CN106244587A - Glyphosate resistant cotton event and for its detection primer and method - Google Patents
Glyphosate resistant cotton event and for its detection primer and method Download PDFInfo
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Abstract
The invention discloses Cotton Transformation event aQ51-1 of resistance glyphosate and characteristic sequence thereof and the primer detected for it and method.The vegetable lamb carrying transformation event aQ51-1 comprises described event i.e. external source insertion DNA sequence and the joint of cotton genomic DNA sequence on the second group chromosome.Utilize external source to insert the DNA sequence of bonding land in DNA sequence and flank cotton gene group thereof, detection primer can be designed, for the specific detection for aQ51-1 event.Detection method for aQ51-1 event can be the means that this specific gene insertion event is followed the trail of in the application offer utilizing this plant event to carry out breeding easily, and this specific gene inserts event and can use to improve breeding work efficiency as molecular marker.
Description
Technical field
The invention belongs to field of plant molecular biology, especially the transgenic crop breeding field in agricultural biotechnologies research, particularly relate to the Cotton Transformation event with glyphosate resistance.
Background technology
Population in the world is increasing rapidly, it is estimated that will be increased to 8,500,000,000 by 7,000,000,000 in 2014 to population in the world in 2025, world food faces safely huge challenge.It is true that improved by conventional breed improvement and cultivation technique, particularly utilization of pesticides, grain-production has significantly improved in the past few decades.Cytogenetics and cytobiology technology that the sixties in last century rises have caused " Green revolution ", make the yield of cereal crops significantly improve, disease resistance and insect resistace are remarkably reinforced.But, the widely used of these technology serves new problem, as the use of pesticide and chemical fertilizer causes Soil erosion to aggravate to environment and human health band the most simultaneously.Meanwhile, after these technology use 20 years, the contribution to increases in grain production also significantly reduces.For overcoming these problems, the scientists coming from government, university, research institution and company is seeking new technology and method.Along with the development of sufficient DNA technique, having been developed over some new GENERALIZATION OF MODERN BREEDING TECHNIQUE and methods to the scientists eighties in last century, the transgenic technology wherein utilizing agrobacterium mediation converted and via Particle Bombardment Transformation is the most successful one.
Going through to start from the commercialization of the first in the world genetically modified crops in 1996, the cultivated area of whole world genetically modified crops adds 106 times, and within 2014, global genetically modified crops cultivated area is record-breaking 1.815 hundred million hectares.The country of plantation genetically modified crops is also added to 28.Wherein the U.S., Argentina, Brazil, Canada and China are primary transgenic crop-planting countries.For transgene traits, imported by transgenic technology at present and the most important character of commercial development surely belongs to antiweed, genetically modified crops (mainly including Semen sojae atricolor, Semen Maydis, Cotton Gossypii, the Brassica campestris L) cultivated area of antiweed in 2014 is about 101,600,000 hectares, accounts for the 56% of whole world genetically modified crops cultivated area；Next to that insect resistace, anti-pest GM crop (mainly Semen Maydis, Cotton Gossypii and other crops) area is about 27,220,000 hectares, accounts for 15%；Complex character genetically modified crops account for 28%.The initial data that Klumper and Qaim (2014) utilizes the farm carried out all over the world investigation or field test to draw has carried out comprehensive analysis to 147 the known genetically modified crops researchs of past 20 years, and reports the impact in terms of crop yield, the use of pesticide and peasant's profit of genetically engineered soybean, Semen Maydis or Cotton Gossypii.This conclusion comprehensively analyzed is that " employing of transgenic technology makes the use of chemical pesticide decrease 37%, and crop yield increases by 22%, and peasant's profit increases by 68%.
Cotton Gossypii is global most important Plant fiber crop, mainly plants in tropical or semi-tropical arid area.The quality and yield of Cotton Gossypii in crop smothering serious threat.Promoting the use of of herbicide, can be greatly reduced cotton field management recruitment, reduce labor intensity.The cotton variety promoted at present does not have resistance to herbicide, poisoning phenomenon often occurs, and cotton plants causes certain damage, and cultivating Resistant Herbicide Crops kind is to prevent the maximally effective approach of herbicide damage.Therefore, this area persistently needs to cultivate the Resistant Herbicide Crops kind more preventing herbicide damage.
Summary of the invention
The present inventor obtains a kind of transformation event by transgenic method, and inventor is by its named aQ51-1.This transformation event has stable high-resistance glyphosate character.Its representative Gossypium hirsutum L. (Gossypium hirsutum) seed was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC on 05 28th, 2015, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica postcode: 100101), deposit number is: CGMCC No10490.
First aspect present invention provides a kind of Cotton Transformation event aQ51-1, it is characterized in that its DNA sequence as shown in SEQ ID No:25, it is by the T-DNA insertion sequence of 1154-9044bp, the upstream flank cotton gene group sequence of 1-1153bp and the downstream flank cotton gene group Sequence composition of 9045-10295bp.
Second aspect present invention provides a DNA sequence, and described DNA sequence comprises the T-DNA insertion sequence (i.e. SEQ ID No:25 1154-9044bp) described in first aspect present invention and described flank cotton gene group sequence (i.e. the upstream flank cotton gene group sequence of SEQ ID No:25 1-1153bp and the downstream flank cotton gene group sequence of 9045-10295bp).
Third aspect present invention provides a kind of recombinant vector, and described recombinant vector contains the T-DNA insertion sequence (i.e. SEQ ID No:25 1154-9044bp) described in first aspect present invention.In one embodiment, the pBI121-BHR4-BHR5 carrier during described carrier is Fig. 2.
Fourth aspect present invention provides a kind of reconstitution cell, and it contains the recombinant vector described in third aspect present invention.Described reconstitution cell preferred bacterium.In one embodiment, described reconstitution cell is the recombinational agrobacterium cell containing the recombinant vector described in third aspect present invention.
Fifth aspect present invention provides the primer pair for detecting the Cotton Transformation event described in first aspect present invention, and it is made up of the second primer of the T-DNA insertion sequence described in the first primer of the flanking sequence of the either side described in specific recognition first aspect present invention and specific recognition first aspect present invention.In some embodiments, the sequence of described first primer is SEQ ID NO:26, and the sequence of described second primer is SEQ ID NO:27.In other embodiments, the sequence of described first primer is SEQ ID NO:28, and the sequence of described second primer is SEQ ID NO:29.
Sixth aspect present invention provides a kind of identifies the method for transformation event aQ51-1 in Cotton Gossypii biological sample, comprising:
A () is from Cotton Gossypii extraction from biological material DNA sample to be identified；
B (), with the DNA sample extracted as template, uses primer described in fifth aspect present invention to carrying out PCR amplification；
C () detection pcr amplification product, if amplified production length is consistent with theoretical length between the sequence of described PCR primer pair on SEQ ID NO:25, then shows the existence of aQ51-1 transformation event in described Cotton Gossypii biological sample.
Seventh aspect present invention provides the method for transfer in different cotton breeding materials of Cotton Transformation event described in first aspect present invention, and described method includes:
After cotton material containing the transformation event described in first aspect present invention and other cotton breeding materials are hybridized, backcross further, it is thus achieved that containing the new material of the transformation event described in first aspect present invention；
In hybridization and backcross process, the method described in sixth aspect present invention is utilized to carry out Screening and Identification, the existence of confirmation transformation event described in first aspect present invention in progeny population.
Eighth aspect present invention provides the recombinant vector described in the transformation event described in first aspect present invention, the DNA molecular described in second aspect present invention, third aspect present invention or the method described in the reconstitution cell described in fourth aspect present invention, the present invention the 6th and the 7th aspect for improving Resistance Strain of Cotton glyphosate character, carrying out plant breeding and be used as the purposes of molecular marker.
Accompanying drawing explanation
Fig. 1 shows the structure flow process of plant expression vector pBI121-BHR4-BHR5.
Fig. 2 shows plant expression vector pBI121-BHR4-BHR5.
Fig. 3 shows with acceptor material Ji cotton 14DNA as template, uses sequence to be respectively SEQ ID No:23 and the primer pair of SEQ ID No:24, and amplification obtains the amplified production that size is about 420bp, with the result of the flanking sequence comparison of aQ51-1 after order-checking.
Fig. 4 shows the result utilizing cotton samples that primer expands aQ51-1T1 (1-9), Ji cotton 14-1, Ji cotton 14-2 respectively to aQ51-1-RB5 ' (SEQ ID NO:26)/aQ51-1-RB3 ' (SEQ ID NO:27), aQ51-1LB5 ' (SEQ ID NO:28)/aQ51-1LB3 ' (SEQ ID NO:29).In the drawings: M:marker, λ DNA/HindIII+EcoRI；1-9:aQ51-1-RB5 '/aQ51-1-RB3 ' amplification aQ51-1T1 (1-9)；10: Ji cotton 14-1；11: Ji cotton 14-2；12: negative control, positive amplification band 1529bp；13-24:aQ51-1-RB5 '/aQ51-1-RB3 ' amplification aQ51-1T1 (13-9)；22: Ji cotton 14-1；23: Ji cotton 14-2；24: negative control, positive amplification band 880bp.As follows: M:Marker/DL2000；1:aQ51-1-1；2:aQ51-1-2；3:aQ51-1-3；4:aQ51-1-4；5:aQ51-1-5；6:aQ51-1-6；7:aQ51-1-7；8:aQ51-1-8；9:aQ51-1-9；10:J14；11:J14；12:ddH2O；13:aQ51-1-1；14:aQ51-1-2；15:aQ51-1-3；16:aQ51-1-4；17:aQ51-1-5；18:aQ51-1-6；19:aQ51-1-7；20:aQ51-1-8；21:aQ51-1-9；22:J14；23:J14；24:ddH2O.
Detailed description of the invention
In the present invention, " transformation event " refers to be transformed in cotton cells exogenous array by Agrobacterium tumefaciens-mediated Transformation method (as well known to those skilled in the art), and the event that in the transgenic cotton plant obtained further, exogenous array target location in cotton gene group is inserted and integrated；The carrier that " transformation event " is not a kind of plant cell or plant, plant cell or plant are only transformation event existence；The core feature of " transformation event " is the exogenous array external source insertion sequence that the insertion of target location is formed in Plant Genome and one section of characteristic DNA sequence of cotton gene group sequence connection.
Embodiment
Below in conjunction with non-limiting example, the present invention is further described.
Embodiment 1. plant expression vector construction
According to announce OK sequence (Richards et al., 1987, Eur.J.Biochem.84,513-519；Kozak M; 1986, Cell, 44; 283-92) and PS sequence (Guo three heap etc.; 2000, Chinese agriculture science and technology Leader, 2; 21-26) synthesis OK-Pst I-Xho I-PS fragment; two ends band BamH I respectively and Sac I restriction enzyme site, be connected with Pst I, Xho I two restriction enzyme site between OK with PS, inserts protection base and be easy to follow-up enzyme action structure between two restriction enzyme sites.Being template amplification Tnos fragment with pBI121 (purchased from ocean Science and Technology Ltd. of Beijing China), two ends band Sac I respectively and Hind III digestion site, primer sequence is as shown in SEQ ID No:1 and SEQ ID No:2.BamH I and Sac I is utilized by OK-Pst I-Xho I-PS enzyme action and to utilize Hind III and Sac I by described Tnos fragment enzyme action, it is building up in pUC57 (purchased from Jin Sirui biotechnology Co., Ltd), it is thus achieved that recombiant plasmid OK-PS-Tnos-pUC57.Synthetic BHR4 fragment, its sequence, as shown in SEQ ID No:3, utilizes the restriction enzyme site at two ends to be connected in OK-PS-Tnos-pUC57 by BHR4, it is thus achieved that recombiant plasmid OK-BHR4-PS-Tnos-pUC57.Being template amplification 35S fragment with pCambia2300 (purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd), two ends band Xba I respectively and BamH I restriction enzyme site, primer sequence is as shown in SEQ ID No:5 and SEQ ID No:6.BamH I and HindIII is utilized by OK-BHR4-PS-Tnos-pUC57 enzyme action and to utilize Xba I and BamH I by described 35S fragment enzyme action, it is building up to pBS (pBulescript, purchased from Fermentas company) in, it is thus achieved that recombiant plasmid 35S-OK-PS-BHR4-Tnos-pBS.
Synthesis 35S changes promoter, and its sequence, as shown in SEQ ID No:7, utilizes the restriction enzyme site at two ends to be changed by 35S and is connected into 35S-OK-PS-BHR4-Tnos-pBS, it is thus achieved that recombiant plasmid 35S changes-OK-PS-BHR4-Tnos-pBS.PCR expands BHR5 sequence fragment, and its sequence is as shown in SEQ ID No:8, and primer sequence is as shown in SEQ ID No:9 and SEQ ID No:10.Described BHR5 sequence fragment is connected in OK-PS-Tnos-pUC57 by the restriction enzyme site utilizing two ends, obtain recombiant plasmid OK-BHR5-PS-Tnos-pUC57, utilize Pst I and HindIII by OK-BHR5-PS-Tnos, it is building up to 35S and changes in-OK-PS-BHR4-Tnos-pBS, it is thus achieved that recombiant plasmid 35S changes-OK-PS-BHR5-Tnos-pBS.
Mixed in equal amounts linker+ and linker-(sequence is respectively as shown in SEQ ID No:11 and SEQ ID No:12), 70 DEG C be incubated 10 minutes, after be slowly decreased to room temperature.Utilize Hind III and EcoR I, be building up to carrier pBI121, it is thus achieved that pBI121-2.Utilize Spe I and EcoR I that 35S is changed-OK-PS-BHR5-Tnos and be building up to pBI121-2, it is thus achieved that pBI121-BHR5.Utilize Xba I and Kpn I that 35S-OK-PS-BHR4-Tnos is building up to pBI121-BHR5, it is thus achieved that pBI121-BHR4-BHR5.Building flow process and see Fig. 1, the plant expression vector pBI121-BHR4-BHR5 of synthesis is shown in Fig. 2.
The genetic transformation of embodiment 2. Gossypium hirsutum L. (Gossypium hirsutum)
Utilize agrobcterium-mediated transformation, with pBI121-BHR4-BHR5 vector Ji cotton 14 (National Cotton storehouse in mid-term obtains unit Cotton institute, Unified number: the ZM-30270) hypocotyl obtained in embodiment 1.
Picking contains the Agrobacterium LBA4404 of pBI121-BHR4-BHR5 carrier (purchased from Biovector Science Lab, Inc), it is seeded to containing kanamycin (kanamycin, km) 50mg/L, rifampicin (rifampicin, rif) 50mg/L and streptomycin (streptomycin, S/Sm) the LB fluid medium (yeast extract (Yeast extract) 5g/L, tryptone (Tryptone) 10g/L, sodium chloride (NaCl) 10g/L) of 50mg/L, 28 DEG C of vibration light culture overnight arrive bacterial growth logarithmic (log) phase.With bacterium solution: ratio LB of culture medium 1:50-1:100 or body culture medium dilution bacterium solution, then shaken cultivation 4-6h, bacterium solution is diluted to OD600 value 0.8-1.0.
Transgenic acceptor is Ji cotton 14, take the growth aseptic seedling hypocotyl of 3-4 days, it is cut into the section of 0.6-0.8cm, contaminate 10-15min, take out hypocotyl section, put and co-culture culture medium (MSB (Murashige and Skoog salt culture medium)+KT (kinetins) 0.1mg/L+2,4-D (2,4-dichlorphenoxyacetic acid) 0.1mg/L), 22 DEG C-25 DEG C co-culture 2 days.nullGo to calli induction media (MSB+KT 0.1mg/L+2,4-D 0.1mg/L+Kan (kanamycin) 50mg/L) 20-30 days subcultures are once，Wound healing proliferated culture medium (MSB+KT 0.1mg/L+2 it is forwarded to after 90 days,4-D 0.05mg/L+Kan 50mg/L)，20-30 days subcultures are once，After growing embryo callus subculture，By embryo callus subculture subculture to germination medium (MSB+KT 0.1mg/L+Kan 50mg/L)，The green bud of picking sprouting in about 40 days screens to root media (SH (Schenk and Hildebrandt Medium)+Kan 50mg/L)，And then obtain seedling and can grafting resistant plant 300 strain.Resistant plant grafting transplanting after PCR identifies, it is thus achieved that aQ series Cotton Gossypii totally 219 strain.
The Screening and Identification of embodiment 3. transgenic line
Glyphosate tolerant cotton event aQ51-1 is selected from a lot of tolerance glyphosate trophisms and the transgenic cotton event of genitality injury.In the present invention, aQ51-1 is by an event in 219 T0 events (these events obtain in example 2) of the most different DNA construct conversions including pBI121-BHR4-BHR5.Screened by a series of analysis of molecules and glyphosate tolerant and select aQ51-1 event from many events.Specific as follows:
T0 screens for Cotton Gossypii glyphosate tolerant, and in warmhouse booth glyphosate tolerant is tested, trophism and genitality to plant are marked, thus filter events.All plants all grow in warmhouse booth, spray Roundup (antiweed containing glyphosate, 41% glyphosate isopropylamine salt water solution), Roundup stock solution consumption 250g/ mu in 8 leaf phases subsequently.After using glyphosate 7 days, the vegetalitas that evaluation plant is subject to injures.There is the event continued growth of good nutrition patience to ripe, and observe genitality.There is the transformation event of good trophism and genitality for further being tested.
The copy number of the event of the tolerance standard of Roundup trophism and genitality is met by southern blotting technique analysis.The list copy event representing existing well tolerable property at T0 is carried out field resistance test further.Roundup (antiweed containing glyphosate, 41% glyphosate isopropylamine salt water solution), Roundup stock solution consumption 250g/ mu is sprayed in 2-4 leaf phase, 6-8 leaf phase, the envelope departure date, 60% knot bell phase.Obtain the transformation event aQ51-1 with good trophism and genitality.
Embodiment 4. flanking sequence analysis
Prepared by sample: extract the genomic DNA of the cotton material containing aQ51-1 transformation event with Method of Plant DNA Extraction commonly used in the art, takes 2.5 μ g DNA, and Hind III digests 6-8 hour respectively, and alcohol precipitation adds suitable quantity of water after purification and dissolves.
Jointing: analyze according to carrier restriction enzyme site, separately designs and synthesizes two butt joints:
GenomeWalker Adaptor+Hind III and GenomeWalker Adaptor Hind III (sequence is respectively as shown in SEQ ID No:13 and SEQ ID No:14), wherein 5 ' the ends of SEQ ID No:14 have carried out phosphorylation, and 3 ' ends add amino.
Mixed in equal amounts GenomeWalker Adaptor+Hind III and GenomeWalker Adaptor Hind III, 70 DEG C be incubated 10 minutes, after be slowly decreased to room temperature.The DNA taking 4 μ l digestion purification is added to containing 1.9 μ l GenomeWalker Adaptor (25 μMs), 1.6 μ l 10X connect buffer, 0.5 μ l T4DNA ligase (6 units/μ l), incubated overnight at 16 DEG C, stopped reaction, water-bath 5min at 70 DEG C, in each pipe, adding 72 μ l TE (10/1, pH7.5), vibrate 5-10sec under the low speed.
Use Clontech GenomeWalkerTMUniversal test kit, utilizes primer AP1 and GSP1-Hind III (sequence is respectively as shown in SEQ ID No:15 and SEQ ID No:16), to connect product as template, carries out first round amplification: 7 circulations: 94 DEG C of 25sec, 72 DEG C of 6min；32 circulations: 94 DEG C of 25sec, 67 DEG C of 6min；It is incubated 7 minutes then at 67 DEG C after last circulation.PCR primer dilutes after 50 times, carries out second with AP2 and GSP2-Hind III (sequence is respectively such as SEQ ID No:17 and SEQ ID No:18, shown) and takes turns PCR amplification, and PCR program is as follows: 5 circulate: 94 DEG C of 25sec, 72 DEG C of 5min；20 circulations: 94 DEG C of 25sec, 67 DEG C of 5min；It is incubated 10 minutes then at 67 DEG C after last circulation.Product reclaims order-checking.
Left margin (LB) terminal Sequence Analysis to aQ51-1 event, obtain the nucleotide sequence (sequence is as shown in SEQ ID No:19) of 2583bp altogether, it is the partial sequence of BHR5 including 1bp-243bp, 244bp-717bp is PS-Tnos sequence, 718bp-1332bp is BHR5 expression cassette to the carrier sequence inside LB, and 1333bp-2583bp is cotton DNA sequence.
Sequence illustrates:
RB terminal Sequence Analysis, according to the Cotton Gossypii flanking sequence obtained, diploid Cotton Gossypii (Gossypium raimondii) the D chromosome set genome sequence (http://www.phytozome.net/cotton.php) announced is utilized to be analyzed, design downstream primer RBflank51 ' (SEQ ID NO:20).With aQ51-1 genomic DNA as template, carrying out PCR amplification with RBflank51 ' and GSP2-NPTII31 ' (shown in SEQ ID No:21), PCR program is as follows: 35 circulations: 94 DEG C of 25sec, 67 DEG C of 5min；It is incubated 10 minutes then at 67 DEG C after last circulation.Product reclaims order-checking.
RB terminal Sequence Analysis to aQ51-1 event, obtain 2438bp nucleotide sequence (sequence such as SEQ ID No:22) altogether, including, 1bp-1153bp is cotton DNA sequence, 1154bp-1459bp is Pnos promoter sequence, 1460bp-1473bp is the catenation sequence between Pnos and NPTII, and 1474bp-2268bp is NPTII sequence, and 2269bp-2438bp is the catenation sequence between NPTII and Tnos.
Sequence illustrates:
With acceptor material Ji cotton 14DNA as template, respectively at RB and the LB side wing cotton gene group primers (sequence is respectively as shown in SEQ ID No:23 and SEQ ID No:24) of insertion sequence, obtain the amplified production that size is about 420bp, flanking sequence comparison with aQ51-1 finds, this event replaces what 60bp base in protogene group sequence obtained by insertion sequence.Comparison result is shown in Fig. 3.
According to result above, those skilled in the art can easily draw the characteristic DNA sequence (SEQ ID NO:25) of aQ51-1 event, as follows, underlining shown partially is T-DNA insertion sequence, and not underlining shown partially is the flank cotton genomic DNA sequence of insertion sequence.
Embodiment 5. transformation event detects
Using DNA primer to expand carrying out PCR to detect aQ51-1 event, described primer forms by the second primer of the arbitrary flanking sequence of insertion sequence described in the first primer of specific recognition T-DNA of the present invention insertion sequence and specific recognition.AQ51-1 insertion sequence and qualification primer are as described herein.Such as, when the first primer is aQ51-1-RB5 ' (SEQ ID NO:26), the second primer is aQ51-1-RB3 ' (SEQ ID NO:27)；When the first primer is aQ51-1LB5 ' (SEQ ID NO:28), the second primer is aQ51-1LB3 ' (SEQ ID NO:30).
Utilize above-mentioned primer to carrying out the method that PCR identifies that use is commonly used in the art.Utilize primer aQ51-1-RB5 ' (SEQ ID NO:26)/aQ51-1-RB3 ' (SEQ ID NO:27), aQ51-1LB5 ' (SEQ ID NO:28)/aQ51-1LB3 ' (SEQ ID NO:29) are expanded respectively aQ51-1T1 (1-9), Ji cotton 14-1, Ji cotton 14-2 cotton samples result as shown in Figure 4.
Embodiment 6. chromosome mapping
According to the Cotton Gossypii flanking sequence obtained, diploid Cotton Gossypii (Gossypium raimondii) the D chromosome set genome sequence (http://www.phytozome.net/cotton.php) announced is utilized to be analyzed, understand in aQ51-1 event, the Cotton Gossypii flanking DNA sequence engaged with insertion sequence and the sequence very high homology on D2 chromosome, thus, it can be known that the integration site of foreign DNA insertion sequence is positioned on D genome the 2nd group chromosome of receptor tetraploid cotton in aQ51-1 event.Its representative Cotton Gossypii (Gossypium raimondii) seed was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC on 05 28th, 2015, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica postcode: 100101), deposit number is: CGMCC No10490.
Other sequences are as follows:
Claims (10)
1. a Cotton Transformation event, it is characterised in that its DNA sequence is as shown in SEQ ID No:25, and it is by
The T-DNA insertion sequence of 1154-9044bp, the upstream flank cotton gene group sequence of 1-1153bp and
The downstream flank cotton gene group Sequence composition of 9045-10295bp.
2. a DNA sequence, described DNA sequence comprises the T-DNA of SEQ ID No:25 1154-9044bp
Insertion sequence and the flank cotton gene group sequence of SEQ ID No:25 1-1153bp and 9045-10295bp.
3. a recombinant vector, it contains the T-DNA insertion sequence of SEQ ID No:25 1154-9044bp；Example
As, described carrier is the pBI121-BHR4-BHR5 carrier in Fig. 2.
4. a reconstitution cell, containing the recombinant vector described in claim 3；Such as, described reconstitution cell is for containing having the right
Profit requires the recombinational agrobacterium cell of the recombinant vector described in 3.
5. being used for test right and require the primer pair of the Cotton Transformation event described in 1, it is by specific recognition SEQ ID No:
First primer of the flank cotton gene group sequence of 25 1-1153bp or 9045-10295bp and specific recognition SEQ
Second primer composition of the T-DNA insertion sequence of ID No:25 1154-9044bp.
6. the primer pair described in claim 5, the sequence of wherein said first primer is SEQ ID NO:26, described second
The sequence of primer is SEQ ID NO:27.
7. the primer pair described in claim 5, the sequence of wherein said first primer is SEQ ID NO:28, described second
The sequence of primer is SEQ ID NO:29.
8. the method identifying in Cotton Gossypii biological sample Cotton Transformation event described in claim 1, comprising:
A () is from Cotton Gossypii extraction from biological material DNA sample to be identified；
B (), with the DNA sample extracted as template, uses primer described in any one of claim 5-7 to carrying out PCR
Amplification；
(c) detection pcr amplification product, if amplified production length and described PCR primer pair on SEQ ID NO:25
Sequence between theoretical length consistent, then show the existence of transformation event in described Cotton Gossypii biological sample.
9. the method obtaining transgenic glyphosate resistant cotton material, described in include:
After cotton material containing the transformation event described in claim 1 and other cotton breeding materials are hybridized, enter
One step backcrosses, it is thus achieved that containing the new material of the transformation event described in claim 1；
In hybridization and backcross process, the method described in claim 8 is utilized to carry out Screening and Identification in progeny population, really
Recognize the existence of transformation event described in claim 1.
10. described in the transformation event described in claim 1, the DNA sequence described in claim 2, claim 3
Reconstitution cell described in recombinant vector, claim 4, the method described in claim 8 or 9 are used for improving Resistance Strain of Cotton grass
Sweet phosphine character, carry out cotton breeding and be used as molecular marker purposes.
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| CN103827305A (en) * | 2011-07-28 | 2014-05-28 | 吉奈克蒂夫公司 | Glyphosate tolerant corn event vco-phi1981-5 and kit and method for detecting the same |
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