CN104726489B

CN104726489B - The method that agriculture bacillus mediated live body rataria conversion obtains transgene cotton

Info

Publication number: CN104726489B
Application number: CN201510148155.4A
Authority: CN
Inventors: 俞嘉宁; 蔡云巧
Original assignee: Shaanxi Normal University
Current assignee: Shaanxi Normal University
Priority date: 2015-03-31
Filing date: 2015-03-31
Publication date: 2018-07-10
Anticipated expiration: 2035-03-31
Also published as: CN104726489A

Abstract

A kind of method that agriculture bacillus mediated live body rataria conversion obtains transgene cotton, by the stripping of sterile embryo, prepare conversion fluid, concussion is infected, prepare transgenosis cotton, transfer-gen plant authentication step form.In the stripping of sterile embryo, 30% hydrogen peroxide and sterile water are configured to thimerosal, cotton-seed ginning is put into thimerosal, 25 DEG C, impregnate 24 hours, seed is taken out to be placed in culture dish, kind of skin and 1/3~1/2 cotyledon are cut away, remaining cotton embryo is put into the culture dish containing basal medium, makes cotton embryo injured；In conversion fluid step is prepared, induction liquid is made of acetosyringone and magnesium sulfate, and acetosyringone can induce Agrobacterium Vir gene activations, improve the efficiency of exogenous origin gene integrator；Earthquake is infected in step, and Silwet L 77 and MS fluid nutrient mediums are added in conversion fluid, makes Agrobacterium is wider to be covered on cotton embryo.The present invention has many advantages, such as to be easy to screen, the growth cycle of cotton is short, easily operated, transformation efficiency.

Description

The method that agriculture bacillus mediated live body rataria conversion obtains transgene cotton

Technical field

The invention belongs to transgene cotton technical fields, and in particular to be turned to a kind of using agriculture bacillus mediated live body rataria Change the foundation for obtaining transgene cotton method.

Background technology

Cotton is important fibre crops, and fiber production accounts for the 40% of world's total fiber yield, and the plantation amount of upland cotton reaches To the 90% of total cotton.Cottonseed be only second to soybean, vegetable seed the vegetable seeds containing oils, therefore cotton accounts in industrial crops Critical role.

The quality of cotton fiber is improved by transgenic technology, it is to cultivate the important hand of new cotton variety to improve cottonseed oil productivity Section.1987, cotyledon Regenerated from Hypocotyl Explants method is infected by Agrobacterium, obtains transgenic cotton plant for the first time, then grain occur again Son bombardment embryo suspension cell method and pollen tube passage method obtain transgenic cotton.Wherein Agrobacterium infects cotyledon Regenerated from Hypocotyl Explants method not Limited by time, season, the advantages that foreign gene multi-copy integration probability is low, and transformation efficiency is higher and by numerous researchers blueness It looks at and more common a kind of cotton transgenic method at present, but this method also has certain limitation, such as：Easily by cotton seed Strain limits, and the cotton regenerated ability of different lines is different；The tissue growth period is long, and 8 are usually needed from hypocotyl to regeneration plant It was differed by 18 months；And deformity seedling is more, is susceptible to the variants such as cytometaplasia, organ variation；The seed vitality of regeneration plant Low defect.Therefore, it is necessary to a kind of convenient, fast, the cotton transgenic new method that transformation efficiency is high, applicability is wide.

Invention content

The shortcomings that technical problems to be solved by the invention are to overcome existing cotton transgenic technology provides a kind of side Just, fast, the method that the high agriculture bacillus mediated live body rataria conversion of transformation efficiency obtains transgene cotton.

Technical solution is made of following step used by solving above-mentioned technical problem：

(1) sterile embryo stripping

It is 1 by volume by 30% hydrogen peroxide and sterile water：9 are hybridly prepared into thimerosal, and cotton-seed ginning is put into thimerosal In, 25 DEG C, impregnate 24 hours, take out seed be placed in culture dish, cut away kind of skin and 1/2 cotyledon, remaining cotton embryo, which is put into, to be contained Have in the culture dish of basal medium, 1000mL basal mediums are by following raw material proportionings：

Mixing, tune pH for 5.8~6.3,0.07MPa, 115 DEG C sterilize 15 minutes in high-pressure steam sterilizing pan, be divided in In culture dish, natural cooling is prepared into basal medium.

(2) conversion fluid is prepared

100mg/L kanamycins is added in LB fluid nutrient mediums, 50mg/L rifampins, 100mg/L streptomysins, is contained The Agrobacterium LBA4404 of GFP expression vectors, LB fluid nutrient mediums and 50mg/mL kanamycins, 50mg/mL rifampins, 50mg/ ML streptomysins, Agrobacterium LBA4404 containing GFP expression vectors volume ratio be 1：0.002：0.001：0.002：0.1,28 DEG C, 200 revs/min of shaken cultivations 22~24 hours, be prepared into OD₆₀₀For 1.2~1.6 bacterium solution, 5000 revs/min of centrifugations 10 Minute, supernatant is removed, collects thalline, adds in MS fluid nutrient mediums and induction liquid, mixing, thalline and MS fluid nutrient mediums lure The volume ratio of drain is 1：10：0.1,28 DEG C, 200 revs/min of shaken cultivations 20 minutes, obtain conversion fluid.

GFP expression vectors are transformed into Agrobacterium LBA4404 with recombinant plasmid pBI121-GFP and obtain recombinational agrobacterium, institute It is that the small fragment between Xba I and Sac the I digestion recognition sites by pBI121 is substituted by GFP to state recombinant plasmid pBI121-GFP The obtained recombinant plasmid of sequence, the LB fluid nutrient mediums of above-mentioned 100mL are by following raw material proportionings：

Mixing, tune pH is 6.5,0.07MPa, 115 DEG C sterilize 15 minutes and be made in high-pressure steam sterilizing pan；Above-mentioned 100mL MS fluid nutrient mediums are by following raw material proportionings：

Sucrose 2g

MS culture mediums 0.443g

Water adds to 100mL

Mixing, tune pH is 5.8~6.3,0.07MPa, 115 DEG C sterilize 15 minutes and be made in high-pressure steam sterilizing pan；On The induction liquid stated is 1 by volume by the acetosyringone of 100 μm of ol/L and the magnesium sulfate of 10mmol/L：1 is mixed.

(3) concussion is infected

Silwet L-77 and MS fluid nutrient mediums are added in conversion fluid, Silwet L-77 and MS fluid nutrient mediums turn The volume ratio for changing liquid is 1：4000~6000：4000~6000 are configured to infect liquid, and the cotton embryo of preparation is put into and infects liquid, and 28 DEG C, 200 revs/min of shaken cultivations 3 hours, obtain the cotton embryo containing Agrobacterium.

(4) prepare transgenosis cotton

Basal medium sterilizes 15 minutes in 0.07MPa, 115 DEG C of high-pressure steam sterilizing pan, is cooled to 45~50 DEG C, Add in 100mg/L kanamycins, 500mg/L cephalosporins, mixing, basal medium and 50mg/mL kanamycins, 500mg/mL The volume ratio of cephalosporin is 1：0.002：0.001, screening and culturing medium is configured to, is distributed into culture dish, Agrobacterium will be contained Cotton embryo moves into culture dish, be put into 24 ± 1 DEG C, intensity of illumination 24000lux, humidity be 51% incubator in, illumination 16 is small When, it is 8 hours dark, it cultivates 2~3 days, the cotton seedling for selecting growing way excellent is moved into the tissue culture bottle for promoting root media, the training of growth-promoting root It supports base to be sterilized 15 minutes in 0.07MPa, 115 DEG C of high-pressure steam sterilizing pan by basal medium, is cooled to 45~50 DEG C, adds Enter 1.25mg/L 6- benzyl purines, 0.5mg/L methyl α-naphthyl acetates, 100mg/L kanamycins, 500mg/L cephalosporins, mixing, basis Culture medium and 1.25mg/mL 6- benzyl purines, 0.5mg/mL methyl α-naphthyl acetates, 50mg/mL kanamycins, 500mg/mL cephalosporins Volume ratio be 1：0.001：0.001：0.002：0.001, it after the completion of preparation, is dispensed into tissue culture bottle, kind is had into cotton seedling Tissue culture bottle be put into 24 ± 1 DEG C, intensity of illumination 24000lux, humidity be 51% incubator in, illumination 16 hours, dark 8 Hour, it cultivates 2~3 weeks, the cotton seedling for choosing true leaf as green is grafted onto on the cotton stock of growth 2~3 weeks, and acquisition turns base Because of cotton.

(5) transfer-gen plant is identified

PCR Molecular Detections, sequencing are carried out to the cotton seedling green true leaves extraction DNA of grafting 1~3 month；To grafting 1~ March, the true leaf of cotton seedling extracted RNA, reverse transcription cDNA, carried out PCR Molecular Detections, and it is glimmering to graft cotton seedling progress in 1~3 month Light is observed.

In step (3) is infected in the concussion of the present invention, Silwet L-77 and MS fluid nutrient mediums are added in conversion fluid, The volume ratio of Silwet L-77 and MS fluid nutrient mediums, conversion fluid is 1：5000：5000 are configured to infect liquid, by the cotton of preparation Flower embryo is put into and infects liquid, and 28 DEG C, 200 revs/min of shaken cultivations 3 hours obtain the cotton embryo containing Agrobacterium.

It is 1 by volume by 30% hydrogen peroxide and sterile water in being removed in sterile embryo：9 are hybridly prepared into Thimerosal, cotton-seed ginning are put into thimerosal, 25 DEG C, impregnate 24 hours, take out seed be placed in culture dish, cut away kind of skin and 1/ 3~1/2 cotyledons, remaining cotton embryo are put into the culture dish containing basal medium, since cotton embryo is by cutting damage, are had It adsorbs and infects conducive to Agrobacterium；In conversion fluid step is prepared, induction liquid by 100 μm of ol/L acetosyringone with The magnesium sulfate of 10mmol/L is made, and acetosyringone can induce Agrobacterium Vir gene activations, improves the effect of exogenous origin gene integrator Rate；In step is infected in the concussion of the present invention, Silwet L-77 and MS fluid nutrient mediums are added in conversion fluid, can be increased thin Cellular surface permeability promotes conversion of the Agrobacterium to cotton blastocyte.The present invention overcomes agriculture bacillus mediated to infect under cotyledon The shortcomings that in plumular axis conversion method to the limitation of cotton strain and long growth cycle, the present invention have it is easily operated, be easy to screen, turn The advantages that growth cycle of gene cotton is short, transformation efficiency is high.

Description of the drawings

Fig. 1 be 24 ± 1 DEG C, intensity of illumination 24000lux, humidity be 51% incubator in, illumination 16 hours is black Dark 8 hours, the cotton seedling photo of culture 20 days.

Fig. 2 is that grafting grows 1 month cotton seedling true leaf DNA level PCR testing result photo.

Fig. 3 is that grafting grows 1 month cotton seedling true leaf rna level PCR testing result photo.

Fig. 4 is 1 month cotton seedling inverted fluorescence microscope observation fluorescence results photo of grafting.

Specific embodiment

The present invention is described in more detail with reference to the accompanying drawings and examples, but the present invention is not limited to these Examples.

Embodiment 1

The method that the present embodiment line transformation obtains transgene cotton is made of following step：

1st, sterile embryo stripping

30% hydrogen peroxide 10mL and sterile water 90mL in the triangular flask of sterilizing are mixed, are configured to thimerosal, cotton seed takes off Suede is put into thimerosal, 25 DEG C, impregnate 24 hours, take out seed be placed in culture dish, cut away kind of skin and 1/2 cotyledon, it is remaining Cotton embryo is put into the culture dish containing basal medium, and 1000mL basal mediums are by following raw material proportionings：

2nd, conversion fluid is prepared

20 μ L of 50mg/mL kanamycins, 10 μ L of 50mg/mL rifampins, 50mg/mL are added in LB fluid nutrient mediums 10mL That is mould for 20 μ L of streptomysin, Agrobacterium LBA4404 1000 μ L, LB fluid nutrient mediums and 100mg/L cards containing GFP expression vectors Element, 50mg/L rifampins, 100mg/L streptomysins, GFP expression vectors Agrobacterium LBA4404 volume ratio be 1：0.002： 0.001：0.002：0.1,28 DEG C, 200 revs/min of shaken cultivations 22~24 hours, are prepared into OD₆₀₀For 1.2~1.6 bacterium solution, 5000 revs/min centrifuge 10 minutes, remove supernatant, collect thalline, take 1mL thalline, add in MS fluid nutrient mediums 10mL and lure The volume ratio of 100 μ L of drain, mixing, thalline and MS fluid nutrient mediums, induction liquid is 1：10：0.1,28 DEG C, 200 revs/min is shaken Culture 20 minutes is swung, obtains conversion fluid.

Above-mentioned GFP expression vectors, which are transformed into recombinant plasmid pBI121-GFP in Agrobacterium LBA4404, obtains recombination agriculture bar Bacterium, the recombinant plasmid pBI121-GFP are that the small fragment between Xba I and Sac the I digestion recognition sites by pBI121 replaces The recombinant plasmid that sequence for GFP obtains.PBI121 plant expression vectors are purchased from Beijing Baeyer enlightening Bioisystech Co., Ltd, goods Number：MP-091；The gene order number of GFP is GenBank:AB303068.1.The LB fluid nutrient mediums of above-mentioned 100mL are by following Raw material proportioning：

Mixing, tune pH is 6.5,0.07MPa, 115 DEG C sterilize 15 minutes and be made in high-pressure steam sterilizing pan.Above-mentioned 100mL MS fluid nutrient mediums are by following raw material proportionings：

Sucrose 2g

MS culture mediums 0.443g

Water adds to 100mL

Mixing, tune pH is 5.8~6.3,0.07MPa, 115 DEG C sterilize 15 minutes and be made in high-pressure steam sterilizing pan.On The induction liquid stated is 1 by volume by the acetosyringone of 100 μm of ol/L and the magnesium sulfate of 10mmol/L：1 is mixed.

3rd, concussion is infected

It takes and 2 μ L and MS fluid nutrient mediums 10mL, Silwet L-77 of Silwet L-77 and MS liquid is added in conversion fluid 10mL Body culture medium, conversion fluid volume ratio be 1：5000：5000 are configured to infect liquid, and the cotton embryo of preparation is put into and infects liquid, and 28 DEG C, 200 revs/min of shaken cultivations 3 hours, obtain the cotton embryo containing Agrobacterium.Silwet L-77 are purchased from Canadian GE Healthcare Bio-Sciences AB companies, catalog number MB-K5762.

4th, prepare transgenosis cotton

Basal medium 200mL sterilizes 15 minutes in 0.07MPa, 115 DEG C of high-pressure steam sterilizing pan, it is cooled to 45~ 50 DEG C, add in 50mg/mL kanamycins 0.4mL, 500mg/mL cephalosporin 0.2mL, mixing, basal medium and 100mg/L Kanamycins, 500mg/L cephalosporins volume ratio be 1：0.002：0.001, screening and culturing medium is configured to, is distributed into culture Cotton embryo containing Agrobacterium is moved into culture dish by ware, be put into 24 ± 1 DEG C, intensity of illumination 24000lux, humidity be 51% In incubator, illumination 16 hours is 8 hours dark, cultivates 2~3 days, and the cotton seedling for selecting growing way excellent, which moves into, promotees culture of rootage In the tissue culture bottle of base, promote root media and gone out in 0.07MPa, 115 DEG C of high-pressure steam sterilizing pan by basal medium 200mL Bacterium 15 minutes, is cooled to 45~50 DEG C, add in 1.25mg/mL6- benzyl purine 0.2mL, 0.5mg/mL methyl α-naphthyl acetates 0.2mL, 50mg/mL kanamycins 0.4mL, 500mg/mL cephalosporin 0.2mL, mixing, basal medium and 1.25mg/L6- benzyls are fast Purine, 0.5mg/L methyl α-naphthyl acetates, 100mg/L kanamycins, 500mg/L cephalosporins volume ratio be 1：0.001：0.001： 0.002：0.001, it is configured to, is dispensed into tissue culture bottle, the tissue culture bottle equipped with cotton seedling is put into 24 ± 1 DEG C, intensity of illumination In the incubator for being 51% for 24000lux, humidity, illumination 16 hours is 8 hours dark, cultivates 2~3 weeks, cotton can be observed Seedling true leaf is green, as shown in Figure 1, as seen from Figure 1,24 ± 1 DEG C, intensity of illumination 24000lux, humidity be 51% In incubator, illumination 16 hours is 8 hours dark, and the cotton seedling of culture 20 days can be in the culture medium containing that resistance of 100mg/L cards Tissue culture bottle in grow, can tentatively filter out positive transgenic cotton seedling.By true leaf for green cotton seedling be grafted onto growth 2~ On the cotton stock of 3 weeks, transgene cotton is obtained.

5th, transgenic cotton seedling is identified

PCR Molecular Detections are carried out to cotton seedling true leaf extraction DNA, sequencing is as follows：

It extracts wild type cotton seedling and grafts plant leaf DNA of the cotton seedling true leaf for green, and set simultaneously PBI121 plasmids are positive control, and PCR amplification is carried out according to the GFP genes design pair of primers on plasmid.

PCR amplification primer：

Sense primer：5′-CCGGCTCGTATGTTGTGTGGA-3′；

Downstream primer：5′-AAGTTGGGTAACGCCAGGGT-3′.

PCR amplification system：Each 1 μ L, 2 × Taq PCR Mix, 7.5 μ L, 0.1-0.25 μ g/ μ of upstream and downstream primer 20mmol/L The 1 μ L of template of L, deionized water supply system to 15 μ L.

PCR amplification program：94 DEG C of pre-degenerations 5 minutes, 94 DEG C are denaturalized 1 minute, and 55 DEG C are annealed 30 seconds, and 72 DEG C extend 1 point 30 Second, totally 30 recycle；72 DEG C extend 10 minutes.

The amplified production of PCR is detected with 1% agarose gel electrophoresis, and purposeful band is that the sample of 2016bp contains GFP Gene, the results are shown in Figure 2, and the 1st swimming lane is DNA maker, and from top to bottom stripe size is followed successively by 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp；2nd swimming lane is negative control (24 in wild type)；3rd~8 swimming Road is six plants of positive transgenic cotton seedlings tentatively filtered out；9th swimming lane is positive control (pBI121)；From Figure 2 it can be seen that preliminary sieve The positive transgenic cotton seedling and positive control (pBI121) selected it is amplifiable go out purpose band, by PCR Molecular Detections The cotton seedling that six plants of positives tentatively filtered out turn GFP contains target gene in DNA level, is further determined as positive transgenic cotton Seedling.Purpose band is recycled, sequencing company is sent to be sequenced, sequencing result is shown in SEQ ID No.3.The result shows that external source GFP genes turn Enter in positive transgenic cotton seedling.

RNA, reverse transcription cDNA are extracted to green true leaves, carry out PCR Molecular Detections；

Extraction wild type cotton seedling and grafting cotton seedling true leaf RNA, reverse transcription cDNA, Molecular Detection program are same On, the results are shown in Figure 3, and the 1st swimming lane is DNA maker, and band is same as above；2nd swimming lane is negative control (24 in wild type)；The 3rd, 4 swimming lanes are determined as positive transgenic cotton seedling for wherein two plants；7th swimming lane is positive control (pBI121)；As seen from Figure 3, into The transgenic cotton seedling and positive control (pBI121) that one step filters out it is amplifiable go out purpose band, by PCR Molecular Detections two The positive cotton seedling for turning GFP of strain contains target gene in rna level, and foreign gene can be in RNA in positive transgenic cotton seedling Horizontal expression.

Acquisition one month cotton seedling true leaf of grafting, is placed under inverted fluorescence microscope, is seen under 490nm blue light exciting lights The expression of GFP fluorescence is examined, observed result is as shown in Figure 4.From fig. 4, it can be seen that there is green fluorescence in cotton seedling true leaf, in albumen water GFP successful expressions in flat cotton seedling true leaf green fluorescent protein.

Embodiment 2

In sterile embryo strip step 1,30% hydrogen peroxide 10mL and sterile water 90mL are mixed in the triangular flask of sterilizing, Be configured to thimerosal, cotton-seed ginning is put into thimerosal, 25 DEG C, impregnate 24 hours, take out seed be placed in culture dish, cut away kind Skin and 1/3 cotyledon, remaining cotton embryo are put into the culture dish containing basal medium, the raw material proportioning and system of basal medium Preparation Method is same as Example 1.

In conversion fluid step 2 is prepared, 20 μ L of 50mg/mL kanamycins, 50mg/ are added in LB fluid nutrient mediums 10mL 10 μ L of mL rifampins, 20 μ L of 50mg/mL streptomysins, 1000 μ L, the GFP expression of Agrobacterium LBA4404 containing GFP expression vectors Carrier is transformed into Agrobacterium LBA4404 with recombinant plasmid pBI121-GFP and obtains recombinational agrobacterium, the recombinant plasmid PBI121-GFP is that small fragment between Xba I and Sac the I digestion recognition sites by pBI121 is substituted by the sequence of GFP and obtains Recombinant plasmid, LB fluid nutrient mediums and 100mg/L kanamycins, 50mg/L rifampins, 100mg/L streptomysins, GFP expression carry The volume ratio of the Agrobacterium LBA4404 of body is 1：0.002：0.001：0.002：0.1,28 DEG C, 200 revs/min of shaken cultivations 22 ~24 hours, it is prepared into OD₆₀₀For 1.2~1.6 bacterium solution, 5000 revs/min centrifuge 10 minutes, remove supernatant, collect bacterium Body takes thalline 1mL, adds in MS fluid nutrient mediums 5mL and induction 50 μ L of liquid, mixing, and thalline and MS fluid nutrient mediums induce liquid Volume ratio is 1：5：0.05,28 DEG C, 200 revs/min of shaken cultivations 20 minutes, obtain conversion fluid.LB fluid nutrient mediums, MS liquid Culture medium, the raw material proportioning for inducing liquid and preparation method are same as Example 1.

Earthquake is infected in step 3, takes and 1.25 μ L and MS fluid nutrient mediums of Silwet L-77 are added in conversion fluid 5mL The volume ratio of 5mL, Silwet L-77 and MS fluid nutrient mediums, conversion fluid is 1：4000：4000 are configured to infect liquid, will prepare Cotton embryo be put into and infect liquid, 28 DEG C, 200 revs/min of shaken cultivations 3 hours obtain the cotton embryo containing Agrobacterium.

Other steps are same as Example 1, are prepared into line transformation and obtain transgene cotton.

Embodiment 3

In sterile embryo strip step 1,30% hydrogen peroxide 10mL and sterile water 90mL are mixed in the triangular flask of sterilizing, Be configured to thimerosal, cotton-seed ginning is put into thimerosal, 25 DEG C, impregnate 24 hours, take out seed be placed in culture dish, cut away kind Skin and 1/2 cotyledon, remaining cotton embryo are put into the culture dish containing basal medium, the raw material proportioning and system of basal medium Preparation Method is same as Example 1.

In conversion fluid step 2 is prepared, 20 μ L of 50mg/mL kanamycins, 50mg/ are added in LB fluid nutrient mediums 10mL 10 μ L of mL rifampins, 20 μ L of 50mg/mL streptomysins, 1000 μ L, the GFP expression of Agrobacterium LBA4404 containing GFP expression vectors Carrier is transformed into Agrobacterium LBA4404 with recombinant plasmid pBI121-GFP and obtains recombinational agrobacterium, the recombinant plasmid PBI121-GFP is that small fragment between Xba I and Sac the I digestion recognition sites by pBI121 is substituted by the sequence of GFP and obtains Recombinant plasmid, LB fluid nutrient mediums and 100mg/L kanamycins, 50mg/L rifampins, 100mg/L streptomysins, GFP expression carry The volume ratio of the Agrobacterium LBA4404 of body is 1：0.002：0.001：0.002：0.1,28 DEG C, 200 revs/min of shaken cultivations 22 ~24 hours, it is prepared into OD₆₀₀For 1.2~1.6 bacterium solution, 5000 revs/min centrifuge 10 minutes, remove supernatant, collect bacterium Body takes thalline 1mL, adds in MS fluid nutrient mediums 15mL and induction 200 μ L of liquid, mixing, thalline and MS fluid nutrient mediums, induction liquid Volume ratio be 1：15：0..2,28 DEG C, 200 revs/min of shaken cultivations 20 minutes, obtain conversion fluid.LB fluid nutrient mediums, MS Fluid nutrient medium, the raw material proportioning for inducing liquid and preparation method are same as Example 1.

Earthquake is infected in step 3, takes and 2 μ L and MS fluid nutrient mediums of Silwet L-77 are added in conversion fluid 12mL The volume ratio of 12mL, Silwet L-77 and MS fluid nutrient mediums, conversion fluid is 1：6000：6000 are configured to infect liquid, will prepare Cotton embryo be put into and infect liquid, 28 DEG C, 200 revs/min of shaken cultivations 3 hours obtain the cotton embryo containing Agrobacterium.

Other steps are same as Example 1, are prepared into agriculture bacillus mediated live body rataria conversion and obtain transgene cotton.

Claims

1. a kind of method that agriculture bacillus mediated live body rataria conversion obtains transgene cotton, it is characterised in that by following step group Into：

（1）Sterile embryo stripping

It is 1 by volume by 30% hydrogen peroxide and sterile water：9 are hybridly prepared into thimerosal, and cotton-seed ginning is put into thimerosal, and 25 DEG C, impregnate 24 hours, take out seed and be placed in culture dish, cut away kind of skin and 1/2 cotyledon, remaining cotton embryo is put into containing basic In the culture dish of culture medium, 1000mL basal mediums are by following raw material proportionings：

Sucrose 20g

MS culture mediums 4.43g

Agar 5g

Water adds to 1000mL

Mixing, tune pH for 5.8~6.3,0.07MPa, 115 DEG C sterilize 15 minutes in high-pressure steam sterilizing pan, be divided in culture In ware, natural cooling is prepared into basal medium；

（2）Prepare conversion fluid

100mg/L kanamycins is added in LB fluid nutrient mediums, 50mg/L rifampins, 100mg/L streptomysins, contains GFP tables Up to the Agrobacterium LBA4404 of carrier, LB fluid nutrient mediums and 50mg/mL kanamycins, 50mg/mL rifampins, 50mg/mL strepto-s The volume ratio of Agrobacterium LBA4404 plain, containing GFP expression vectors is 1：0.002：0.001：0.002：0.1,28 DEG C, 200 Rev/min shaken cultivation 22~24 hours, is prepared into OD₆₀₀For 1.2~1.6 bacterium solution, 5000 revs/min centrifuge 10 minutes, go Except supernatant, thalline is collected, adds in MS fluid nutrient mediums and induction liquid, mixing, thalline and MS fluid nutrient mediums, the body for inducing liquid Product is than being 1：10：0.1,28 DEG C, 200 revs/min of shaken cultivations 20 minutes, obtain conversion fluid；

GFP expression vectors are transformed into Agrobacterium LBA4404 with recombinant plasmid pBI121-GFP and obtain recombinational agrobacterium, described heavy It is the sequence that the small fragment between Xba I and Sac the I digestion recognition sites by pBI121 is substituted by GFP to organize plasmid pBI121-GFP Obtained recombinant plasmid is arranged, the LB fluid nutrient mediums of above-mentioned 100mL are by following raw material proportionings：

Tryptone 1g

0.5 g of yeast extract

NaCl 1g

Water adds to 100mL

Sucrose 2g

MS culture mediums 0.443g

Water adds to 100mL

Mixing, tune pH is 5.8~6.3,0.07MPa, 115 DEG C sterilize 15 minutes and be made in high-pressure steam sterilizing pan；Above-mentioned It by the acetosyringone of 100 μm of ol/L and the magnesium sulfate of 10mmol/L is 1 by volume to induce liquid：1 is mixed；

（3）Concussion is infected

Silwet L-77 and MS fluid nutrient mediums, Silwet L-77 and MS fluid nutrient mediums, conversion fluid are added in conversion fluid Volume ratio be 1：4000~6000：4000~6000 are configured to infect liquid, and the cotton embryo of preparation is put into and infects liquid, 28 DEG C, 200 revs/min of shaken cultivations 3 hours, obtain the cotton embryo containing Agrobacterium；

（4）Prepare transgenosis cotton

Basal medium sterilizes 15 minutes in 0.07MPa, 115 DEG C of high-pressure steam sterilizing pan, is cooled to 45~50 DEG C, adds in 100mg/L kanamycins, 500mg/L cephalosporins, mixing, basal medium and 50mg/mL kanamycins, 500mg/ mL heads The volume ratio of p0-357 is 1：0.002：0.001, screening and culturing medium is configured to, is distributed into culture dish, by the cotton containing Agrobacterium Flower embryo moves into culture dish, be put into 24 ± 1 DEG C, intensity of illumination 24000lux, humidity be 51% incubator in, illumination 16 hours, It is 8 hours dark, it cultivates 2~3 days, the cotton seedling for selecting growing way excellent is moved into the tissue culture bottle for promoting root media, promotees culture of rootage Base is sterilized 15 minutes by basal medium in 0.07MPa, 115 DEG C of high-pressure steam sterilizing pan, is cooled to 45~50 DEG C, is added in 1.25mg/L 6- benzyl purines, 0.5mg/L methyl α-naphthyl acetates, 100mg/L kanamycins, 500mg/L cephalosporins, mixing, basis training Support base and 1.25mg/mL 6- benzyl purines, 0.5mg/mL methyl α-naphthyl acetates, 50mg/mL kanamycins, 500mg/mL cephalosporins Volume ratio is 1：0.001：0.001：0.002：0.001, it after the completion of preparation, is dispensed into tissue culture bottle, kind is had into cotton seedling Tissue culture bottle be put into 24 ± 1 DEG C, intensity of illumination 24000lux, humidity be 51% incubator in, illumination 16 hours, dark 8 is small When, it cultivates 2~3 weeks, the cotton seedling for choosing true leaf as green is grafted onto on the cotton stock of growth 2~3 weeks, obtains transgenosis Cotton；

（5）Transfer-gen plant is identified

PCR Molecular Detections, sequencing are carried out to the cotton seedling green true leaves extraction DNA of grafting 1~3 month；To grafting for 1~March True leaf extraction RNA, the reverse transcription cDNA of cotton seedling, carry out PCR Molecular Detections, graft 1~3 month cotton seedling and carry out fluorescence sight It surveys.

2. the method that agriculture bacillus mediated live body rataria conversion according to claim 1 obtains transgene cotton, feature It is：Step is infected in earthquake（3）In, Silwet L-77 and MS fluid nutrient mediums, Silwet L-77 are added in conversion fluid Volume ratio with MS fluid nutrient mediums, conversion fluid is 1：5000：5000 are configured to infect liquid, and the cotton embryo of preparation is put into and is infected Liquid, 28 DEG C, 200 revs/min of shaken cultivations 3 hours, obtains the cotton embryo containing Agrobacterium.