CN104145019A

CN104145019A - Cotton plant event A26-5 and primer and method for use in detection thereof

Info

Publication number: CN104145019A
Application number: CN201280001629.7A
Authority: CN
Inventors: 崔洪志; 何云蔚; 王君丹; 陈文华; 王建胜; 宋辉平
Original assignee: Genesis Seed Industry Co ltd
Current assignee: Genesis Seed Industry Co ltd
Priority date: 2012-05-16
Filing date: 2012-05-16
Publication date: 2014-11-12
Also published as: IN2014MN02540A; WO2013170398A1

Abstract

Provided are bollworm-resistant cotton transformation event A26-5 and a characteristic sequence thereof, and, a primer and method for use in detecting the transformation event. Transformation event A26-5 is located on the eighth set of chromosomes of a cotton plant and comprises a combination of an inserted exogenous DNA sequence and a cotton genome DNA sequence. Also provided is a primer for specificity detection of event A26-5 by utilizing the inserted exogenous DNA sequence and a flanking sequence of a junction area on the cotton genome. The method for detecting event A26-5 can be used as a means to provide use of the event for breeding with convenient tracking of the specific gene insertion event. The A26-5 gene insertion event can serve as a molecular marker for use in increasing breeding efficiency.

Description

Cotton plant event A26-5 and primer and method for use in detection thereof

The invention belongs to field of plant molecular biology for vegetable lamb event A26- 5 and the primer for Qi Jian Measuring and method and technology field, transgenic crop breeding field especially in agricultural biotechnologies research, more particularly to there is the Cotton Transformation event A26-5 of bollworm resistance, and detect the peculiar method of the transformation event.Background technology

Worldwide, insect pest brings larger loss to agricultural production.The Prevention Technique of conventionally employed chemical insecticide, huge effect has been played in traditional agriculture practice.But, the drawbacks of this pest control technology has very big, on the one hand, the use of a large amount of toxic chemical agricultural chemicals not only pollutes environment, and easily remains, and seriously threatens health of people；On the other hand, chemical pesticide causes the outburst of insect pest for a long time largely using that can also cause the resistance to the action of a drug of insect.For example, at the beginning of the nineties in last century, due to the development of bollworm resistance, cause the great outburst of China bollworm, make the major cotton regions of China significantly underproduction or even total crop failure.

At present, transgenic technology causes this global problem of serious harm to provide feasible program to solve insect pest to Cotton Production.People prevent and treat certain class insect pest also by selection plantation insect-resistant transgenic crops.Bacillus thuringiensis (Bacillus thurigiensis, hereinafter referred to as Bt) is a kind of to form the Gram-positive bacillus of gemma, it is known that it can be produced to various insects, such as Lepidoptera（Lepidopterans), coleoptera（) and Diptera Coleopterans（) etc. Dipterans crop pest is virose with spore crystallization of protein (Aronson, Microbiol. Rev. 50: 1-14, 1968).So not poisoned to plant and animal including people, and it is environment acceptable due to the selectivity and high selectivity of Bt toxin desinsections.Therefore, anti insect gene used in the insect-resistant transgenic crops of current commercial applications is mainly Bt insecticidal protein genes.

Nineteen ninety-five, heap of Guo three et al. is optimized in codon, artificial synthesized GFM CrylA killing genes (ZL95119563.8), the main breed for being subsequently introduced several Wilt Pathogens cotton regions using plant, Insect Resistant Cotton is obtained, and commercial application has been carried out in 1997.The pest-resistant cotton transformation events of Μ Ο Π 531 that Monsanto Company obtains were commercialized in 19% year；The pest-resistant cotton transformation events of the double Bt of Monl5985 that subsequent Monsanto Company obtains again（Chinese Patent Application No. 02802047.2, in examining）, it is commercialized in the whole world.It is a current international new trend that intellectual property protection is carried out to transgenic crop by transformation event patent; because a commercialized transformation event of entrance is the unique creation for the biological species that inventor is obtained by Screening and Identification; the integration of foreign gene and acceptor crop gene group ad-hoc location; transformation event is not only set to possess the characterization of molecules of uniqueness, and tool There is the exogenous gene expression rule of uniqueness, be different from the other transformation events obtained in research process, the feature of commercial applications is more suitable for comparing, therefore with obvious novelty, practicality and creativeness.At present, existing commercialized transformation event obtains intellectual property protection in the world, is that trans-corporation is possessed, and has partly also applied for patent in China.

In the Insect Resistant Cotton research of China's early stage, due to converting the limitation of means, the Insect Resistant Cotton obtained, which exists, to be integrated copy number and integrates the uncertain situation in position, and transgene traits are not easy to hold in breeding, and the Insect Resistant Cotton variety resistance of different breeding men seed selection differs greatly.Simultaneously as the characterization of molecules of transformation event gene integration flanking sequence can not be obtained, further new transgene traits polymerization is not easy to, it is further applied and limitation occurs.Therefore, the invention goes out the new cotton transformation event of GFM CrylA killing genes, not only insect resistace is strong, inheritance stability, single copy is integrated, and integration Side wing acid molecules features understand, the transgenic line not only itself has significant application value in Insect Resistant Cotton breeding, and due to unique detection method, it is convenient to it polymerize different commercialization transformation events by way of hybridizing polymerization.Content of the invention the present inventor obtains Cotton Transformation event A26- 5 by transgenic method.The transformation event has stable high resistance helicoverpa armigera character.Its representative seed has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is： CGMCC No. 5966.

First aspect present invention provides Cotton Transformation event A26- 5, its characteristic DNA sequence such as SEQ ID No:Shown in 22, its by the 341st 6078 bp T- DNA insetion sequences, the 1st 340 bp upstream flank cotton gene group sequence and the 6079th 6606 bp downstream flank cotton gene group Sequence composition.

Second aspect of the present invention provides the fragment of the characteristic DNA sequence of the Cotton Transformation event described in first aspect present invention, and the fragment comprises at least the part T-DNA insetion sequences and part the flank cotton gene group sequence.

Third aspect present invention provides a kind of recombinant vector, and it contains the T- DNA insetion sequences described in first aspect present invention.In one embodiment, the carrier is the carriers of T66- 35S- OK-Bt- PS- Tnos- 2300 in accompanying drawing 1.

Fourth aspect present invention provides a kind of recombinant cell, and it contains the recombinant vector described in third aspect present invention.In one embodiment, the recombinant cell is the recombinational agrobacterium cell containing the carrier described in third aspect present invention.

Fifth aspect present invention provides the primer pair for examining the Cotton Transformation event described in Measuring first aspect present invention, its by the either side described in specific recognition first aspect present invention flanking sequence the first primer and specific recognition first aspect present invention described in the second primer of T-DNA insetion sequences constitute.In some embodiments, the sequence of first primer is SEQ ID NO:18 or SEQ ID N0:20, the sequence of second primer is SEQ ID NO:11 or SEQ ID NO: 14₀ In other embodiments, wherein the sequence of first primer is SEQ ID N0:19 or SEQ ID NO:21, the sequence of second primer is SEQ ID N0:12 or SEQ ID N0: 15.

Sixth aspect present invention provides a kind of method for identifying A26-5 transformation events in cotton biological sample, and it includes：

(a) from cotton extraction from biological material DM samples to be identified；

(b) using the DNA sample of extraction as template, enter performing PCR using the primer pair described in fifth aspect present invention and expand：

(c) pcr amplification product is detected, if amplified production length and SEQ ID NO:Theoretical length on 22 between the sequence of the PCR primer pair is consistent, then shows the presence of A26-5 transformation events in the cotton biological sample.

Seventh aspect present invention provides the method that Cotton Transformation event described in first aspect present invention is shifted into different cotton breeding materials, including：Using the cotton material containing the transformation event described in first aspect present invention, after being hybridized with other cotton breeding materials, further it is returned, obtains the new material containing the transformation event described in first aspect present invention；In hybridization and backcross process, Screening and Identification is carried out in progeny population using the method described in sixth aspect present invention, the presence of the transformation event described in first aspect present invention is confirmed.

Eighth aspect present invention, which provides transformation event, the fragment described in second aspect of the present invention, the carrier described in third aspect present invention or the recombinant cell described in fourth aspect present invention described in first aspect present invention, the method described in the aspect of the present invention the 6th and the 7th, to be used to improve cotton bollworm resisting character, carries out cotton breeding or the purposes as molecular labeling.Instep figure illustrates that Fig. 1 shows plant expression vector T66-35S-0K-Bt-PS-Tnos- 2300 structure flow.

Fig. 2 shows the result that the DNA of digoxigenin labeled of the Southern blot analytical techniques with being complementary to Bt gene coded sequences is hybridized.

Fig. 3 shows the experimental result detected using Southern hybridization techniques to the copy numbers of transformation event A26- 5.

1, CK+, T66-35S-0K-Bt-PS-Tnos-2300/EcoRI+Hi ndl 11； , marker , λ DNA/HindIII+EcoRI ;

2, A26-5/EcoRI+HindIII ;3, A26- 5/HindIII; 4, A26- 5/EcoRI.

Fig. 4 is right margin (RB) flanking sequence schematic diagram.

Fig. 5 is left margin (LB ＞ flanking sequence schematic diagrames.

Fig. 6 is shown using the DNA of acceptor material Ji cotton 14 as template, the use of sequence is respectively SEQ ID No:18 and SEQ ID No:19 primer pair, amplification obtains amplified production of the size for 900bp or so, the result compared after sequencing with A26-5 flanking sequence.

Fig. 7 is the insetion sequence of A26-5 events and the schematic diagram for identifying primer.

Fig. 8 show using primer pair GSPl-#/i III/A26-5-3 GSPl-ffind III/A26-5- and GSPl-EcoR I/A26- 5- 1, GSP1- EcoR I/A26- 5- 2 expand the strains of A26- 5, the strains of A2- 6, Ji cotton 14-1, the result of Ji cotton 14-2 cotton samples respectively. M, marker, λ DNA/HindIII+EcoRI 1-4：GSPl-ffind III/A26-5-3 expand A26- 5, A2- 6, Ji cotton 14-1, Ji cotton -2, the Kb of positive amplification band 3. 5 or so； 5-8_:GSP1- goes through '/D III/A26- 5- 4 expand A26-5, A2-6, Ji cotton 14-1, Ji cotton 14- 2, the 6Kp of positive amplification band 3. or so； 12-16：The GSP1- EcoR Ι/amplifications of Α 26- 5- 1 A26- 5, A2- 6, Ji cotton 14-1, Ji cotton 14- 2, the 6Kb of positive amplification band 2. or so； 17-21 :GSPl-EcoR I/A26- 5-2 expand A26- 5, A2- 6, Ji cotton 14- 1, Ji cotton 14-2, in the present invention, " transformation event " refers to external source target gene passing through Agrobacterium tumefaciens-mediated Transformation method the 7Kb of positive amplification band 2. or so embodiment（It is as well known to those skilled in the art）, it is transformed into cotton cells, and the event that ad-hoc location of the external source DM sequences in cotton gene group is inserted and integrated in the transgenic cotton plant further obtained;" transformation event " is not a kind of plant cell or plant, and plant cell or plant are the carriers that transformation event is present；The core feature of transformation event is one section of characteristic DNA sequence of the external source insetion sequence that foreign gene insertion of specific site in Plant Genome is formed and the connection of specific cotton gene group sequence.Embodiment spouts to go with reference to non-limiting example to the present invention to be further illustrated.The plant expression vector construction of embodiment 1.

The 35S promoter containing double enhancers is expanded from carrier pCambia2300, two ends are respectively with l a I and I, primer sequence such as SEQ ID No:L and SEQ ID No:Shown in 2, PCR primer cuts rear clone into pMD ~ 18T through ferment, obtains pMD- 35S recombinant vectors.In order to increase the recombination fraction of expression cassette, clone has the SSR sequence TMB0066 of a large amount of topoisomerase Π recognition sites from cotton gene, builds the plant expression vector using TMB0066 as AR sequences, increases the integration efficiency of insetion sequence.TMB0066 sequences such as SEQ ID No:Shown in 3.Its two ends introduces ^/xi Ι Π and site respectively, is cloned into pJH35S, obtains recombinant vector ρ Α β Η Γ Μ Β 0066- 35S.

OK (Omega ＆ Kozak)-Bt-PS (Processing ＆ Splicing sequence) fragment will be contained（Three's sequence is disclosed in the Chinese patent of Patent No. 95119563. 8, and the patent is disclosed to be included herein in full by reference）The plasmid of combination, using Banii i Sac I digestion O-Bt-PS fragments, is cloned into pMD- 18T, Obtain recombinant plasmid pMD-OK- Bt- PS.Terminator Tnos sequences are cloned into PS downstreams by Sac l+Ecd^ I.T B0066-35S are cloned into 35S upstreams using mnA III+feflH I, recombinant plasmid pMD-T B0066-35S-0K-Bt-PS-Tnos are obtained.Using Η π III and ＆o I, TMB0066-35S-0K- Bt-PS- Tnos are cloned into support C arabia2300, recombinant plant expression vector T66-35S-OK-Bt-PS-Tnos-2300 is obtained, flow is built and sees Fig. 1.The upland cotton of embodiment 2.（Gossypium hirsutum) genetic transformation-utilize agrobcterium-mediated transformation, with the carriers of T66-35S-0K-Bt PS- Tnos- 2300 conversion Ji cotton 14 (National Cotton mid-term storehouse, obtain unit Cotton research institute, Unified number：ZM-30270) hypocotyl.

Picking contains the Agrobacterium LBM404 of the carriers of T66-35S-0K-Bt-PS- Tnos- 2300, is seeded to containing kanamycins

(kanamycin, km) 50 mg/L rifampins (rifampicin, rif) in 50 mg/L and the mg/L of streptomysin (streptomycin, S/Sm) 50 LB fluid nutrient mediums, 28 vibration light cultures arrive bacterial growth logarithmic phase overnight.With bacterium solution：Culture medium 1: 50〜；B 100 ratio dilutes bacterium solution with LB or YEB fluid nutrient mediums, is followed by shaking culture 4 ~ 6 h, bacterium solution is diluted into 0D600 values 0. 8 1. 0.

Transgenic acceptor is Ji cotton 14, takes the aseptic seedling hypocotyl of growth 34 days, is cut into 0. 6 0. 8cm section, is contaminated】(Γ δ ι π η, take out hypocotyl section, put co-cultivation culture medium (8+ 0. 1_1¾/ 1^+2,4-1) 0. 1 more than 1), 22 °C ~ 25 °C co-culture 2 days.Go to calli induction media（The mg/L+Kan50mg/L of MSB+KT 0. 1 rag/L+2,4- DO. 1) 20 30 days subcultures once, callus proliferated culture medium is forwarded to after 90 days（The mg/L+Kan50mg/L of MSB+KT 0. 1 rag/L+2,4-D0. 05), 20 30 days subcultures once, after embryo callus subculture is grown, by embryo callus subculture subculture to germination medium（The rag/L+Kan50mg/L of MSB+KT 0. 1), the green bud that 40 days or so pickings are sprouted to root media（SH+KanSOmg/U is screened, and then obtains 268 plants of seedling and graftable resistant plant.Resistant plant grafting and is transplanted after PCR is identified, obtains serial totally 210 plants of the cottons of A.Screening and Identifications of the T0 of embodiment 3. for transgenic line：

Elisa detections and the Insect resistance assay of pest-resistant egg are carried out to seedling stage, florescence, flower bud phase and bell phase respectively.According to seedling stage

El isa detect that the transgene cotton of insect resistance protein height expression has 125 plants.By Insect resistance assay, resistant to bollworm has 94 plants, wherein stronger to bollworm resistance has 25 plants.Numbering PCR Elisa seedling Elisa flower buds Elisa flower Elisa earworm examination number of times insect resistaces

A6-2 P+ 0.323 0. 181 0. 21 0. 085 6 R

A7-2 P+ 0. 223 0. 321 0. 141 0. 129 6 R

A2-1 P+ 0. 162 0. 151 0. 245 0. 115 6 R

A1-1 P+ 0. 421 0.294 0. 207 0. 101 6 N Righteousness s/u P90 ssld

φ > σϊ σ¾ ςο ^ f to to c o^ oi oo oo m co oo csi L L C OQ LC ci σ¾ CD co CD CM σ¾

o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o

O O O O O O O O O O O O O O O o o o o o o o o o o o o o o o o o o o o 05 Ο 00 εΟ ιΟ ΐΛ ΙΛ Ο O¾ t_O 00 C Cs] -* tQ C^ O Oi OO GO <D iC L C O O O O o o o o o o o o o o o o o o o o o o o cM cNi o o esi —^ o o o o o o o o e i e^ M csi ea o c

v οsdΐ +—

A P25+-

：³^ ：^：^ ^； ^：^：^ ^：：^：^：^：^：^：^ ^；^：^：^：^：^ : ¾：2：：¾：：¾：：25：2：：2：0 5¾3 |50 5^ ：¾ ¾3；30；¾ !¾ !0 ¾3 5« » §§ ：2：：2；；2；：2：：2；：2：：2：

7 Shang 8

1 Α2Θ2Ι u/u/d piozss

Ά Οιιί Ge£

Ί A30-6 P+ 2. 084

P+:PCR positive plants.

Twig and shoot pest-N_:Non-resistant；_;It is resistant； HR/HR-:Stronger resistance combination Elisa results and Insect resistance assay result select the stronger material of 22 insect resistaces to carry out Southern analyses.For Southern analyses, and combine plant fertility situation, DNA is extracted from cotton tissue, digested with EcoRI, and hybridized using the DNA of Southern blot analytical techniques and the digoxigenin labeled for being complementary to Bt gene coded sequences, as a result further screened as shown in Fig. 2 filtering out 13 single copy cotton events.The Screening and Identification of the F1 generation of embodiment 4. or T1 for transgenic line：

Collect A19- 2, A2- 6, A3- 6, A3- 7, A6- 7, A7-6, A9- 7, A10- 5, All- 1, A19-5 A19-7,

A19-8, A20 ~ 8, A26- 5 the difference selfings of totally 14 cotton events and with other cotton culture kinds（LG6035、 LG6036

LG610K LG6118, LG6162) hybridization, selfed seed and cenospecies are harvested respectively.The seed of harvest is sowed, to T1 generations（Or F1 generation）Cotton plants, second true leaf in top is selected in 4 leaf phases, blade is carried out with 2000ppm kanamycins solution and smears screening, retains kanamycins and handles 7 days non-discoloring plant of rear blade, Elisa detections and Insect resistance assay are carried out respectively at seedling stage, flower bud phase, florescence.Insect resistance assay wins down two leaves respectively, connects worm 12, and a repetition is set per sample, in observing result after 5 days, is compared with compareing, and calculates corrected mortality.Calculation formula is as follows：

Average mortality（%)：Dead ^ ^ die rate² χ ΐ∞%

12x2

^ average mortalities-warship average mortality_ΛηηΜ

The calibration death rate (% }=X 100%

¹；100-to) average mortality

Plant numbers explanation：

F1 generation hybridization number format is " AXB- m "." A " is that, as maternal cotton material, " B " is the cotton material as male parent, and " m " numbers for individual plant；

T1 selfings number format is " A- ι η ", and " Α " is the cotton material of selfing, and " m " numbers for self progeny's individual plant, "（1) " represent without experiment repetition is carried out, be otherwise the average data of three repetitions；As a result it is as follows_:

(1) seedling stage result:

2) according to seedling worm result, choose the pest-resistant plant done very well and carry out flower bud phase Insect resistance assay, as a result as follows-

The result detected according to Tl I Fl for Insect resistance assay and Elisa, filters out 5 two the pest-resistant of transformation event of A2-6 and A26- and puts up the best performance.Continue further experimental analysis.The identification of the backcross progeny material of embodiment 5.

The purpose hybridized in previous embodiment 4 is that transformation event is transferred in other cotton breeding materials, subsequently by the row continuous backcross that further spouts, obtain that economical character is consistent with backcross parent and new material containing the transformation event, to accelerate the process of Breeding Application.In hybridization or backcross process, the method for blade identification or the detection method of the embodiment of the present invention 8 are smeared using foregoing kanamycins solution（It is more accurate compared with the former）Screening and Identification is carried out in progeny population, the presence of the transformation event is confirmed.Such as find that transformation event is lost, then eliminate it.

Plant numbers explanation：

Backcross progeny number format is " AXB BCmFl- x-y... "." A " is cotton material of the Fl generations as female parent, and " B " is the cotton material for making male parent in F1 generations, and the numbering material of wherein LG beginnings is backcross parent；" BC " represents backcrossing (Backcross), and " m " is backcrossing generation；"-x-y... " numbers for individual plant.

At present, if using thousand cotton backcross transformation parent materials of planted in different ecological areas, having been obtained for the backcrossing four generations material of the two transformation events.

Following table lists the insect resistace appraising datum of tri- key breeding resources material backcross progeny material different growth phases of 5 two transformation events of A2-6 and A26- and LG6101, LG6036, LG6035, with other transformation events（Data are unlisted）Compare, insect resistace is good, and more stable in different developmental phases, very good-

The transformation event A26-5 copy number Jian Measuring of embodiment 6.

Detected using Southern hybridization techniques to turning the copy numbers of transformation event A26- 5.

Sample preparation：The TO of transformation event A26- 5 are taken for young tender plant tissue 4. 0_gLeft and right, extracts plant genome DNA, comprises the following steps that：It is ground into powder in liquid nitrogen.Extraction buffer 15ml is preheated in 65'C water-baths, will grind to form and added after uniform powdery in Extraction buffer, vibration is mixed, during which 65 water-bath 45min shake up 2- 3 times, fully cracking.Add 1/3 volume 5mol/L KAc to turn upside down mixing, ice bath about 2-h3,4V, 12000rpm, centrifuge lOmin;Supernatant is taken, 5% CTAB Buffer of 1/5 volume are added, turn upside down abundant mixing, 65 water-baths about 20min_;To be cooled put is stopped up after temperature, adds isometric chloroform/isoamyl alcohol（24:1) extract 3 times, room temperature 12000rpm centrifugation 5min, such as interface muddiness extracts one to twice again；Supernatant is taken, 2/3 volume isopropanol is added, abundant mixing of turning upside down, room temperature places lOrain, room temperature, 12000rpm centrifuges lOrain;Supernatant is abandoned, precipitation is washed with 70% ethanol twice.Drain precipitation, plus the dissolving of 500ul aqua sterilisas；Plus 1/10 volume Nase processing DM samples, 37 30 min;Plus 1/10 volume 3BIO1/L NaAc (pH5. 2), 2 times of volume absolute ethyl alcohols are mixed, -20'C places 10min, 4 °C, 12 OOOrpm, centrifuge 5niin, paint precipitation is washed with 70% ethanol twice, precipitation, plus appropriate ddH20 dissolvings is drained, DNA concentration and purity are measured using ultraviolet specrophotometer, 100 200 μ are taken₈D with alcohol precipitation after restriction enzyme ferment EcoR I, Hind III, EcoR 1+ Hind III digestion overnight, is added in 0. 8% Ago-Gel after being dissolved with appropriate ddffiO, stayed overnight with IV/cm electrophoresis respectively.Vacuum is transferred on nylon membrane, UV-crosslinked fixation.

It is prepared by probe：Plasmid using the-Bt genes of Κ containing Ο is template, with 0KF and BtR primers（Sequence such as SEQ ID No:4 and SEQ ID No:Shown in 5）, the specific probe of high zinc mark is prepared using PCR methods, PCR system is as follows：

The μ 1 of 10 X amplification buffers 5. 0

DNTP (0. 7 Let ol/L DIG-ll-dUTP,

L 3 draws o】/ L dTTP, other dNTP concentration are 2 BBBOI/L) Ι Ο Ι

5, the μ 1 of primer (10 rool/L) 2.0

3, primer (10 BK) 1/L) 2. 01

Template DNA (the genes of Bt containing OK- Zhi） 1.0 μ 1

Taq DNA Polymerase 2 U

Plus sterilized water is to the μ 1 of cumulative volume 50

PCR programs： 94. 0Γ 5min;30 circulations： 94. 0.C 30s, 53.0.C 30s, 72. 0.C 30s ； 72. 0°C 5mir

Hybridization check：Nylon membrane is put into hybrid pipe, plus certain volume hybridization solution (10ffil/100_CM2), 65 °C, The h of prehybridization 3;95 denaturation DIG label probes (25ng/ml) lOmin, are immediately placed in cooling lOmin in frozen water and thoroughly become；Denatured probe is added rapidly to hybrid pipe（3. 5ml/100cm2 membrane), mix, 65 °C of hybridized overnights（>10h).Nylon membrane is taken out, film is washed in progress.At room temperature, the 1%SDS of 30ml 2XSSC/0. vibrations washing 2X5min.Under 50, film is transferred to equipped with vibration washing 5min in 20ml lavation buffer solutions by the SDS of 0. 1 X SSC I 0. 1 vibration washing 2X 15min.Hybridization and it is rigorous wash paint after, film is put and washes paint buffer solution infiltration l 5min;Fu educates 30min in 20 30ml confining liquids;Die of hunger in lOral antibody liquids and educate 3ftnin;Washed with 20 30ml cleaning solutions in 2X 15mi 15ml detection liquid and balance 2 5min;Now match somebody with somebody 20ml chromogenic substrates（NBT/BCIP) dark place stands colour developing；50ml aqua sterilisas or TE wash film 5min color development stoppings, preservation of taking pictures.Testing result such as Fig. 3 such as shows, only there is a hybridization signal band in two groups of single endonuclease digestion results, it is single copy insertion to show A26-5, the hybridizing band pattern of the DNA and T66-35S- OK- Bt- PS- Tnos- 2300 of digestion A26- 5 through Hind III+ FcdR I is basically identical, and it is single insertion for copying and be complete TDNA areas to show A26-5 events.The side Side sequence analyses of embodiment 7.

Sample preparation：With well known to a person skilled in the art the genomic DNA that Method of Plant DNA Extraction extracts the cotton material containing A26-5 transformation events, 2. 5 μ are taken_δDNA, uses Ec I, Η η III to digest 68 hours, alcohol precipitation adds suitable quantity of water to dissolve after purification respectively.

Jointing：According to carrier restriction enzyme site analyze, separately design synthesis two butt joints-

GenomeWalker Adaptor+EcdR I, GenomeWalker Adaptor-EccR I (sequence such as SEQ ID No:6, SEQ ID No:Shown in 7）With Genome lker Adaptor+Η ι III, GenomeWalker Adaptor-ffind III (sequence such as SEQ ID No:8, SEQ ID No:Shown in 9）, wherein to SEQ ID No:7 and SEQ ID No:9 5' ends have carried out phosphorylation, 3' ends ammonification base.

Mixed in equal amounts GenomeWalker Adaptor+FcdR I and Genomeliali er Adaptor-EccR I, GenomeWalker Adaptor+Hind 111 and GenomeWalker Adaptor-Hind III, 70 respectively.C be incubated 10 minutes, after be slowly decreased to room temperature.The DNA of 4 μ 1 digestion purifying is taken to be added to the GenomeWalker Adaptor containing 1. 9 μ 1 (25 Μ), 1. 6 μ 10X connection buffer solutions, the D of 0. 5 μ, 1 Τ 4 connect enzyme（6 units/), in 16 times incubated overnights, stop reaction, 5 min are cultivated under 70'C, in each pipe, adding the TE of 72 μ 1, (10/1, pH 7. 5), and the sec of 5- 10 are vibrated under the low speed.

Drug box is tried using Clontech GenoBteffalker Universal, primer API and GSP1- EcdR I, GSPl-ffi l III (sequence such as SEQ ID No are utilized: 10, SEQ ID No : ll, SEQ ID No:Shown in 12）, using connection product as template, carry out first round amplification：7 circulations： 94'C 25S, 72 6min_;32 circulations：94 °C of 25S, 67 6 min_;After last circulation 7 minutes are incubated then at 67 Γ.PCR primer is diluted after 50 times, with AP2 and GSP2-fcoR I, GS2 ~ ffind III (sequence such as SEQ ID No: 13, SEQ ID No: 14, SEQ ID No:Shown in 15）Carry out second and take turns PCR amplifications, PCR programs are as follows：5 circulations： 94Ό 25S, 72V 5min_;20 cycles: 94 V 25S, 67°C 5 niin;Then at 67 insulation 10 minutes after last circulation.Product reclaims sequencing.

To the right margin of the events of A26- 5（RB) terminal Sequence Analysis as shown in figure 4, obtain 1539 bp nucleotide sequence altogether（Sequence such as SEQ ID No:Shown in 16）, include the bp of lbp 340 cotton gene group sequence, the bp of the 341st bp 475 are the carrier sequence between RB and TMB0066, the bp of 476bp 752 are TMB0066 sequences, the bp of the 753rd bp ~ 1539 is CaMV 35S sequences.Sequence explanation：

1-340 cotton DNAs

Carrier sequence between 341-475 RB and TO066

476-752 T0066

Left margins of the 753-1539 CaMV 35S to A26-5 events（LB) terminal Sequence Analysis as shown in figure 5, obtain 1651 bp nucleotide sequence altogether（Sequence such as SEQ ID No:Shown in 17）, include the bp of lbp 798 NPTII sequences, 799bp 1011bp CaMV 35SpolyA sequences, the bp of the 1097th bp 1122 TW (left margins）With 1123b_P1651bp cotton gene group

Sequence explanation-

1-798 NPTII

799-1011 CaMV 35SpolyA

1097-1122 left margins

1123-1651 cotton DNAs are using the D of acceptor material Ji cotton 14 as template, respectively in RB the and LB sides wing cotton gene group primers of insetion sequence（Sequence such as SEQ ID No:18, SEQ ID No:Shown in 19）, amplified production of the size for 900bp or so is obtained, Fa Now are compared with A26- 5 flanking sequence, the event replaces what 206bp bases in protogene group sequence were obtained by insetion sequence.Comparison result is shown in Fig. 6.

According to result above, those skilled in the art can easily draw characteristic DNA sequence (the SEQ ID N0 of A26-5 events:22), as follows, it is T-DNA insetion sequences to underline shown partially, and not underlining shown partially is The flank cotton genomic DNA sequence of insetion sequence

I CTACCTAMA TCTT.4TATTT TCCT CCTT ATCCATCGAC GAGT.4GAACT TTTTC G

61 AMAACATTA TTCGMTTTT TATTCATAM CTCATGATGT TTAAAACTGT TTCATAAAM 121 ATGMTTGTT AAAGAG,\AAG AAGCTTTT AATTAACGTA GATGGTGCGA ACGGA ATG

181 TATACAACGT CTATTTTAAC AACTCAAATA CTTAAATAM ATATTCGMT AATTTAAATA

241 CAATTTTGTA ACTHTTGAA ATTAMAAAC TAA CATAT AATTTAATGT AATTTACTTA

301 AAAATTMCT GTTA.AATTTG AAAAA T TATTGCCGCG ACTGTTt^GA AGGGCGATCG

361 GTGCGGGCCT CTTOiCTATT ACGCCAGCTG GCGAMGGGG GATGTGCTGC AAGGCGATTA 421 AGTTGGGTAA CGCCAGCGTT HCCCAGTCA CGACGTTGTA AAACGACGGC CAGTGCCAAG

481 CTTATCGCCA TCAACGCHA TATGTATTTA ATCCGTAATC TCAGTCCGGC TCTCGCCACC

541 AGCTACGCTT ACGTTAACCC GGTGGTCGCG GTCTTGCTGG GTACGGGACT 6GGTGGAGAA

601 ACACTGTCGA AGAHGAATG GCTGGCGCTC GGCGTMTTG TCTTCG(¾GT GGTACTGGTC

662 ACGTTGGGAA MTATCTCTT CCCGGCAAAA CCCGTAGHG CGCCAGTTAT TCAGGACGCA ?21 TCMGCGAGT AAATGAATCC CCTGCGTGM TCTCTAGAGG ATCACGACAC TCTCGTCTAC

781 TCCMG TA TCAAAGATAC AGTCTCAGM GACCAAAGGG CTATTGAGAC TTTTCAACAA

841 AGCGTAATAT CCGCAAACCT CCTCGGATTC CATTGCCCAG CTATCTGTCA CTTCATCAAA

901 AGGACAGTAG AAMGGAAGG TGCCA CTAC AAATGCCATC ATTGCGATAA AGGAMGGCT

961 ATCGTTCAAG ATGCCTCTGC CGACAGTGGT OCCAAAGATG GACCCCCACC CACGAGGAGC 1021 ATCGTGGAAA AAGAAGACGT TCCAACCA(¾ TCTTCAAAGC MGTGGATTG ATGTGMCAT

1081 GGTGGAGCAC GACACTCTCG TCTACTCCAA GAATATCAAA GATACAGTCT CAGAAGACCA

1141 MGGGCTATT GAGACTTTTC AACAMGGGT TATCGGGA CCTCCTCG GATTCCATTG

1201 CCCAGCTATC TGTCACTTCA TCAA GGAC AOTAGAMAG GAAGGTGGCA CCTACA TG

1261 CCATCATTGC GATAAAGGM AGGCTATCGT TCAAGATGCC TCTGCCGACA GTGGTCCCAA 1321 AGAT6GACCC CCACCCACGA GGAGCATCGT GCAAAAAGAA GACGTTCC CCACGTCTTC

1381 AMGC GTG GATTGATGTG ATATCTCCAC TGA(¾TAAGG GATGACGCAC TCCCACTA

1441 TCCTTCGCAA GACCCTTCCT CTATATAAGG AAGTTCATTT CATTTGGAGA GGACACGCTG

1501 AAATCACCAG TCTCTCTCTA CAAATCTATC TCTGGATCCC TCTGGATCCT ATTTTTACM

1561 CAATTACCAA CMCMCAAA CMCAMCM CATTAC TT ACTATTTACA AT CMTGG 1621 ACTGCAGGCC ATACMCTGC TTGAGTAACC CAGAAGTTGA AGTACTTGGT GGAGAACGCA

1681 TTGAAACCGG TTACACTCCC ATCGACATCT CCTTGTCCTT GACACACTTT CTGCTCAGCG

1741 AGTTCGTGCC AGGTGCTGGG TTCGTTCTCG GACTAGTTGA CATCATCTGG GGTATCTTTG

1801 GTCCATCTCA ATGGGATGCA TTCCTCGTGC AMTTGAGCA GTTGATCAAC CAGAGGATCG

1861 MGAGTTCGC CAGGMCCAG GCCATCTCTA GGTTGGMGG ATTGAGCAAT CTCTACCAAA 1921 TCTATGCAGA GAGCTTCAGA GAGTGGGMG CCGATCCTAC TAACCCAGCT CTCCGCGAGG

1981 A TGCGTAT TCAATTCAAC GACATGAACA GCGCCTTGAC CACAGCTATC CCATTGTTCG

2041 CAGTCCAGM CTACCAAGTT CCTCTCTTGT CCGTGTACGT TCAAGCAGCT MTCTTCACC

2101 TCAGCGTGCT TCGAGACGTT AGCGTGTTTG GCCAAAGGTG GGGATTOAT GCTGCMCCA

2161 TCMTAGCCG TTACAACGAC CTTACTAGGC TGATTGGAAA CTACACCGAC CACGCTGTTC 2221 GnGGTACAA CACTGGCTTG GAGCGTGTCT GGGGTCCTGA TTCTAGAGAT TGGATTAGAT

2281 ACMCCAGTT CAGGAGAGAA TTGACCXTTCA CAGTTTTGGA CATTGTGTCT CTCTTCCCGA

2341 ACTATGACTC CAGMCCTAC CCTATC^TA CAGTGTCCCA ACTTACCAGA GAAATCTATA

2401 CTAACCCAGT TCTTGAGAAC TTCGACGGTA GCTTCCGTGG TTCTGCCCAA GGTATCG G

2461 GCTCCATCAG GAGCCCACAC TTGATGGACA TCTTGAACAG CATMCTATC TACACCGATG 2521 CTCACAGAGG AGAGTAWAC TGGTCTGCAC ACCAGATCAT 6GCCTCTCCA GTTGGATTCA

2581 GCGGGCCCGA GTTTACCTTT CCTCTCTATG GAACTATGGG AMCGCCGCT CCACAACAAC

2641 GTATCGTTGC TCMCTAGGT CAGGGTGTCT ACAGMCCTT GTCTTCCACC TTGTACAGM

2701 GACCCTTCAA TATCGGTATC MCAA(¾:AGC AACTTTCCGT TCTTGACGGA ACAGAGTTCG

2761 CCTATGGMC CTCTTCT C TTGCC CCG CTGTTTACAG AAAGAGCGGA ACCGTTGATT 2821 CCTTGGACGA AATCCCACCA CAGAACAACA ATGTGCCACC CAGGCAAGGA TTCTCCCACA

2881 GCTTGAGCCA CGTGTCCATG TTCCGTTC(¾ GATTCAGCAA CAGTTCCGT6 AGCATCATCA

2941 GGGCTCCTAT GTTCTCTTGG ATACAC(¾TA GTGCTGAGTT CMCAACATC ATCGCATCCG 3001 ATAGTATTAC TCAAATCCCT GCAGTGMGG GAAACTTTCT OTCAACGGT TCTGTCATTT

3061 CAGGACCAG6 ATTCACTGGT GGAGA TTCG TTAGACTCM CAGCAGTGGA MCAACATTC

3121 AGAATAGGAG GTATATTGAA GTTCCAATTC ACTTCCCATC CACATCTACC AGATATAGAG

3181 TTCGTGTGAG GTATGCTTCT GTGACCCCTA TTCACTrCM CGnMTTGG GGTAATTCAT

3241 CCATCTTCTC C TACAGTT CCAGCTACAG CTACCTCCTT GGATMTCTC C TCCAGCG

3301 ATTTCGG™ CTTTGAMGT GCCAATGCTT TTACATCTTC ACTCGGTAAC ATCGTGGGTG

3361 TTAGMACTT TAGTGGGACT GCTGGAGTGA TTATCGACAG ATTCGAGTTC ATTCCAGTTA

3421 CTGC CACT CGAGGCTGAG TAAGGTT C TTTGAGTATT ATGGCAHGG AAMGCCATT

3481 GTTCTGCTTG TMTTTACTG TGTTCTTTCA GTTTTGTTTT CGGACATCAA GTTAACMM

3541 AAAAAAAAAA AAAAAAAA ATTTMCMA AAMAAAAAA AAAAAAAAAA TTTAACAAAA

3601 AAAAAAAAM AAAAAAAAAT TTAAAGAGCT CGAATTTCCC CGATCGTTCA AACATTTGGC

3661 MTA GTTT CTTAAGAHG AATCCTGTTG CCGGTCTTGC GATGATTATC ATATAATTTC

3721 TGTTG TTA CGTTAAGCAT GTAATMTTA ACATGTMTG CATGACGHA TTTATGAGAT

3781 GGGTTTTTAT GATTAGAGTC CCGCAATTAT ACATTTMTA CGCGATAGAA MCAAMTAT

3841 AGCGCGCMC TAGGATAAAT TATCGCGCGC GGTGTCATCT ATGTTA AG ATCGGGMTC

3901 CGTMTCATC GTCATAGCTG TTTCCTGTGT GAAATTGHA TCCGCTCACA ATTOACACA

3961 ACATACGAGC CGGAAGC MGTGTAMG CCTGGGGTGC CT TGAGTG AGCTMCTCA

4021 CATTMTTGC GTTGCGCTCA CTGCCCGCTT TCCAGTC¾GG AMCCTGTCG TGCCAGCT6C

4081 ATTAATGAAT CGGCCAACGC GCGGGGAGAG GCGGTTTGCG TATTGGCTAG AGCAGCTTGC

4141 CMCATGGTG GAGCACGACA CTCTCGTCTA CTCC GAAT ATCAAAGATA CAGTCTCAGA

42Q1 AGACCAMGG GCTATTGAGA CTTTTCAACA MGGGTAATA TO¾GAAACC TCCTCGGATT

4261 CCATTGCCCA GCTATCTGTC ACTTCATCAA AAGGACAGTA CAAMGGAAG GTGGCACCTA

4321 CAMTGCCAT CATTGCGATA AAGGAAAGGC TATi¾TTCM GATGCCTCTG CCGACAGTGG

4381 TCCCA GAT GGhCCCCCAC CCACGAGGAG CATCGTGGM AMGMGACG TTCCMCCAC

4441 GTCTTCA G CMGTGGATT GATGTGATM CATGGTGGAG CACGACACTC TCGTCTACTC

4501 C GMTATC AGATACAG TCTCAG GA CC AGGGCT mGAGACTT TTCAACAMG

4561 GGTMTATCG GGAAACCTCC TCGGATTCCA TTGCCCAGCT ATCTCTCACT TCATC AAC

4621 GACAGTAGM AAGGAAGGTG GCACCTACAA ATGCCATCAT TGCGATAAAG GAAAGGCTAT

4681 CCTTC GAT GCCTCTGCCG ACAGTGGTCC CA GATGGA CCCCCACCCA CGAGGAGCAT

4741 CGTGGAAAAA GMGACGTTC C CCACGTC TTCMAGCM GTGGATTGAT GTGATATCTC

4801 CACTGACGTA AGGGATGACG CACAATCCCA CTATCCTTCG CMGACCTTC CTCTATATAA

4861 GGAAGTTCAT TTCATTTGGA GAGGACACGC TCAMTCACC AGTCTCTCTC TACAAATCTA

4921 TCTCTCTCGA GCTTTCGCAG ATCTGTCGAT CGACCATGGG GAHGAACAA GATGGATTGC

•498】 ACGCAGGTTC TCCGGCCGCT TGGGTGGAGA GGCTATTCGG CTATGACTGG GCACMCAGA

5041 CAATCGGCTG CTCTGATGCC GCCGTGTTCC GGCTCTCAGC GCAGGGGCGC CCGGTTCTTT

5101 TTGTCMGAC CGACCTGTCC GGTGCCCTGA ATGAACTCCA GGACGAGGCA GCGCGGCTAT

5161 CGTGGCTGGC CACGACGGGC GTTCCT GCG CAGCTGTGCT CGACGTTGTC ACTGAAGO¾

5221 GAAGGGACTG GCTGCTATTG GGCXJMCTGC (X¾¾«:AGGA TCTCCTGTCA TCTCACCTTG

5281 CTCCTGCCGA GMAGTATCC ATCATGGCTG ATGCAATGCG GCGGCTGCAT ACGCTTGATC

5341 CGGCTACCTG CCCATTCGAC CAa GCGA MCATCGCAT TOAGCGAGCA CGTACTCGGA

5401 TGGMGCCGG TCTTGTCGAT CAGGATGATC TGGACG GA GCATCAGGGG CTOJCGCCAG

5461 CCGAACTCTT CGCCAGGCTC AAGGCGCGCA TGCCCGACGG CGAGGATCTC GT(ETGACAC

5521 ATGGOJATGC CTGCTTGCCG AATATCATGG TCGMAATGG C X TTTCT GGATTCATCG

5581 ACTGTGGCCG GCTGGGTGTG GCGGACCGCT ATCAGGACAT AGCGHGGCT ACCCGTGATA

5641 nGCTG GA GCTTGGCGGC GAATGGGCTG AC JCTTCCT CGTGCTTTAC GGTATCGC(¾

5701 CTCCCGATTC GCAGCGCATC GCCTTCTATC GCCTTCTTGA CGAGTTCTTC TGAGCGGGAC

5761 TCTGGGGTTC GGATCGATCC TCTAGCTAGA GTOATffiAC GCTCGAGT TTCTCCATAA

5821 TMTGTGTGA GTAGTTCCCA GATMGGGAA TTAGGGTTCC TATAGGGTTT CGCTCATGTG

5881 TTGAGCATAT MGAMCCCI TAGTATGTAT TTGTATTTGT AAAATACTTC TATCAATAAA

5941 ATTTCTMTT CCTMAACCA AAATCCAGTA CTAAAATCCA GATCCCCCGA ATTMHCGG

6001 CGTTAAHCA CTA VTTA MCGTCCGCA ATGTGTTATT AAGHGTCTA AGCGTCMTT

6061 TGTTTACACC ACAATATACC ATATCTTAAA CG ATCTAC CCGTTCTTCC CACATMTAA

6121 TGTAGGTAAT TATTTGA T TGGATGAA TTTMATG ATATGTTAAA TTTMTACAT 6181 TMCAT TA ATTCATGTGT ATTTCACTGT TGATATATAT ΤΑΤΤΤΪΤΤΑΑ ACATTCTTTT

62 1 ATGAACTTTG AAATTTGTT6 AATTTTT Fine TATTTTTAH TATTTTTGTA Μ Α Τ Τ Τ Τ Τ Α

6301 TAATTAATAT CGTTAAATTT TGAATAATTT AATTAACTTT TTCATCTTAA AA ACCAGT

6361 TTGACT AT TITTATTAAG CAGGCA CA CC TTT6AC AMAAAATAT GAATMTAM

6421 ΑΑΤΤΛΑΑΤΤΤ ATCATTATGC CTATTATATA ΤΑΤ ΠΑΤΤ ATTTTTTCTT TTTTAAA A

6481 GGTGT TAT GTA ACTTT GGTCTATTTA TAA ATTAA ATTTGCACCA AA MAGTA

6541 ACATMAT TATGTACTTG G6TTCAAATT ATTATGAMA AAMGGAAAT AGGATTTATA

The transformation event of 6601 TCGTTA embodiments 8. is detected

Using DNA primer to entering performing PCR amplification to examine the events of Measuring A26- 5, second primer of any flanking sequence of insetion sequence is constituted described in the first primer and specific recognition of the primer pair by specific recognition T-DNA insetion sequences of the present invention.A26-5 insetion sequences and identification primer are as shown in Figure 7.For example, when the first primer is A26-5_3 (SEQ ID N0:Or A26-5-4 (SEQIDTO 21):19) when, the second primer can be GSP1- ffi2d III (SEQIDN0:Or GSP2- ' xi III (SEQ ID Books 12)：15);When the first primer is A26- 5- 1 (SEQ ID ^):Or 26 ~ 5-2 (SEQ ID NO 20):18) when, the second primer can be GSP1- ￡ o I (SEQ ID NO:Or I (SEQ ID N0 11):14).

The method for entering performing PCR identification using above-mentioned primer is well-known to those skilled in the art.Using primer pair GSPl-ffind III/ A26- 5- 3, GSPl-i '/3d III/ A26- 5-4 and GSP foretell EcoR 1/ A26- 5- 1, the A26- 5- 2 of GSPl-EcoR 1/ expand respectively A26-5 events, the events of A2- 6, Ji cotton 14- 1, Ji cotton 14- 2 cotton samples result it is as shown in Figure 8.The chromosome mapping of embodiment 9.

According to the cotton flanking sequence obtained, the diploid cotton iGossypium raimondi D genome genome sequences of announcement are utilized（http：〃 www. phytozome. net/cotton, php) it is analyzed, understand in A26-5 events, the cotton Side wing DM sequences engaged with insetion sequence and the sequence very high homology on D8 chromosomes, thus, it can be known that the integration site of exogenous DNA insetion sequence is located on the 8th group chromosome of acceptor tetraploid cotton in A26-5 events.

P international application no 201,2/0 00673 explanations on microbial preservation of KT/CN of applicant or attorney docket FP " " 2056

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City of BeiJing, China is towards area North Star West Road】Number Institute of Microorganism, Academia Sinica of institute 100101

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Claims

Claims

L Cotton Transformation events A26- 5, its feature DM sequences as shown in SEQ ID Not 22, its by 341-6078 bp T-DNA insetion sequences, 1-340 bp upstream flank cotton gene group sequence and the 6079th 6606 bp downstream flank cotton gene group Sequence composition.

2. the fragment of the characteristic DNA sequence of the Cotton Transformation event described in claim 1, the DNA fragmentation comprises at least the part T-DNA insetion sequences and part the flank cotton gene group sequence.

3.-kind of recombinant vector, it contains the T- DNA insetion sequences described in claim i；For example, the carrier is the T66-35S- OK- Bt- PS- Tnos-2300 carriers in accompanying drawing 1.

4. a kind of recombinant cell, contains the carrier described in claim 3；For example, the recombinant cell is the recombinational agrobacterium cell containing the carrier described in claim 3.

5. for the primer pair of the Cotton Transformation event described in test right requirement 1, its by the either side described in specific recognition claim 1 flanking sequence the first primer and specific recognition claim 1 described in the second primer of T-DNA insetion sequences constitute.

6. the primer pair described in claim 5, wherein the sequence of first primer is SEQ ID NO:18 or SEQ ID):20, the sequence of second primer is SEQ ID N0:11 or SEQ ID N0: 14_o

7. the primer pair described in claim 5, wherein the sequence of first primer is SEQ ID NO:19 or SEQ ID N0:21, the sequence of second primer is SEQ ID NO:12 or SEQ ID MO: 15ο

8. the method for Α 26-5 transformation events in-kind of identification cotton biological sample, it includes：

<A) from cotton extraction from biological material DM samples to be identified；

(b) using the DNA sample of extraction as template, usage right requires that the primer pair described in any one of 5-7 enters performing PCR amplification；

(c) pcr amplification product is detected, if amplified production length and SEQ ID NO:Theoretical length on 22 between the sequence of the PCR primer pair is consistent, then shows the presence of A26-5 transformation events in the cotton biological sample.

9. a kind of method for obtaining transgenic pest-resistant cotton material, including：Using the cotton material containing the transformation event described in claim 1, after being hybridized with other cotton breeding materials, further it is returned, obtains the new material containing the transformation event described in claim 1；In hybridization and backcross process, Screening and Identification is carried out in progeny population using the method described in claim 8, the presence of the transformation event described in claim 1 is confirmed.

10. the method described in the fragment described in transformation event, claim 2, the carrier described in claim 3, the recombinant cell described in claim 4, claim 8 or claim 9 described in claim 1 is used to improve cotton bollworm resisting character, carries out cotton breeding or the purposes as molecular labeling.