it is being
compared, i.e., the target sequence, under high stringency
conditions. Appropriate stringency conditions which promote DNA
hybridization, for example, 6.0.times. sodium chloride/sodium
citrate (SSC) at about 45.degree. C., followed by a wash of
2.0.times.SSC at 50.degree. C., are known to those skilled in the
art or can be found in Current Protocols in Molecular Biology, John
Wiley & Sons, N.Y., 6.3.1-6.3.6., 1989. For example, the salt
concentration in the wash step can be selected from a low
stringency of about 2.0.times.SSC at 50.degree. C. to a high
stringency of about 0.2.times.SSC at 50.degree. C. In addition, the
temperature in the wash step can be increased from low stringency
conditions at room temperature, about 22.degree. C., to high
stringency conditions at about 65.degree. C. Both temperature and
salt may be varied, or either the temperature or the salt
concentration may be held constant while the other variable is
changed. In a preferred embodiment, a nucleic acid of the present
invention will specifically hybridize to one or more of the nucleic
acid molecules set forth either in SEQ ID NO:1 or SEQ ID NO:2 or
complements thereof or fragments of either under moderately
stringent conditions, for example at about 2.0.times.SSC and about
65.degree. C. In a particularly preferred embodiment, a nucleic
acid of the present invention will specifically hybridize to one or
more of the nucleic acid molecules set forth either in SEQ ID NO:1
or SEQ ID NO:2 or complements or fragments of either under high
stringency conditions. A nucleic acid of the present invention that
hybridizes to a nucleic acid sequence comprising SEQ ID NO:1 or to
a nucleic acid sequence comprising SEQ ID NO:3 will not necessarily
hybridize to a nucleic acid sequence comprising SEQ ID NO:2 or to a
nucleic acid sequence comprising SEQ ID NO:4, and vice versa.
[0051] In one aspect of the present invention, a preferred marker
nucleic acid molecule of the present invention has the nucleic acid
sequence set forth in SEQ ID NO:1 or in SEQ ID NO:2 or complements
thereof or fragments of either. In another aspect of the present
invention, a preferred marker nucleic acid molecule of the present
invention shares between 80% and 100% or between 90% and 100%
sequence identity with the nucleic acid sequence set forth in SEQ
ID NO:1 and SEQ ID NO:2 or complement thereof or fragments of
either. In a further aspect of the present invention, a preferred
marker nucleic acid molecule of the present invention shares
between 95% and 100% sequence identity with the sequence set forth
in SEQ ID NO:1 and SEQ ID NO:2 or complement thereof or fragments
of either. SEQ ID NO:1 and SEQ ID NO:2 may be used as markers in
plant breeding methods to identify the progeny of genetic crosses
similar to the methods described for simple sequence repeat DNA
marker analysis, in "DNA markers: Protocols, Applications, and
Overviews, 173-185, Cregan, et al., eds., Wiley-Liss NY, 1997. The
hybridization of the probe to the target DNA molecule can be
detected by any number of methods known to those skilled in the
art, these can include, but are not limited to, fluorescent tags,
radioactive tags, antibody based tags, and chemiluminescent
tags.
[0052] Regarding the amplification of a target nucleic acid
sequence (e.g., by PCR) using a particular amplification primer
pair, "stringent conditions" are conditions that permit the
individual primers in a primer pair to hybridize only to the
individual and unique target nucleic-acid sequence to which each
primer, comprising the corresponding wild-type sequence (or its
complement), would bind, and preferably to produce a unique
amplification product, the amplicon, in a DNA thermal amplification
reaction.
[0053] As used herein, the term "transformation" refers to the
transfer of a nucleic acid fragment into the genome of a host
organism such as a host plant, resulting in genetically stable
inheritance. Host plants containing the transformed nucleic acid
fragments are referred to as "transgenic plants".
[0054] As used herein, "amplified DNA" or "amplicon" refers to the
product of nucleic-acid amplification of a target nucleic acid
sequence that is part of a nucleic acid template. For example, to
determine whether the corn plant resulting from a sexual cross
contains transgenic event genomic DNA from the corn plant MON863 of
the present invention, DNA extracted from a corn plant tissue
sample may be subjected to a nucleic acid amplification method
using a primer pair that includes a primer derived from the
flanking sequence in the genome of the plant adjacent to the
insertion site of the inserted heterologous DNA, and a second
primer derived from the inserted heterologous DNA to produce an
amplicon that is diagnostic for the presence of the event DNA. The
amplicon is of a length and has a sequence that is also diagnostic
for the event. The amplicon may range in length from the combined
length of the primer pairs plus one nucleotide base pair,
preferably plus about fifty nucleotide base pairs, more preferably
plus about two hundred-fifty nucleotide base pairs, and even more
preferably plus about four hundred-fifty nucleotide base pairs.
Alternatively, a primer pair can be derived from the flanking
sequence on both sides of the inserted DNA so as to produce an
amplicon that includes the entire insert nucleotide sequence. A
member of a primer pair derived from the plant genomic sequence may
be located in a distance from the inserted DNA sequence, this
distance can range from one nucleotide base pair up to about twenty
thousand nucleotide base pairs. The use of the term "amplicon"
specifically excludes primer dimers that may be formed in the DNA
thermal amplification reaction.
[0055] Nucleic-acid amplification can be accomplished by any of the
various nucleic-acid amplification methods known in the art,
including the polymerase chain reaction (PCR). A variety of
amplification methods are known in the art and are described, inter
alia, in U.S. Pat. Nos. 4,683,195 and 4,683,202 and in PCR
Protocols: A Guide to Methods and Applications, ed. Innis et al.,
Academic Press, San Diego, 1990. PCR amplification methods have
been developed to amplify up to 22 kb of genomic DNA and up to 42
kb of bacteriophage DNA (Cheng et al., Proc. Natl. Acad. Sci. USA
91:5695-5699, 1994). These methods as well as other methods known
in the art of DNA amplification may be used in the practice of the
present invention. The sequence of the heterologous DNA insert or
the flanking sequence from the corn event MON863 can be verified
(and corrected if necessary) by amplifying such sequences from the
event using primers derived from the sequences provided herein
followed by standard DNA sequencing of the PCR amplicon or of the
cloned DNA.
[0056] The amplicon produced by these methods may be detected by a
plurality of techniques. One such method is Genetic Bit Analysis
(Nikiforov, et al. Nucleic Acid Res. 22:4167-4175, 1994) where a
DNA oligonucleotide is designed which overlaps both the adjacent
flanking genomic DNA sequence and the inserted DNA sequence. The
oligonucleotide is immobilized in wells of a microwell plate.
Following PCR of the region of interest (using one primer in the
inserted sequence and one in the adjacent flanking genomic
sequence), a single-stranded PCR product can be hybridized to the
immobilized oligonucleotide and serve as a template for a single
base extension reaction using a DNA polymerase and labelled ddNTPs
specific for the expected next base. Readout may be fluorescent or
ELISA-based. A signal indicates presence of the insert/flanking
sequence due to successful amplification, hybridization, and single
base extension.
[0057] Another method is the Pyrosequencing technique as described
by Winge (Innov. Pharma. Tech. 00:18-24, 2000). In this method an
oligonucleotide is designed that overlaps the adjacent genomic DNA
and insert DNA junction. The oligonucleotide is hybridized to
single-stranded PCR product from the region of interest (one primer
in the inserted sequence and one in the flanking genomic sequence)
and incubated in the presence of a DNA polymerase, ATP,
sulfurylase, luciferase, apyrase, adenosine 5' phosphosulfate and
luciferin. DNTPs are added individually and the incorporation
results in a light signal that is measured. The light signal
indicates the presence of the transgene insert/flanking sequence
due to successful amplification, hybridization, and single or
multi-base extension.
[0058] Fluorescence Polarization as described by Chen, et al.,
(Genome Res. 9: 492-498, 1999) is a method that can be used to
detect the amplicon of the present invention. Using this method an
oligonucleotide is designed which overlaps the genomic flanking and
inserted DNA junction. The oligonucleotide is hybridized to
single-stranded PCR product from the region of interest (one primer
in the inserted DNA and one in the flanking genomic DNA sequence)
and incubated in the presence of a DNA polymerase and a
fluorescent-labeled ddNTP. A single base extension results in
incorporation of the ddNTP. Incorporation can be measured as a
change in polarization using a fluorometer. A change in
polarization indicates the presence of the transgene
insert/flanking sequence due to successful amplification,
hybridization, and single base extension.
[0059] Taqman.RTM. (PE Applied Biosystems, Foster City, Calif.) is
described as a method of detecting and quantifying the presence of
a DNA sequence and is fully understood in the instructions provided
by the manufacturer. Briefly, a FRET oligonucleotide probe is
designed which overlaps the genomic flanking and insert DNA
junction. The FRET probe and PCR primers (one primer in the insert
DNA sequence and one in the flanking genomic sequence) are cycled
in the presence of a thermostable polymerase and dNTPs.
Hybridization of the FRET probe results in cleavage and release of
the fluorescent moiety away from the quenching moiety on the FRET
probe. A fluorescent signal indicates the presence of the
flanking/transgene insert sequence due to successful amplification
and hybridization.
[0060] Molecular Beacons have been described for use in sequence
detection as described in Tyangi, et al., (Nature Biotech. 14:
303-308, 1996). Briefly, a FRET oligonucleotide probe is designed
that overlaps the flanking genomic and insert DNA junction. The
unique structure of the FRET probe results in it containing
secondary structure that keeps the fluorescent and quenching
moieties in close proximity. The FRET probe and PCR primers (one
primer in the insert DNA sequence and one in the flanking genomic
sequence) are cycled in the presence of a thermostable polymerase
and dNTPs. Following successful PCR amplification, hybridization of
the FRET probe to the target sequence results in the removal of the
probe secondary structure and spatial separation of the fluorescent
and quenching moieties. A fluorescent signal results. The
fluorescent signal indicates the presence of the flanking/transgene
insert sequence due to successful amplification and hybridization,
and is diagnostic for the corn event MON863 nucleic acid in a
sample.
[0061] All of the above methods can be modified to determine the
zygosity of a particular sample of nucleic acids derived from a
single source. For example, a corn event MON863 plant which is
homozygous for the event 863 allele contains within its genome two
copies of the event 863 allele characteristic of and diagnostic for
the corn event MON863 genome, and thus when selfed would breed
true. Alternatively, a corn event MON863 homozygous plant can be
crossed with another variety of corn, and the result of that cross
would be plants that were heterozygous for the event MON863 allele.
Methods are envisioned in which one skilled in the art could
determine the zygosity of a particular plant with reference to the
event MON863 allele.
[0062] For example, the use of three different primers in an
amplification reaction with corn event MON863 DNA as a template,
and in a separate and parallel amplification reaction with negative
control corn DNA that is not MON863, i.e., that does not contain
the inserted DNA present within MON863 DNA, would result in two
different outcomes depending on the zygosity of the corn DNA
containing the corn event MON863 DNA. Exemplary primers could be
selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10,
and SEQ ID NO:12. Amplification of non-MON863 DNA with this group
of primers would result in primer pair SEQ ID NO:10 and SEQ ID
NO:12 producing a first amplicon corresponding to the contiguous
corn genome sequence into which the PV-ZMIR13 sequence was
inserted, that amplified sequence corresponding substantially to
the linked combination of SEQ ID NO:5 and SEQ ID NO:6. This first
amplicon would be expected in a plant that was heterozygous for the
corn event MON863 allele, however, a heterozygote would also
produce a second amplicon corresponding to SEQ ID NO:3 from the
extension of the primer pair corresponding to SEQ ID NO:9 and SEQ
ID NO:10. A corn plant containing DNA that was homozygous for the
MON863 allele would only produce the second amplicon.
[0063] Similarly, a third amplicon would be produced from a thermal
amplification reaction that used the primers SEQ ID NO:10, SEQ ID
NO:11, and SEQ ID NO:12 with template DNA from a MON863 corn plant,
this third amplicon corresponding to SEQ ID NO:4. This third
amplicon would be the only amplicon produced using this particular
combination of primers and template DNA if the plant was homozygous
for the MON863 allele, however, heterozygote template DNA would
result in the amplification of the first and the third amplicons,
and non-MON863 template DNA would result in the amplification of
only the first amplicon.
[0064] Herein, the inventors have determined as judged by molecular
characterization that corn event MON863 contains a primary
functional insert containing a significant portion of the
transformation plasmid, PV-ZMIR13. This segment is detectable and
diagnostic for the event MON863 nucleic acid sequences in a sample,
in particular in plants that have been selfed since the origination
of the MON863 event.
[0065] There are many methods for transforming the Cry3Bb nucleic
acid molecules into plant cells such as maize plant cells to
produce a desired event such as MON863. Suitable methods are
believed to include virtually any methods by which nucleic acid
molecules may be introduced into the cells, such as by
Agrobacterium infection or direct delivery of nucleic acid
molecules that may include PEG-mediated transformation,
electroporation and acceleration of DNA coated particles, etc.
(Pottykus, Ann. Rev. Plant Physiol. Plant Mol. Biol. 42:205-225,
1991; Vasil, Plant Mol. Biol. 25: 925-937, 1994). For example,
electroporation has been used to transform Zea mays protoplasts
(Fromm et al., Nature 312:791-793, 1986). In general, the following
are four most commonly used general methods for delivering a gene
into cells: (1) chemical methods (Graham and van der Eb, Virology,
54:536-539, 1973); (2) physical methods such as microinjection
(Capecchi, Cell 22:479-488, 1980), electroporation (Wong and
Neumann, Biochem. Biophys. Res. Commun. 107:584-587, 1982; Fromm et
al., Proc. Natl. Acad. Sci. (USA) 82:5824-5828, 1985; U.S. Pat. No.
5,384,253); and the gene gun (Johnston and Tang, Methods Cell Biol.
43:353-365, 1994); (3) viral vectors (Clapp, Clin. Perinatol.
20:155-168, 1993; Lu et al., J. Exp. Med. 178:2089-2096, 1993;
Eglitis and Anderson, Biotechniques 6:608-614, 1988); and (4)
receptor-mediated mechanisms (Curiel et al., Hum. Gen. Ther. 3:
147-154, 1992; Wagner et al., Proc. Natl. Acad. Sci. (USA) 89:
6099-6103, 1992).
[0066] Transformation of plant protoplasts can be achieved using
methods based on calcium phosphate precipitation, polyethylene
glycol treatment, electroporation, and combinations of these
treatments. See for example (Potrykus et al., Mol. Gen. Genet.,
205:193-200, 1986; Lorz et al., Mol. Gen. Genet., 199:178, 1985;
Fromm et al., Nature, 319:791, 1986; Uchimiya et al., Mol. Gen.
Genet.: 204:204, 1986; Callis et al., Genes and Development, 1183,
1987; Marcotte et al., Nature, 335:454, 1988). Application of these
systems to different plant strains depends upon the ability to
regenerate that particular plant strain from protoplasts. Among
them are the methods for corn (U.S. Pat. No. 5,569,834, U.S. Pat.
No. 5,416,011; McCabe et al., Biotechnology 6:923, 1988; Christou
et al., Plant Physiol., 87:671-674, 1988). Illustrative methods for
the regeneration of cereals from protoplasts are also described
(Fujimura et al., Plant Tissue Culture Letters, 2:74, 1985;
Toriyama et al., Theor. Appl. Genet. 205:34, 1986; Yamada et al.,
Plant Cell Rep. 4: 85, 1986; Abdullah et al., Biotechnology,
4:1087, 1986).
[0067] A transgenic plant such as a transgenic corn MON863 plant
formed using transformation methods typically contains a single
added Cry3Bb gene on one chromosome. Such a transgenic plant can be
referred to as being heterozygous for the added Cry3Bb gene. More
preferred is a transgenic plant that is homozygous for the added
Cry3Bb gene; i.e., a transgenic plant that contains two added
Cry3Bb genes, one gene at the same locus on each chromosome of a
chromosome pair. A homozygous transgenic plant can be obtained by
sexually mating (selfing) an independent segregated transgenic
plant that contains a single added Cry3Bb gene, germinating some of
the seeds produced and analyzing the resulting plants produced for
the Cry3Bb gene.
[0068] It is understood that two different transgenic plants can
also be mated to produce offspring that contain two independently
segregating added Cry3Bb genes; Selfing of appropriate progeny can
produce plants that are homozygous for both added Cry3Bb genes that
encode Cry3Bb polypeptides. Backcrossing to a parental plant and
out-crossing with a non-transgenic plant are also contemplated, as
is vegetative propagation.
[0069] Specifically, a method for producing a corn plant that is
resistant to coleopteran insect infestation may be conducted with
the following steps: 1) sexually crossing a first corn plant grown
from the corn seed event MON863 comprising a DNA molecule selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,
SEQ ID NO:4 and SEQ ID NO:20 that confers resistance to coleopteran
insect infestation, and a second corn plant that lacks the
resistance to coleopteran insect infestation, thereby producing a
plurality of first progeny plants; 2) selecting a first progeny
plant that is resistant to coleopteran insect infestation; 3)
selfing said first progeny plant, thereby producing a plurality of
second progeny plants; and 4) selecting from said second progeny
plants a plant resistant to coleopteran insect infestation. The
first progeny plant that is resistant to coleopteran insect
infestation or the second progeny plant that is resistant to
coleopteran insect infestation may be backcrossed to the second
corn plant or a third corn plant resulting in a corn plant that is
resistant to coleopteran insect damage infestation.
[0070] The regeneration, development, and cultivation of plants
such as the MON863 plants from transformants or from various
transformed explants are well known in the art (Weissbach and
Weissbach, In: Methods for Plant Molecular Biology, Eds., Academic
Press, Inc. San Diego, Calif., 1988). This regeneration and growth
process may typically include the steps of selection of transformed
cells containing exogenous Cry3Bb genes, culturing those
individualized cells through the usual stages of embryonic
development through the rooted plantlet stage. Transgenic embryos
and seeds are similarly regenerated. The resulting transgenic
rooted shoots are thereafter planted in an appropriate plant growth
medium such as soil.
[0071] The regeneration of plants containing the foreign, exogenous
gene that encodes a protein of interest is well known in the art.
As described in the present invention, the regenerated plants such
as the regenerated MON863 plants that contain the Cry3Bb nucleic
acids, either wild type or chemically synthesized, that encode for
the Cry3Bb proteins, may be preferably self-pollinated to provide
homozygous transgenic maize plants, as discussed before. Otherwise,
pollen obtained from the regenerated maize plants may be crossed to
seed-grown plants of agronomically important lines. Conversely,
pollen from plants of these important lines is used to pollinate
regenerated plants. A transgenic MON863 plant of the present
invention may be cultivated using methods well known to one skilled
in the art.
[0072] There are a variety of methods for the regeneration of
plants from plant tissue. The particular method of regeneration
will depend on the starting plant tissue and the particular plant
species to be regenerated. Transformation of monocot plants using
electroporation, particle bombardment, and Agrobacterium has also
been reported. Transformation and plant regeneration have been
achieved in many monocot plants that include maize, asparagus,
barley and wheat, etc. (Bytebier et al., Proc. Natl. Acad. Sci. USA
84:5345, 1987; Wan and Lemaux, Plant Physiol 104:37, 1994; Rhodes
et al., Science 240: 204, 1988; Gordon-Kamm et al., Plant Cell,
2:603, 1990; Fromm et al., Bio/Technology 8:833, 1990; Armstrong et
al., Crop Science 35:550-557, 1995; Vasil et al., Bio/Technology
10:667, 1992; U.S. Pat. No. 5,631,152).
[0073] In addition to the above discussed procedures, practitioners
are familiar with the standard resource materials which describe
specific conditions and procedures for the construction,
manipulation and isolation of macromolecules (e.g., DNA molecules,
plasmids, etc.), generation of recombinant organisms and the
screening and isolating of clones (see, for example, Sambrook et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Press, 1989; Mailga et al., Methods in Plant Molecular Biology,
Cold Spring Harbor Press, 1995; Birren et al., Genome Analysis:
Analyzing DNA, 1, Cold Spring Harbor, N.Y., 1997).
[0074] DNA detection kits can be developed using the compositions
disclosed herein and the methods well known in the art of DNA
detection. The kits are useful for identification of corn event
MON863 DNA in a sample and can be applied to methods for breeding
corn plants containing the MON863 DNA. The kits contain one or more
DNA sequences comprising at least 11 contiguous nucleotides
homologous or complementary to sequences selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and complements thereof.
These DNA sequences can be used in DNA amplification reactions or
as probes in a DNA hybridization method.
[0075] The following examples are included to demonstrate examples
of certain preferred embodiments of the invention. It should be
appreciated by those of skill in the art that the techniques
disclosed in the examples that follow represent approaches the
inventors have found function well in the practice of the
invention, and thus can be considered to constitute examples of
preferred modes for its practice. However, those of skill in the
art should, in light of the present disclosure, appreciate that
many changes can be made in the specific embodiments that are
disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention.
EXAMPLES
Example 1
Isolation and Characterization of the DNA Sequences Flanking the
MON863 Insertion Event
[0076] Corn event MON863 was generated through particle
acceleration technology using a 4.7-Kb agarose gel-isolated Mlu I
restriction fragment from the plasmid vector PV-ZMIR13 (pMON25097,
FIG. 1). The plant expression vector pMON25097 contains a first
expression cassette comprising a non-naturally occurring CaMV35S
AS4 promoter sequence (P-CaMV.AS4, SEQ ID NO:17) operably connected
to a wheat chlorophyll A/B binding protein untranslated leader
sequence (L-Ta.hcb1, SEQ ID NO:18) operably connected to a rice
actin intron sequence (I-Os.Act1, SEQ ID NO:19) operably connected
to a non-naturally occurring sequence encoding Cry3Bb variant
protein (SEQ ID NO:20) operably connected to a wheat heat shock
Hsp17 transcription termination and polyadenylation sequence
(T-Ta.Hsp17, SEQ ID NO:21). The plant expression vector pMON25097
contains a second expression cassette linked to the Cry3Bb
expression cassette that confers paromomycin resistance to
transformed plant tissue (i.e. the 3' end of the cry3Bb expression
cassette is linked to the 5' end of the second expression cassette
conferring paromomycin resistance). This resistance cassette
consists of an enhanced CaMV35S promoter sequence (U.S. Pat. No.
5,164,316) that is operably connected to a neomycin
phosphotransferase coding sequence (U.S. Pat. No. 5,569,834) that
is operably connected to a nopaline synthase transcription
termination and polyadenylation sequence (Fraley et al. Proc. Natl.
Acad. Sci. USA 80:4803-4807, 1983). Transgenic corn plants
resistant to paromomycin were derived essentially as described in
U.S. Pat. No. 5,424,412.
[0077] Molecular characterization of the insert in the corn event
MON863 demonstrated that one copy of the DNA fragment used for
transformation is present in the corn event MON863. In order to
develop event-specific PCR identification methods, the sequences of
corn DNA flanking the 5' and 3' ends of the insert in the corn
event MON863 were determined using GenomeWalker.TM. technology
(Clontech Laboratories, Inc.) in accordance with the manufacturer's
instructions. The GenomeWalker.TM. involves first completely
digesting purified corn MON863 DNA with different restriction
enzymes provided in the GenomeWalker.TM. kit that leave blunt ends.
Next, the purified blunt-ended genomic DNA fragments are ligated to
GenomeWalker.TM. Adaptors comprising known nucleic acid fragments.
Each ligation is then amplified in a first PCR reaction using an
outer adaptor primer, SEQ ID NO:22 (5'-GTAATACGACTCACTATAGGGC-3')
provided by GenomeWalker.TM. and an outer, gene-specific primer
(SEQ ID NO:13, 5'-GAACGTCTTCTTTTTCCACGATGCTCC-3', and SEQ ID NO:15,
5'-GCGAGTCTGATGAGACATCTCTGTAT-3', for the 5' and 3' ends of the
transgene insert, respectively). The first PCR product mixture is
then diluted and used as a template for a secondary or nested PCR
with the nested adaptor primer, SEQ ID NO:23
(5'-ACTATAGGGCACGCGTGGT-3') provided by GenomeWalker.TM. and a
nested gene-specific primer (SEQ ID NO:14,
5'-TCGGCAGAGGCATCTTGAATGATAGC-3', and SEQ ID NO:16,
5'-AATTTGGTTGATGTGTGTGCGAGTTCT-3', for the 5' and 3' ends of the
transgene insert, respectively). The secondary PCR product, which
begins with the known gene-specific sequences and extends into the
unknown adjacent genomic DNA, can then be sequenced using methods
well known in the art. Once the flanking corn genomic sequences
were determined, PCR assays capable of detecting the presence of
corn plant PV-ZMIR13 (MON863) DNA in a sample were developed.
[0078] Following this procedure, the nucleotide sequence as set
forth in SEQ ID NO:5 was characterized as the corn genome sequence
that is immediately adjacent to and upstream of the arbitrarily
assigned 5' end of the pMON25097 DNA fragment that was inserted
into the corn genome resulting in the construction and isolation of
transgenic corn event MON863. One skilled in the art, or even one
of ordinary skill in the art, would realize that additional
nucleotide sequence information can readily be obtained that is
even more distal from the junction sequence as set forth in SEQ ID
NO:1 but still within the corn genome than the present 242
nucleotides exemplified herein in SEQ ID NO:5, and from nucleotide
position 267 through nucleotide position 508 as set forth in SEQ ID
NO:3. Also, the nucleotide sequence as set forth in SEQ ID NO:6 was
characterized as the corn genome sequence that is immediately
adjacent to and downstream of the arbitrarily assigned 3' end of
the pMON25097 DNA fragment that was inserted into the corn genome
resulting in the construction and isolation of transgenic corn
event MON863. One skilled in the art will also realize that
additional nucleotide sequence information can readily be obtained
that is even more distal from the junction sequence as set forth in
SEQ ID NO:2 but still within the corn genome than the present 224
nucleotides exemplified herein in SEQ ID NO:6, and from nucleotide
position 361 through nucleotide position 584 as set forth in SEQ ID
NO:4.
Example 2
Detection of the Presence of MON863 DNA in a Sample
[0079] The following provides a non-limiting example of the PCR
assays developed to detect the presence of the MON863 DNA in a
sample.
[0080] DNA was extracted from approximately 100 mg of ground grain
tissue using Qiagen's Dneasy Plant Mini Kit (catalog #68163,
Valencia, Calif.) according to the manufacturer's recommended
protocol with one exception. The grain used was processed prior to
extraction in a -80.degree. C. freezer, and not ground under liquid
nitrogen using a mortar and pestle immediately prior to extraction.
DNA quantitation was conducted using methods well-known in the art,
a Hoefer DNA Quant 200 Fluorometer, and Boehringer Mannheim
(Indianapolis, Ind.) molecular size marker IX as a DNA calibration
standard.
[0081] PCR analysis of the genomic DNA sequences flanking the 5'
end of the insert in MON863 was performed using one primer (primer
A) derived from the 5' genomic flanking sequence (SEQ ID
NO:9,5'-GTCTTGCGAAGGATAGTGGGAT-3') paired with a second primer
(primer B) located near the 5' end of the inserted DNA in the 35S
promoter (SEQ ID NO:10, 5'-CATATGACATAAGCGCTCTTGG-3'), covering a
508-bp region. The PCR analysis for genomic DNA sequences flanking
the 3' end of the MON863 insert was conducted using one primer
(primer D) derived from the 3' genomic flanking sequence (SEQ ID
NO:12, 5'-AGACTCTATGCTCTGCTCATAT-3') paired with a second primer
(primer C) located in the tahsp17 polyadenylation sequence near the
3' end of the insert spanning a 584-bp region (SEQ ID NO:11,
5'-CTGATCATTGGTGCTGAGTCCTT-3') (FIG. 2). The PCR analyses were
conducted using 50 ng of the corn event MON863 genomic DNA or a
MON846 non-transgenic genomic DNA template in a 50 .mu.L reaction
volume containing a final concentration of 1.5 mM Mg.sup.2+, 0.4
.mu.M of each primer, 200 .mu.M each dNTP, and 2.5 units of Taq DNA
polymerase. The reactions were performed under the following
cycling conditions: 1 cycle at 94.degree. C. for 3 minutes; 38
cycles of 94.degree. C. for 30 seconds, 60.degree. C. for 30
seconds, 72.degree. C. for 1.5 minutes; 1 cycle at 72.degree. C.
for 10 minutes.
[0082] The PCR products (20 .mu.L) of the expected sizes
representing the genomic sequence flanking the 5' and 3' ends of
the insert were isolated by gel electrophoresis on a 2.0% agarose
gel at 60 V for .about.1 hour and visualized by ethidium bromide
staining. The PCR fragments representing the 5' and 3' flanking
sequences were excised from the gel and purified using the QIAquick
Gel Extraction Kit (Qiagen, catalog #28704) following the procedure
supplied by the manufacturer. The purified PCR products were then
sequenced with the initial PCR primers using dye-terminator
chemistry.
[0083] The control reactions containing no template as well as the
reactions containing non-transgenic corn DNA did not generate a PCR
product with either primer set, as expected. PCR analysis of the
corn rootworm event MON863 DNA generated the expected size products
of 508 bp representing the 5' flanking sequence (SEQ ID NO:3) when
using primers A and B having SEQ ID NOs: 9 and 10 and 584 bp
representing the 3' flanking sequence (SEQ ID NO:4) when using
primers D and C having SEQ ID NOs: 11 and 12.
[0084] Sequence data indicated that the 5' amplicon, i.e., SEQ ID
NO:3, consisted of 266 bp of the 5' end of the 35S promoter at the
5' end of the insert followed by 242 bp of corn genomic flanking
DNA. Sequence data indicated that the 3' amplicon, i.e., SEQ ID
NO:4, consisted of 360 bp of the tahsp17 3' polyadenylation
sequence which defines the 3' end of the insert, immediately
followed by 224 bp of corn genomic flanking DNA.
[0085] Agronomically and commercially important products and/or
compositions of matter including but not limited to animal feed,
commodities, and corn products and by-products that are intended
for use as food for human consumption or for use in compositions
that are intended for human consumption including but not limited
to corn flour, corn meal, corn syrup, corn oil, corn starch,
popcorn, corn cakes, cereals containing corn and corn by-products,
and the like are intended to be within the scope of the present
invention if these products and compositions of matter contain
detectable amounts of the nucleotide sequences set forth herein as
being diagnostic for the corn event MON863.
[0086] Seed comprising the MON863 corn event have been deposited by
the Applicant with American Type Culture Collection (ATCC), 10801
University Boulevard, Manassas, Va., USA ZIP 20110-2209 on Oct. 17,
2000. The ATCC provided the Applicant with a deposit receipt,
assigning the ATCC Deposit Accession No. PTA-2605 to the corn Zea
mays event MON863 PV-ZMIR13.
[0087] Those of skill in the art, in light of these examples,
should appreciate that many changes can be made to the foregoing
assays to detect DNA derived from corn event MON863 in a sample.
For example, a primer set comprising one primer complementary to
corn genome DNA and another primer complementary to sequences
within the insert are envisioned. Furthermore, any of various
hybridization assays described earlier using DNA probes
complementary to the novel nucleic acid sequences located at
transgene/genome junctions are envisioned as well.
[0088] Having illustrated and described the principles of the
present invention, it should be apparent to persons skilled in the
art that the invention can be modified in arrangement and detail
without departing from such principles. We claim all modifications
that are within the spirit and scope of the appended claims.
[0089] All publications and published patent documents cited in
this specification are incorporated herein by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
Sequence CWU 1
1
21120DNAArtificial Sequence5' junction sequence 1agcttggtac
actttggggt 20220DNAArtificial Sequence3' junction sequence
2ggaattcggt ctccctatag 203508DNAArtificial Sequence5' genome +
insert sequence 3gtcttgcgaa ggatagtggg attgtgcgtc atcccttacg
tcagtggaag tatcacatca 60atccacttgc tttgaagacg tggttggaac gtcttctttt
tccacgatgc tcctcgtggg 120tgggggtcca tctttgggac cactgtcggc
agaggcatct tgaatgatag cctttccttt 180atcgcaatga tggcatttgt
aggtgccacc ttccttttct actgtccttt tgatgaagtg 240acaggtagga
tcggaaagct tggtacactt tggggtgaac acccatccga acaagtaggg
300tcaatagttc agcatttagg ccgtaacatt tagcaaaaaa ctaatcttaa
acccaacaag 360tgctctccga accaagctag atagtctcct atcactaggc
tcaccaacca acctggactt 420tgattctttc ttattattct aaccgggata
taaaaaccat aaggattgtt tccagccaag 480agttcccata tgacataagc gctcttgg
5084584DNAArtificial Sequence3' insert + genome sequence
4ctgatcattg gtgctgagtc cttcgtctcc aacgagaaga tctacatcga caagatcgag
60ttcatccccg tccagctgtg ataggaactc tgattgaatt ctgcatgcgt ttggacgtat
120gctcattcag gttggagcca atttggttga tgtgtgtgcg agttcttgcg
agtctgatga 180gacatctctg tattgtgttt ctttccccag tgttttctgt
acttgtgtaa tcggctaatc 240gccaacagat tcggcgatga ataaatgaga
aataaattgt tctgattttg agtgcaaaaa 300aaaaggaatt agatctgtgt
gtgttttttg gatccccggg gcggccgcgg ggaattcggt 360ctccctatag
agcagagcat agtgacaaaa gttccattta gatatggttg tatcatatgt
420atataaagaa tgtactcgca atgaactggc taagtccaac caaccatgat
ggcagcctgc 480ccccctatag ccaaagcaag cgatagcaaa tagtgatttt
atggagtaag cttcgctccg 540cgccaattag aaaaaagtga aaagactcta
tgctctgctc atat 5845242DNAcornDNA(1)..(242)5' corn genome sequence
5actttggggt gaacacccat ccgaacaagt agggtcaata gttcagcatt taggccgtaa
60catttagcaa aaaactaatc ttaaacccaa caagtgctct ccgaaccaag ctagatagtc
120tcctatcact aggctcacca accaacctgg actttgattc tttcttatta
ttctaaccgg 180gatataaaaa ccataaggat tgtttccagc caagagttcc
catatgacat aagcgctctt 240gg 2426224DNAcornDNA(1)..(224)3' corn
genome sequence 6ctccctatag agcagagcat agtgacaaaa gttccattta
gatatggttg tatcatatgt 60atataaagaa tgtactcgca atgaactggc taagtccaac
caaccatgat ggcagcctgc 120ccccctatag ccaaagcaag cgatagcaaa
tagtgatttt atggagtaag cttcgctccg 180cgccaattag aaaaaagtga
aaagactcta tgctctgctc atat 2247266DNAArtificial Sequence5' insert
sequence 7gtcttgcgaa ggatagtggg attgtgcgtc atcccttacg tcagtggaag
tatcacatca 60atccacttgc tttgaagacg tggttggaac gtcttctttt tccacgatgc
tcctcgtggg 120tgggggtcca tctttgggac cactgtcggc agaggcatct
tgaatgatag cctttccttt 180atcgcaatga tggcatttgt aggtgccacc
ttccttttct actgtccttt tgatgaagtg 240acaggtagga tcggaaagct tggtac
2668360DNAArtificial Sequence3' insert sequence 8ctgatcattg
gtgctgagtc cttcgtctcc aacgagaaga tctacatcga caagatcgag 60ttcatccccg
tccagctgtg ataggaactc tgattgaatt ctgcatgcgt ttggacgtat
120gctcattcag gttggagcca atttggttga tgtgtgtgcg agttcttgcg
agtctgatga 180gacatctctg tattgtgttt ctttccccag tgttttctgt
acttgtgtaa tcggctaatc 240gccaacagat tcggcgatga ataaatgaga
aataaattgt tctgattttg agtgcaaaaa 300aaaaggaatt agatctgtgt
gtgttttttg gatccccggg gcggccgcgg ggaattcggt 360922DNAArtificial
Sequence5' 5' primer 9gtcttgcgaa ggatagtggg at 221022DNAArtificial
Sequence5' 3' primer 10catatgacat aagcgctctt gg 221123DNAArtificial
Sequence3' 5' primer 11ctgatcattg gtgctgagtc ctt
231222DNAArtificial Sequence3' 3' primer 12agactctatg ctctgctcat at
221327DNAArtificial Sequence5' genome walker primer 1 13gaacgtcttc
tttttccacg atgctcc 271426DNAArtificial Sequence5' genome walker
primer 2 14tcggcagagg catcttgaat gatagc 261526DNAArtificial
Sequence3' genome walker primer 1 15gcgagtctga tgagacatct ctgtat
261627DNAArtificial Sequence3' genome walker primer 2 16aatttggttg
atgtgtgtgc gagttct 2717416DNACauliflower mosaic
virusDNA(1)..(416)CaMV35S AS4 promoter sequence 17ttctagagga
tcagcatggc gcccaccgtg atgatggcct cgtcggccac cgccgtcgct 60ccgttcctgg
ggctcaagtc caccgccagc ctccccgtcg cccgccgctc ctccagaagc
120ctcggcaacg tcagcaacgg cggaaggatc cggtgcatgc aggtaacaaa
tgcatcctag 180ctagtagttc tttgcattgc agcagctgca gctagcgagt
tagtaatagg aagggaactg 240atgatccatg catggactga tgtgtgttgc
ccatcccatc ccatcccatt tcccaaacga 300accgaaaaca ccgtactacg
tgcaggtgtg gccctacggc aacaagaagt tcgagacgct 360gtcgtacctg
ccgccgctgt cgaccggcgg gcgcatccgc tgcatgcagg ccatgg
4161875DNATriticum aestivumDNA(1)..(75)L-Ta.hcb1 untranslated
leader sequence 18ctagaaccat cttccacaca ctcaagccac actattggag
aacacacagg gacaacacac 60cataagatcc aaggg 7519804DNAOryza
sp.DNA(1)..(804)I-OS.Act1 rice actin intron sequence 19accgtcttcg
gtacgcgctc actccgccct ctgcctttgt tactgccacg tttctctgaa 60tgctctcttg
tgtggtgatt gctgagagtg gtttagctgg atctagaatt acactctgaa
120atcgtgttct gcctgtgctg attacttgcc gtcctttgta gcagcaaaat
atagggacat 180ggtagtacga aacgaagata gaacctacac agcaatacga
gaaatgtgta atttggtgct 240tagcggtatt tatttaagca catgttggtg
ttatagggca cttggattca gaagtttgct 300gttaatttag gcacaggctt
catactacat gggtcaatag tatagggatt catattatag 360gcgatactat
aataatttgt tcgtctgcag agcttattat ttgccaaaat tagatattcc
420tattctgttt ttgtttgtgt gctgttaaat tgttaacgcc tgaaggaata
aatataaatg 480acgaaatttt gatgtttatc tctgctcctt tattgtgacc
ataagtcaag atcagatgca 540cttgttttaa atattgttgt ctgaagaaat
aagtactgac agtattttga tgcattgatc 600tgcttgtttg ttgtaacaaa
atttaaaaat aaagagtttc ctttttgttg ctctccttac 660ctcctgatgg
tatctagtat ctaccaactg acactatatt gcttctcttt acatacgtat
720cttgctcgat gccttctccc tagtgttgac cagtgttact cacatagtct
ttgctcattt 780cattgtaatg cagataccaa gcgg 804201984DNAArtificial
Sequencenon-native Cry3Bb variant 11231mv1 20ccatggccaa ccccaacaat
cgctccgagc acgacacgat caaggtcacc cccaactccg 60agctccagac caaccacaac
cagtacccgc tggccgacaa ccccaactcc accctggaag 120agctgaacta
caaggagttc ctgcgcatga ccgaggactc ctccacggag gtcctggaca
180actccaccgt caaggacgcc gtcgggaccg gcatctccgt cgttgggcag
atcctgggcg 240tcgttggcgt ccccttcgca ggtgctctca cctccttcta
ccagtccttc ctgaacacca 300tctggccctc cgacgccgac ccctggaagg
ccttcatggc ccaagtcgaa gtcctgatcg 360acaagaagat cgaggagtac
gccaagtcca aggccctggc cgagctgcaa ggcctgcaaa 420acaacttcga
ggactacgtc aacgcgctga actcctggaa gaagacgcct ctgtccctgc
480gctccaagcg ctcccagggc cgcatccgcg agctgttctc ccaggccgag
tcccacttcc 540gcaactccat gccgtccttc gccgtctcca agttcgaggt
cctgttcctg cccacctacg 600cccaggctgc caacacccac ctcctgttgc
tgaaggacgc ccaggtcttc ggcgaggaat 660ggggctactc ctcggaggac
gtcgccgagt tctaccgtcg ccagctgaag ctgacccaac 720agtacaccga
ccactgcgtc aactggtaca acgtcggcct gaacggcctg aggggctcca
780cctacgacgc atgggtcaag ttcaaccgct tccgcaggga gatgaccctg
accgtcctgg 840acctgatcgt cctgttcccc ttctacgaca tccgcctgta
ctccaagggc gtcaagaccg 900agctgacccg cgacatcttc acggacccca
tcttcctgct cacgaccctc cagaagtacg 960gtcccacctt cctgtccatc
gagaactcca tccgcaagcc ccacctgttc gactacctcc 1020agggcatcga
gttccacacg cgcctgaggc caggctactt cggcaaggac tccttcaact
1080actggtccgg caactacgtc gagaccaggc cctccatcgg ctcctcgaag
acgatcacct 1140cccctttcta cggcgacaag tccaccgagc ccgtccagaa
gctgtccttc gacggccaga 1200aggtctaccg caccatcgcc aacaccgacg
tcgcggcttg gccgaacggc aaggtctacc 1260tgggcgtcac gaaggtcgac
ttctcccagt acgatgacca gaagaatgaa acctccaccc 1320agacctacga
ctccaagcgc aacaatggcc acgtctccgc ccaggactcc atcgaccagc
1380tgccgcctga gaccactgac gagcccctgg agaaggccta ctcccaccag
ctgaactacg 1440cggagtgctt cctgatgcaa gaccgcaggg gcaccatccc
cttcttcacc tggacccacc 1500gctccgtcga cttcttcaac accatcgacg
ccgagaagat cacccagctg cccgtggtca 1560aggcctacgc cctgtcctcg
ggtgcctcca tcattgaggg tccaggcttc accggtggca 1620acctgctgtt
cctgaaggag tcctcgaact ccatcgccaa gttcaaggtc accctgaact
1680ccgctgcctt gctgcaacgc taccgcgtcc gcatccgcta cgcctccacc
acgaacctgc 1740gcctgttcgt ccagaactcc aacaatgact tcctggtcat
ctacatcaac aagaccatga 1800acaaggacga tgacctgacc taccagacct
tcgacctcgc caccacgaac tccaacatgg 1860gcttctcggg cgacaagaat
gaactgatca ttggtgctga gtccttcgtc tccaatgaaa 1920agatctacat
cgacaagatc gagttcatcc ccgtccagct gtgataggaa ctctgattga 1980attc
198421234DNATriticum aestivumDNA(1)..(234)T-Ta.Hsp17 termination
and polyadenylation sequence 21aattctgcat gcgtttggac gtatgctcat
tcaggttgga gccaatttgg ttgatgtgtg 60tgcgagttct tgcgagtctg atgagacatc
tctgtattgt gtttctttcc ccagtgtttt 120ctgtacttgt gtaatcggct
aatcgccaac agattcggcg atgaataaat gagaaataaa 180ttgttctgat
tttgagtgca aaaaaaaagg aattagatct gtgtgtgttt tttg 234
* * * * *