CN111172158B - Glyphosate-resistant cotton transformation event KJC017 and application thereof - Google Patents

Abstract

The glyphosate-resistant cotton transformation event KJC017 is characterized in that a characteristic gene sequence comprises a transgenic expression cassette, and the nucleotide sequence of the transgenic expression cassette is SEQ ID NO. 9. The invention also provides a primer pair for detecting the glyphosate-resistant transformation event KJC017 of cotton and a detection method. The cotton of the glyphosate-resistant transformation event KJC017 can tolerate high-dose glyphosate, and boll formation and boll opening are not influenced, so that the control of field weeds is facilitated, and the cultivation efficiency and the yield are improved.

Description

Glyphosate-resistant cotton transformation event KJC017 and application thereof

Technical Field

The invention relates to the technical field of plant breeding, and mainly relates to a glyphosate-resistant cotton transformation event KJC017 and application thereof in cotton breeding.

Background

Cotton (upland cotton) is an important commercial crop in many parts of the world and biotechnological methods have been applied to this crop in order to produce cotton with desirable traits. This desirable trait is herbicide tolerance. Expression of the herbicide tolerance transgene in a plant can confer the plant with desirable herbicide tolerance traits, but expression of the transgene may be affected by the chromosomal location and genomic outcome of the transgene insertion. For example, it has been observed in plants that there is often variation in the level and pattern of expression of a transgene between individual events that differ in the chromosomal insertion site of the transgene but are otherwise identical. There may also be unwanted and/or wanted phenotypic or agronomic differences between events. Thus, in order to select events that have both the desired trait and the optimal phenotypic and agronomic characteristics needed to make them suitable for commercial purposes, it is often necessary to generate and analyze a large number of individual plant transformation events. Such a selection typically requires greenhouse and field trials of many events over many years, at multiple sites and under a variety of different conditions so that a large amount of agronomic, phenotypic and molecular data can be collected. The resulting data must then be analyzed by scientists and agriculturists for the purpose of selecting commercially suitable events. Such an event, once selected, may then be used to introgress the desired trait into other genetic backgrounds using plant breeding methods, and thereby produce a large number of different crop varieties that contain the desired trait and are suitably adapted to specific local growth conditions.

Disclosure of Invention

In the research process, the applicant finds a transgenic expression cassette which can enable cotton to obtain the glyphosate-resistant character, and transgenic cotton plants can still grow normally under the condition of high dose of glyphosate.

One aspect of the invention provides a cotton glyphosate-resistant transformation event KJC017, which is characterized in that a characteristic gene sequence comprises a transgenic expression cassette, and the nucleotide sequence of the transgenic expression cassette is SEQ ID NO. 9.

In one embodiment according to the invention, the signature gene sequence further comprises a 5 'linker sequence, the 5' linker sequence being selected from any one of SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5 or SEQ ID NO 7.

In one embodiment according to the invention, the signature gene sequence further comprises a 3 'linker sequence, the 3' linker sequence being selected from any one of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6 and SEQ ID NO 8.

In one embodiment according to the invention, the characteristic gene sequence is the gene sequence having the nucleotide sequence SEQ ID NO. 10 or the complement thereof.

In one embodiment according to the invention, the signature sequence is located on chromosome 5 of the D genome of tetraploid cotton.

Another aspect of the present invention is to provide a primer pair for detecting the above-mentioned cotton glyphosate resistant transformation event KJC017, the primer pair comprising a first primer pair consisting of SEQ ID NO:11 and SEQ ID NO:12 (P681: 5'-CCAGAATCCTTGTCAGATT-3' and D5-660F: 5'-AAAGAATAGGTATTG GGC-3') and/or a second primer pair specifically recognizing flanking sequences; the second primer pair consists of SEQ ID NO 13 and SEQ ID NO 14. FMV 1: 5'-ATAAAGGCAAAGATGCTTTTGGTAGGTG-3' and D5-R2: 5'-ATGCATGCAGCAATTCATTT-3' are provided.

The invention further provides a method for identifying the glyphosate-resistant cotton transformation event KJC017 in a cotton biological sample, which comprises the steps of (a) extracting a DNA sample from the cotton biological sample to be identified; (b) performing PCR amplification using the primer pair of claim 6 using the extracted DNA sample as a template; (c) the length of the PCR amplification product was compared to the length of the theoretical nucleotide sequence in the glyphosate resistant transformation event KJC 017. If the length of the nucleotide sequence of the amplification product obtained by amplification with the first primer pair is 1082bp, or the length of the nucleotide sequence of the amplification product obtained by amplification with the second primer is 891bp, the glyphosate-resistant conversion event KJC017 exists in the sample.

Further, the applications comprise cotton plants, cotton plant cells, seeds, progeny plants, hybrid plants, cotton oil and cotton protein with glyphosate-resistant transformation event KJC 017.

Another aspect of the present invention is to provide the use of the above-described transformation event KJC017 to improve glyphosate resistance in cotton, to perform cotton breeding or as a molecular marker.

Drawings

FIG. 1 shows the structure of a gene expression cassette in cotton glyphosate resistant gene transformation event KJC017 in the genome of a cotton plant containing event KJC 017.

Fig. 2 is a test of glyphosate resistance of cotton at the generation KJC 017T 1, wherein the left side is the non-transgenic new-land early 45 of the control group and the right side is the cotton at the generation T1 of the glyphosate resistance transformation event of the present invention.

FIG. 3 is a photograph showing the normal boll formation and boll opening of cotton of KJC017 resisting the glyphosate transformation event when glyphosate is sprayed in the boll opening period of 3 months in 2019.

FIG. 4 is a graph showing the results of glyphosate tolerance tests, wherein a Control (CK) is 45% of new land of a non-transgenic receptor, and the other 4 experimental strains with KJC017 event are sprayed with 1 x (840g ae/ha), 4 x, 8 x and 12 x glyphosate respectively at the 4-leaf stage of cotton, and 1 x glyphosate is sprayed on the control, and pictures are taken after 10 days of spraying.

Fig. 5 is a graph of the detection result of cotton glyphosate resistant transformation event KJC017, where M: trans 2K plus II with molecular weight standard of 8kb, 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp and 100bp from top to bottom;

lanes 1-7 show the amplification products (1082bp) of 7 individuals of KJC 017T 3 generation DNA as templates, lane 8 shows the amplification products of Xinluaozao 45DNA as a control template, and lane 9 shows the PCR product with ddH2O as a template;

fig. 6 is a graph of the detection result of cotton glyphosate resistant transformation event KJC017, where M: trans 2K plus II with molecular weight standard of 8kb, 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp and 100bp from top to bottom; lanes 1-7 show the amplification products (891bp) from 7 individuals of KJC 017T 3 generation as templates, lane 8 shows the amplification products from Xinluo Zao 45DNA as templates, and lane 9 shows the PCR product from ddH2O as templates.

Detailed Description

The following examples are intended to illustrate the present application but are not intended to limit the scope of the present application.

Specific embodiments of the present application will be described in more detail below. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the present application, but is made for the purpose of illustrating the general principles of the application and not for the purpose of limiting the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.

"primers" are typically highly purified isolated polynucleotides designed for use in specific annealing or hybridization methods involving thermal amplification. A pair of primers can be used in a thermal amplification, such as a Polymerase Chain Reaction (PCR), with a template DNA, such as a cotton genomic DNA sample, to produce amplicons, wherein the amplicons produced from such a reaction have DNA sequences corresponding to the sequence of the template DNA between the two sites where the primers hybridize to the template. As used herein, an "amplicon" is a fragment or fragment of DNA synthesized using amplification techniques. The amplicons of the invention comprise at least one nucleotide sequence set forth in SEQ ID NO: 1-10. A primer is typically designed to hybridize to a complementary target DNA strand to form a hybrid between the primer and the target DNA strand, and the presence of the primer is a recognition site for a polymerase to initiate primer extension (i.e., polymerization of additional nucleotides into an elongated nucleotide molecule) using the target DNA strand as a template. As used herein, a primer pair is intended to refer to the use of two primers that bind to opposite strands of a double-stranded nucleotide fragment for the purpose of linearly amplifying a polynucleotide fragment between positions targeted by the binding of the individual members of the primer pair, typically in a thermal amplification reaction or other conventional nucleic acid amplification method.

The term "isolated" herein means that a molecule is at least partially separated from other molecules with which it is normally associated in nature or state. In one embodiment, the term "isolated" refers to a DNA molecule that is at least partially separated from nucleic acids that normally adjoin the DNA molecule in a natural or native state. Thus, for example, a DNA molecule fused to a regulatory or coding sequence with which it is not normally associated is considered herein to be isolated as a result of recombinant technology. Such molecules are considered isolated even when integrated into the chromosome of the host cell or present in nucleic acid solution with other DNA molecules.

The DNA molecules disclosed in the present invention or fragments thereof can be isolated and manipulated using a number of methods well known to those skilled in the art. For example, PCR (polymerase chain reaction) techniques can be used to amplify a particular starting DNA molecule and/or to generate variants of the original molecule. The DNA molecule or fragment thereof may also be obtained by other techniques, for example by direct synthesis of the fragment using chemical means, as is commonly achieved by using an automated oligonucleotide synthesizer.

Example 1: transformation and KJC017 event selection of Cotton

This example describes the generation, analysis and selection of transformation event KJC 017.

Glyphosate tolerant transgenic cotton transformation event KJC017 was generated by agrobacterium mediated transformation of cotton using a plant transformation vector containing the expression cassette shown in figure 1. To generate the KJC017 event, new grand early 45 cotton material was used for plant transformation. Cotton cells are transformed and regenerated into whole cotton plants. Rooted plants with normal phenotypic characteristics were selected and transferred to soil for growth and further evaluation.

T0 plants were transferred to soil and T0 plants containing the T-DNA expression cassette were identified and selected by PCR. The T-DNA expression cassette contains the P-FMV tsf1 promoter, which comprises: the Figwort Mosaic Virus (FMV) enhancer, the arabidopsis thaliana transcription factor 1 promoter and its 5' UTR regulatory sequence; operably linked to a DNA molecule (CTP2) encoding the N-terminal chloroplast transit peptide of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Arabidopsis thaliana; operably linked to a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding a polypeptide from Agrobacterium tumefaciens CP 4; operably linked to the 3' UTR DNA molecule (T-E9) of the rbcs 2E 9 gene of pea;

herbicide screening is carried out for T1 generation at a field application rate (840g ae/ha) of 1 Xof glyphosate, and resistant single plants are selfed to collect seeds. The tolerance of the T2 generation was tested with 1 ×,4 ×, 8 ×, 12 × glyphosate, respectively. The most resistant transformation event KJC017 was screened. The T3 generation Taqman analysis CP4-EPSPS was a single copy insertion.

Example 2: characterization of the sequence of KJC017 events of transformation

Molecular DNA probes comprising the entire coding region of the plant expression cassette and its corresponding regulatory elements, promoters, introns and polyadenylation sequences are used. Analysis showed that KJC017 contained a single transgene DNA insert with one copy of the expression cassette. Hi-Tail PCR and DNA sequence analysis were performed to determine the insert-the 5 'and 3' junctions of the plant genome, confirm the organization of the elements within the insert (FIG. 1), and determine the complete DNA sequence of the insert in cotton plants containing transformation event KJC017 (provided herein as SEQ ID NO: 9). A cotton plant comprising in its genome the linked transgenic genetic element shown in figure 1 and resistant to glyphosate herbicide is an aspect of the invention.

SEQ1SEQ ID NO:1 is a 20 nucleotide sequence representing the 5' junction region of cotton genomic DNA with the integrated transgene expression cassette. SEQ ID NO:1 is located in SEQ ID NO: 661-680 nucleotide positions in 10.

SEQ ID NO:2 is a 20 nucleotide sequence representing the 3' junction region of cotton genomic DNA with the integrated transgene expression cassette. SEQ ID NO:2 is located in SEQ ID NO: nucleotide positions 4930-4949 of 10.

SEQ ID NO:3 is a 60 nucleotide sequence representing the 5' junction region of cotton genomic DNA with the integrated transgene expression cassette.

SEQ ID NO:4 is a 60 nucleotide sequence representing the 3' junction region of cotton genomic DNA with the integrated transgene expression cassette.

SEQ ID NO:5 is a 100 nucleotide sequence representing the 5' junction region of cotton genomic DNA and the integrated transgene expression cassette.

SEQ ID NO:6 is a 100 nucleotide sequence representing the 3' junction region of cotton genomic DNA and the integrated transgene expression cassette.

SEQ ID NO:7 is the 5' sequence of the insert DNA flanking KJC017 up to and including a part of the integrated transgene expression cassette.

SEQ ID NO:8 is the 3' sequence of the insert DNA flanking KJC017 up to and including a part of the integrated transgene expression cassette.

SEQ ID NO:9 is the sequence of the integrated transgene expression cassette, where the 1bp "G" is randomly introduced upon T-DNA integration.

SEQ ID NO:10 is a contiguous nucleotide sequence flanked by the 5 'sequence of the inserted DNA (SEQ ID NO: 7), the integrated transgene expression cassette (SEQ ID NO: 9), and the 3' sequence of the inserted DNA (SEQ ID NO: 8). SEQ ID NO:10 comprises SEQ ID NO: 1-9.

Example 3 Cotton transformation event KJC017 Screen identification

1. Testing the glyphosate-resistant transformation event KJC 017T 1 generation glyphosate of cotton:

test time: 12 months 2018, test site: the test control of the southern Binhain area is 45 of the non-transgenic receptor Xinluzao, 1 × glyphosate (41% glyphosate isopropylamine aqua, 840g ae/ha) is sprayed at the 4-leaf stage of cotton, the cotton is recorded and photographed after being sprayed for 10 days, and the growth condition of the cotton is tracked at the whole growth stage.

The field was sprayed with 1 × Glyphosate (840g ae/ha) for 10 days (2018.12).

And (3) test results: the results are shown in FIG. 2. When 1 Xglyphosate (840g ae/ha) is used in the 4-leaf stage, the KJC017 event leaves have no withered spots, the growing points are normal, and the leaves are green, which shows that the glyphosate-resistant plant has good resistance.

The transgenic cotton sprayed with glyphosate normally bolls and bolls, which shows that glyphosate has no influence on the growth of cotton in the whole growth period, as shown in fig. 3.

2. Transformation event KJC 017T 2 generation glyphosate tolerance test

Test time: year 2019, month 5, test site: a test base of Xin village company of Zhao quan Zhengzhen of Beijing cis-sense region is compared with a non-transgenic receptor Xin Lu Zao 45, and a KJC017 event is sprayed with 1 x (840g ae/ha), 4 x, 8 x and 12 x glyphosate at the 4-leaf stage of cotton, and is sprayed with 1 x glyphosate in a comparison manner, and the record and the picture are recorded after 10 days of spraying.

And (3) test results:

the results are shown in FIG. 4: spraying glyphosate with the concentration of 1 x (840g ae/ha), 4 x, 8 x and 12 x at the leaf stage of cotton 4, wherein the maximum 12 x (10080g ae/ha) at the leaf stage of a transformation event KJC 0174, and the new leaves, the old leaves and the growing points are normal, and the phytotoxicity index is 0; and the control new-terrestrial-early plants all die at 1 × concentration, which indicates that the transformation event KJC017 has good resistance to glyphosate and can tolerate glyphosate at 12 × concentration.

Example 4 Taqman analysis

Genomic DNA of cotton material containing transformation event KJC017 was extracted by a plant DNA extraction method commonly used in the art and dissolved in 50ul of water. Taking 1ul as template DNA, taking (SEQ ID NO:15) 5'-GGCAGCCTTCGTATCGGAG-3' and (SEQ ID NO:16) 5'-CTCGTGTCGGAAAACCCTGT-3' as primers and (SEQ ID NO:17) FAM-CAG GTCCATGAACTCCGGGAAGCTC-BHQ1 as a probe, and detecting the copy number of CP 4-EPSPS. Transformation event KJC017 was analyzed as a single copy.

Example 5 analysis of flanking sequences

The flanking sequences of the KJC017 event insert were obtained according to the Hi-tail pcr method of the reference (Liu Y G, et al, 2007). 3 specific primers were designed on the CP4-EPSPS gene, SP3(SEQ ID NO: 18): 5'-AACTCTCCGATACGAAGGCTGCCTGA-3', SP2(SEQ ID NO: 19): 5'-ACGATGGACTCCAGTCGGTGGACGATGCC ACGATGATCG-3', SP1(SEQ ID NO: 20): 5'-GATCACCGCATCGCCAT GAGCTT-3', degenerate primers as described in the literature (Liu Y G, Chen Y. high-efficiency thermal asymmetric PCR for amplification of unknown cloning sequences [ J ]. BioTechniques,2007,43(5): 649-656).

Hi-Tail PCR amplification system and amplification process

Table 1 amplification system:

table 2 amplification procedure:

the products of the Tail II PCR and Tail III PCR were analyzed by electrophoresis on a 1% agarose gel, and the band of greater than 1000bp in length and larger than the Tail II PCR product was selected for sequencing. LB of the T-DNA was found to be adjacent to the unknown sequence of 300bp by sequencing. Comparative analysis was performed using the published genomic sequence of cotton (Gossypium hirsutum) (https:// www.cottongen.org /) Gossypium hirsutum (AD1) TM-1genome ZJU-Improved v2.1_ a1, which is highly homologous to the sequence on chromosome D05.

Primers D5-660F and D5-R2 were designed upstream and downstream of the homologous sequence with reference to the genome sequence, and then KJC017 genome DNA was PCR amplified separately using D5-660F (SEQ ID NO:12) and P681(SEQ ID NO:11) (primers inserted into sequence T-E9), D5-R2(SEQ ID NO:14) and FMV1(SEQ ID NO:13) (primers inserted into sequence P-FMV) to obtain 1082bp and 891bp fragments. Sequencing to obtain cotton flanking DNA sequences joined to the inserted sequences.

Example 6 chromosomal mapping

According to the obtained cotton flanking sequences, by using a published cotton (Gossypium hirsutum) genome sequence (https:// www.cottongen.org /) Gossypium hirsutum (AD1) TM-1genome ZJU-Improved v2.1_ a1 for comparative analysis, the cotton flanking DNA sequences joined with the insertion sequences in the transformation event KJC017 are highly homologous with the sequences on the D05 chromosome, so that the integration sites of the exogenous DNA insertion sequences in the transformation event KJC017 are located on the 5 th group of chromosomes of the D genome of the receptor tetraploid cotton.

Example 7 detection of transformation event KJC017

The KJC017 event was detected by PCR amplification using DNA primer pair, annealing at 55 ℃ and extension at 72 ℃ for 1min with the DNA of the transformation event KJC017 as template and the primers P681(SEQ ID NO:11) and D5-660F (SEQ ID NO:12), and the Huada 2 x Super PCR Mix was amplified to obtain T-E9 to LB flanking sequence fragment 1082 bp. FMV1(SEQ ID NO:13) using KJC017 event DNA as a template: and D5-R2(SEQ ID NO: 14): annealing at 55 ℃ and extending at 72 ℃ for 1min as a primer, and amplifying by using Huada 2 Xsuper PCR Mix to obtain a P-FMV-RB flanking sequence fragment 891 bp.

PCR identification using the above primer pairs was performed using methods commonly used in the art. The results of the amplification of transformation event KJC017, new land early 45 cotton samples using primer pairs are shown in fig. 5 and 6, respectively.

Although the present application has been described in detail with respect to the general description and the specific examples, it will be apparent to those skilled in the art that certain changes and modifications may be made based on the present application. Accordingly, such modifications and improvements are intended to be within the scope of this invention as claimed.

Sequence listing

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ttacccaatc acaattcaaa tcgacataca tcttaaatac gcaaatcaac aagttcactc 2880

gtatatagat ccaccaacca acaatcaatc gaacaaatta aaatctcaaa acaagatcga 2940

tcagaatggg aaaatagttt catgaataaa ttatcgttta gaagcagtga aacgttgata 3000

ttaacaatcg ataaaatcta aacaacagat cagaattttc agaacgatca atcaacaaag 3060

agcatcgaat ccaacaatca tcgaaataat atgagcaacg atcagcagac gaaaaagaaa 3120

aagaacgatc taagggtacg ttaacgctta cctcagagat atcgcagaaa agtgtgtagc 3180

ggctagggtc aataagaacg ataaatggaa tcagcaacga gagtggtttt atttatagcg 3240

ttgaatccga aaccaatgtt agggtttaac ataaaataat atcattcttt aaattaccat 3300

tatgcccctg tttagccgtg aatactcaat acctcttggg ttggttgtgt tttatgaatg 3360

tttagtacta catgttacaa acatcattta atcacacctt ttaattaagg ttattcaagc 3420

aaatggttat acatgttcgt ctcccaaaaa tttcattgat tgtggaccat caatctatat 3480

gacactcttc tctagagtaa atatttgata tctcaatgaa tgcagattat caagagccat 3540

ttatatcatt tattcaaaca ccaaataaaa taatgaacaa gtaaagtata tattaacctc 3600

aaaatctcat gacaatggaa atggtttatt ataacaacca tatgtgtccc attccacgag 3660

caacctccct caagagagaa acttccagat ctcttcctta caaacctttg aggatgggag 3720

ttccttcttg gttttggcga taccaatttg aataaagtga tatggctcgt accttgttga 3780

ttgaacccaa tctggaatgc tgctaaatcc tgagctcaag ctaattcttt tgtggtcgtc 3840

actgcgttcg tcatacgcat tagtgagtgg gctgtcagga cagctctttt ccacgttatt 3900

ttgttcccca cttgtactag aggaatctgc tttatctttg caataaaggc aaagatgctt 3960

ttggtaggtg cgcctaacaa ttctgcacca ttcctttttt gtctggtccc cacaagccag 4020

ctgctcgatg ttgacaagat tactttcaaa gatgcccact aactttaagt cttcggtgga 4080

tgtctttttc tgaaacttac tgaccatgat gcatgtgctg gaacagtagt ttactttgat 4140

tgaagattct tcattgatct cctgtagctt ttggctaatg gtttggagac tctgtaccct 4200

gaccttgttg aggctttgga ctgagaatta agctgctaga ttgtcgtttc ccgccttcag 4260

tttaaacta 4269

<210> 10

<211> 5503

<212> DNA

<213> Artificial Sequence

<400> 10

aaagaatagg tattgggcta tacttaatga attaatatta aatataagat ttaaaaaaac 60

ccaaattaaa agaaatgtat tgttttcaat taataaaact aaaattaatt gagcatatag 120

aatacaaaaa aaaatcatat ataaattgat tatttttgct acatcataaa atataacatt 180

gttagtatat tggagtaatt cataaaacgt ttgacaagta ccaaatcgaa caaaaaaaga 240

gcaggtacca aattaggaaa aagagtcgaa ttgaagcact agattggacc cccaaaaagt 300

ttaagtatca acttaagaaa aagttgtcaa gtttaaatac cgaatattat attaaaccta 360

ttattttatt aaatttaaaa tatttattaa ttacattcat atatatatat atcactttaa 420

ttgattaatt agtgtatttg atctataaaa tagtaaatgc ttataattaa atatctattt 480

aacacgaatt tgtataatct atttttagtc ttgagttgaa ttccagctat atatatttat 540

ttataaattg gatgggtggt tttgtgtgaa ttttgttttg gaagccgata gatactagag 600

agtagttttc atttaatttt ggatattcgt taggtgtaaa ttaatgaagt gaagcaatgc 660

aaatattgta gatggcttca tgtccgggaa atctacatgg atcagcaatg agtatgatgg 720

tcaatatgga gaaaaagaaa gagtaattac caattttttt tcaattcaaa aatgtagatg 780

tccgcagcgt tattataaaa tgaaagtaca ttttgataaa acgacaaatt acgatccgtc 840

gtatttatag gcgaaagcaa taaacaaatt attctaattc ggaaatcttt atttcgacgt 900

gtctacattc acgtccaaat gggggcttag atgaggcgcg ccggtaccgt cgacattgat 960

gcatgttgtc aatcaattgg caagtcataa aatgcattaa aaaatatttt catactcaac 1020

tacaaatcca tgagtataac tataattata aagcaatgat tagaatctga caaggattct 1080

ggaaaattac ataaaggaaa gttcataaat gtctaaaaca caagaggaca tacttgtatt 1140

cagtaacatt tgcagctttt ctaggtctga aaatatattt gttgcctagt gaataagcat 1200

aatggtacaa ctacaagtgt tttactcctc atattaactt cggtcattag aggccacgat 1260

ttgacacatt tttactcaaa acaaaatgtt tgcatatctc ttataatttc aaattcaaca 1320

cacaacaaat aagagaaaaa acaaataata ttaatttgag aatgaacaaa aggaccatat 1380

cattcattaa ctcttctcca tccatttcca tttcacagtt cgatagcgaa aaccgaataa 1440

aaaacacagt aaattacaag cacaacaaat ggtacaagaa aaacagtttt cccaatgcca 1500

taatactcaa actcagtagg attctggtgt gtgcgcaatg aaactgatgc attgaacttg 1560

acgaacgttg tcgaaaccga tgatacgaac gaaagctgaa ttctcaggca gccttcgtat 1620

cggagagttc gatcttcgcg cccagcccgg ccatcaggtc catgaactcc gggaagctcg 1680

tggcgatcat cgtggcatcg tccaccgtga cagggttttc cgacacgagg cccatgacga 1740

ggaagctcat ggcgatgcgg tgatcgagat gggtggcgac ggcggcgccc gaggcgttgc 1800

cgagcccctt gccgtcaggg cggccacgca cgacgagcga cgtctcgccc tcatcgcaat 1860

ccacgccatt gagcttgagg ccattggcga cggccgagag gcggtcgctt tccttgacgc 1920

ggagttcttc cagaccgttc atcacggtcg ccccttccgc gaaggcggcg gcgacagcga 1980

gaatcggata ttcgtcgatc atcgaaggcg cgcggtcttc cggcaccgtg acgcccttca 2040

gcgtggagga gcgaacgcgc aggtccgcca cgtcttcgcc gccggcaagg cgcgggttga 2100

tgacttcgat gtcggcgccc atttcctgca gcgtcaggat gaggccggtg cgggtggggt 2160

tcatcagcac gttgaggatg gtgacgtcgg agcccggaac aagcagggcc gcaaccagcg 2220

ggaaggccgt cgaggacggg tcgcccggca cgtcgatgac ttggccggtg agcttgccgc 2280

ggccttccag gcggatggtg cgcacgccgt ccgcatccgt ctcgacggta aggttggcgc 2340

caaagccctg cagcatcttt tccgtatgat cgcgcgtcat gatcggctcg atgaccgtcg 2400

tgatgccggg cgtgttgagg ccggcgagca gcacggcgga cttcacctgt gcggaggcca 2460

tcggcacgcg gtaggtgatc ggcgtcggcg tcttcggccc gcgcaaggta acgggaagac 2520

ggtcaccgtc ttccgatttc acctgcacgc ccatttcgcg cagcgggttc aacacgcggc 2580

ccatcgggcg ctttgtgagc gaggcgtcgc cgatgaaggt gctgtcgaaa tcgtagaccc 2640

cgacgaggcc catcgtcagg cggcagcccg tggcggcatt gccgaaatcg agcggcgcct 2700

caggcgccag gaggccgcca ttgccgacgc catcgatgat ccaggtgtcg ccttccttac 2760

ggatgcgggc gcccatcgcc tgcatggcct tgcccgtatt gatgacgtcc tcgccttcca 2820

gaaggccggt gatgcgcgtt tcaccgctcg cgagaccgcc gaacatgaag gaccggtggg 2880

agatcgactt gtcgccggga atgcggacgg ttccggaaag gccagaggat ttgcgggcgg 2940

ttgcgggccg gctgcttgca ccgtgaagca tgcacgccgt ggaaacagaa gacatgacct 3000

taagaggacg aagctcagag ccaattaacg tcatcccact cttcttcaat ccccacgacg 3060

acgaaatcgg ataagctcgt ggatgctgct gcgtcttcag agaaaccgat aagggagatt 3120

tgcgttgact ggatttcgag agattggaga taagagatgg gttctgcaca ccattgcaga 3180

ttctgctaac ttgcgccatg gtagttctct ggtcaacaaa tctgcaaaaa aaagaacaat 3240

caaatattga atcaataaaa acagtagaca tatataacca ttatgaaaat catctttaac 3300

acaaaacaat catagtaaaa agccgctatc ggagtcaaac cgatttctac cacaaaaatt 3360

gtggtgctct atcatctgag ctaagcgagc taaattcaat caaatcggca atttcaagag 3420

atataaagca aacaatccga gaattaccct tagaaaaagg gaacactgga tttgttatgc 3480

tactccagaa ttacccaatc acaattcaaa tcgacataca tcttaaatac gcaaatcaac 3540

aagttcactc gtatatagat ccaccaacca acaatcaatc gaacaaatta aaatctcaaa 3600

acaagatcga tcagaatggg aaaatagttt catgaataaa ttatcgttta gaagcagtga 3660

aacgttgata ttaacaatcg ataaaatcta aacaacagat cagaattttc agaacgatca 3720

atcaacaaag agcatcgaat ccaacaatca tcgaaataat atgagcaacg atcagcagac 3780

gaaaaagaaa aagaacgatc taagggtacg ttaacgctta cctcagagat atcgcagaaa 3840

agtgtgtagc ggctagggtc aataagaacg ataaatggaa tcagcaacga gagtggtttt 3900

atttatagcg ttgaatccga aaccaatgtt agggtttaac ataaaataat atcattcttt 3960

aaattaccat tatgcccctg tttagccgtg aatactcaat acctcttggg ttggttgtgt 4020

tttatgaatg tttagtacta catgttacaa acatcattta atcacacctt ttaattaagg 4080

ttattcaagc aaatggttat acatgttcgt ctcccaaaaa tttcattgat tgtggaccat 4140

caatctatat gacactcttc tctagagtaa atatttgata tctcaatgaa tgcagattat 4200

caagagccat ttatatcatt tattcaaaca ccaaataaaa taatgaacaa gtaaagtata 4260

tattaacctc aaaatctcat gacaatggaa atggtttatt ataacaacca tatgtgtccc 4320

attccacgag caacctccct caagagagaa acttccagat ctcttcctta caaacctttg 4380

aggatgggag ttccttcttg gttttggcga taccaatttg aataaagtga tatggctcgt 4440

accttgttga ttgaacccaa tctggaatgc tgctaaatcc tgagctcaag ctaattcttt 4500

tgtggtcgtc actgcgttcg tcatacgcat tagtgagtgg gctgtcagga cagctctttt 4560

ccacgttatt ttgttcccca cttgtactag aggaatctgc tttatctttg caataaaggc 4620

aaagatgctt ttggtaggtg cgcctaacaa ttctgcacca ttcctttttt gtctggtccc 4680

cacaagccag ctgctcgatg ttgacaagat tactttcaaa gatgcccact aactttaagt 4740

cttcggtgga tgtctttttc tgaaacttac tgaccatgat gcatgtgctg gaacagtagt 4800

ttactttgat tgaagattct tcattgatct cctgtagctt ttggctaatg gtttggagac 4860

tctgtaccct gaccttgttg aggctttgga ctgagaatta agctgctaga ttgtcgtttc 4920

ccgccttcag tttaaactaa gcaggcgttc cttattaact tatcctttct tttttctcat 4980

attaaccacc atgaccaaaa atgttcatgc tcattttctt tcattttact ttttatattt 5040

cctatcaacc accctcaatt tttctttact ttttattagt caaaatttaa attaaaaatt 5100

ttcaacaaaa atgaatatag aaaaaagaaa aaagaaccta aaatccctaa aaagtaaaac 5160

tcgtccattc ctctcgtgga atccatagtc atttttgacc gtcctaaatc cgaaagtctt 5220

aaattaagtt agaggtttat gatgtacaaa tagatgtgta aaattttaaa tttatgaaat 5280

gaaaacaagc acctagtagg tttttaaaga aaaatgtcaa aactcacttt agagagcatt 5340

aagatttaaa ttaaatattg agaatttttt atatataaaa aaaactagaa taagactttt 5400

aaaaccaaag tttttaatta acggaaattg ttaacctcac attaattttt tttatttact 5460

caattttaaa tattataaag acgaaatgaa ttgctgcatg cat 5503

<210> 11

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 11

ccagaatcct tgtcagatt 19

<210> 12

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 12

aaagaatagg tattgggc 18

<210> 13

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 13

ataaaggcaa agatgctttt ggtaggtg 28

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 14

atgcatgcag caattcattt 20

<210> 15

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 15

ggcagccttc gtatcggag 19

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 16

ctcgtgtcgg aaaaccctgt 20

<210> 17

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 17

caggtccatg aactccggga agctc 25

<210> 18

<211> 26

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 18

aactctccga tacgaaggct gcctga 26

<210> 19

<211> 39

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 19

acgatggact ccagtcggtg gacgatgcca cgatgatcg 39

<210> 20

<211> 23

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 20

gatcaccgca tcgccatgag ctt 23

Claims (6)

1. A characteristic gene sequence of glyphosate-resistant cotton transformation event KJC017 is characterized in that the nucleotide sequence of the characteristic gene sequence is the gene sequence of SEQ ID NO. 10 or the complementary sequence thereof.

2. The glyphosate resistant cotton transformation event KJC017 signature gene sequence according to claim 1 characterized in that said signature sequence is located on the D genome group 5 chromosome of tetraploid cotton.

3. Primer pair for detecting a characteristic gene sequence of glyphosate resistant cotton transformation event KJC017 according to claim 1 or 2, characterized in that the primer pair comprises a first primer pair and/or a second primer pair specifically recognizing flanking sequences, the first primer pair consisting of SEQ ID No. 11 and SEQ ID No. 12; the second primer pair consists of SEQ ID NO 13 and SEQ ID NO 14.

4. Use of the gene sequence characteristic of the glyphosate resistant cotton transformation event KJC017 of claims 1 or 2 for improving the glyphosate resistant property of cotton, breeding cotton or as a molecular marker.

5. The use of claim 4, wherein the genetic material harboring said glyphosate resistant transformation event KJC017 comprises cotton plants, cotton plant cells, seeds, progeny plants, hybrid plants, cotton oil, cotton protein.

6. A method for identifying a characteristic gene sequence of a cotton glyphosate-resistant transformation event KJC017 in a cotton biological sample comprises the steps of (a) extracting a DNA sample from the cotton biological sample to be identified; (b) using the extracted DNA sample as a template and using the primer pair of claim 3 to carry out PCR amplification, (c) detecting the length of the PCR amplification product and comparing the length with the length of a theoretical nucleotide sequence in the glyphosate-resistant transformation event KJC 017.

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