CN102533845A

CN102533845A - Agrobacterium tumefaciens-medicated genetic transformation method of wheat

Info

Publication number: CN102533845A
Application number: CN2011104593604A
Authority: CN
Inventors: 李新玲; 闫玉清; 徐香玲
Original assignee: Harbin Normal University
Current assignee: Harbin Normal University
Priority date: 2011-12-31
Filing date: 2011-12-31
Publication date: 2012-07-04
Anticipated expiration: 2031-12-31
Also published as: CN102533845B

Abstract

The invention discloses an agrobacterium tumefaciens-medicated genetic transformation method of wheat. The agrobacterium tumefaciens-medicated genetic transformation method comprises the following steps of: firstly, inoculating recombinant Agrobacterium tumefaciens containing a target DNA (Deoxyribonucleic Acid) into wheat immature embryos; and then co-culturing the inoculated wheat immature embryos for enabling the T-DNA of the recombinant Agrobacterium tumefaciens to be integrated with a chromosome of the wheat; and secondly, directly inoculating the wheat immature embryos co-cultured in the first step in a long-seedling culture medium for culturing to obtain transgenic wheat seedlings with roots, stems and leaves. The method disclosed by the invention has the advantages that the applicability is wide, so that the method is suitable for transforming various wheat species; the method is simple in operation, and thus a transgenic plant can be obtained without inducing a tissue culture and regenerating the plant; and the transformation period is short, i.e. the process from the infection to the obtaining of the transgenic wheat seedlings with the roots, the stems and the leaves only requires 11-14 days. The method has very important significance for genetic improvement on the wheat species and the obtaining of new species of the transgenic wheat.

Description

A kind of agriculture bacillus mediated wheat genetic method for transformation

Technical field

The present invention relates to a kind of agriculture bacillus mediated wheat genetic method for transformation.

Background technology

Wheat is one of important cereal crop of depending on for existence of the mankind, and worldwide cultivated area and ultimate production have all surpassed other farm crop, and its genetic improvement is the important goal that people make great efforts all the time.Traditional wheat breeding technology exists limitation such as breeding cycle is long partially, yield potential is limited, along with development of biology, utilizes genetic engineering technique that wheat is carried out breed improvement and more and more comes into one's own.Aspect the research of plant gene conversion system, wheat shows hysteresis quality and difficulty with respect to other cereal crop such as paddy rice, corn etc.The wheat genetic Study on Transformation starts from phase late 1980s; Because wheat is not the natural host of Agrobacterium; And wheat protoplast regeneration is difficult relatively again; So successfully GUS and Bar gene are imported the wheat callus through particle bombardment up to people such as Vasil in 1992, and obtain after the transfer-gen plant, the report of relevant wheat transgenic just begins to increase.At present, transgenic paddy rice has got into semipilot and safety evaluation stage, and the research of wheat transgenic obviously falls behind, and also is in the foundation of transformation system and improves the stage.

In the plant genetic engineering, utilizing agriculture bacillus mediated transgenosis is maximum, the clearest, the most sophisticated gene transformation approach of technological method of transformation mechanism of research at present.Compare with the particle gun conversion method, the Agrobacterium-mediated Transformation method has a lot of advantages, and as can importing big fragment goal gene, and mostly goal gene is single copy or low copy; Get rid of redundant plasmid dna sequence easily; Insert the border sequence mostly border is T-DNA, the rearrangement rate is low etc.Can this method is used for monocotyledonous transgenosis such as wheat becomes the focus of research before and after the nineties.At that time, a kind of tendentious viewpoint was to think that monocotyledons is not the natural host of Agrobacterium, utilized the agrobacterium mediation converted monocotyledons maybe or not have possibility hardly.The representative figure of this viewpoint is the Switzerland scientist Potrykus that achievement was once arranged at cereal crop tissue culture such as wheat and particle gun conversion aspect Zhuo, and he has delivered his pessimistic point of view on internal authority magazine Bio/Technology and Nature.Acquisition along with agriculture bacillus mediated officinalis transfer-gen plant; The particularly agriculture bacillus mediated transgenic paddy rice and the acquisition of transgenic corn plant; Not only having changed monocotyledons is not the viewpoint of Agrobacterium natural host, and the proof agrobacterium-mediated transformation can be used for the genetic transformation of gramineous crop fully.Yet the Agrobacterium-mediated Transformation of wheat remains a difficult problem in the world up to now.In recent years; The research group of lot of domestic and international has carried out a large amount of research and exploration from multi-angles such as wheat genotypes, agrobacterium strains, common culture condition, explant type; Propose a lot of cover genetic conversion systems, but all had no a cover to be used widely.

At present, mostly the wheat explant that is used for agrobacterium mediation converted is rataria and the formed embryo callus of mature embryo, and they all need pass through in vitro tissue and cultivate the transfer-gen plant of regenerating.Yet the different genotype of wheat has very big influence for the regeneration of plant, and this has further limited through the tissue culture approach and has obtained transgenic wheat.

Summary of the invention

The purpose of this invention is to provide a kind of agriculture bacillus mediated wheat genetic method for transformation, this method is without the step that callus forms, and has wide adaptability, simple to operate, advantage that the transformation period is short.

Method provided by the present invention is to change target DNA over to wheat cell through agriculture bacillus mediated, comprises the steps:

The reorganization Agrobacterium that 1) will contain target DNA is inoculated in wheat immature embryo, then postvaccinal said wheat immature embryo is carried out common cultivation and makes the T-DNA of said reorganization Agrobacterium and the chromosomal integration of said wheat;

2) will pass through the said wheat immature embryo direct inoculation that step 1) cultivates altogether and in long seedling substratum, cultivate, promptly obtain having root, the seedling of stem and leaf.

In aforesaid method, after Agrobacterium is infected, be prone to brownization death in order to prevent wheat immature embryo, before the said reorganization Agrobacterium that will contain target DNA of step 1) is inoculated in wheat immature embryo, said wheat immature embryo also pass through 12-24 hour, like 24 hours preparatory cultivations;

Said preparatory cultivation specifically can be carried out according to the method that comprises the steps: with said wheat immature embryo with preparatory culture medium 25 ℃ down dark cultivate 12-24 hour, as 24 hours; The basic medium of said preparatory culture medium can be the MS substratum.

In aforesaid method, said Agrobacterium can be Agrobacterium rhizogenes, like Agrobacterium rhizogenes R1000.

In aforesaid method, said wheat immature embryo is 13-15 days the said wheat immature embryo in back of blooming.

In aforesaid method, the said reorganization Agrobacterium that will contain target DNA of step 1) is inoculated in wheat immature embryo, carries out according to the method that comprises the steps: said wheat immature embryo was soaked in the bacterium liquid of said reorganization Agrobacterium 5～10 minutes, as 5 minutes; The OD of said reorganization Agrobacterium bacterium liquid ₆₀₀Be 0.5～0.6, as 0.6.

In aforesaid method, between said soak period, contain the said bacterium liquid 20～30s of said wheat immature embryo with ultrasonication, like 20s, said frequency of ultrasonic is that 27kHz, power are 80w.

In aforesaid method; The said cultivation altogether of step 1) specifically can be carried out according to the method that comprises the steps: the said postvaccinal said wheat immature embryo of step 1) is cultivated with being total to culture medium; In order to prevent said postvaccinal said wheat immature embryo browning; In the said substratum of cultivating altogether, can add inhibitor, said inhibitor specifically can be WR 34678; In order to promote the said T-DNA of Agrobacterium and the chromosomal integration of said wheat of recombinating, in the said substratum of cultivating altogether, also can add Syringylethanone.

In aforesaid method, the said time of cultivating altogether of step 1) was 2-3 days, as 3 days.

In aforesaid method; In order to remove non-transformed cell; In step 2) can contain the selective pressure of screening transformant in the described long seedling substratum, in order to suppress growth of Agrobacterium, in step 2) also can add the microbiotic that suppresses the Agrobacterium growth in the described long seedling substratum.

In aforesaid method, said preparatory culture medium specifically can be and contains hydrolysis casein 1.0g/L and 2, the MS substratum of 4-D 2mg/L, pH5.8; The said substratum of cultivating altogether specifically can be the MS substratum that contains Syringylethanone 150 μ mol/L and WR 34678 1g/L, pH5.2; Said long seedling substratum specifically can be the MS substratum that contains cephamycin 200mg/L and Totomycin 100mg/L, pH5.8.

Do not comprise the step that forms callus in the method for the present invention.

Method of the present invention can be applicable to all wheats, is particularly suitable for common wheat.

Method of the present invention has the following advantages: wide adaptability is suitable for the conversion of a plurality of wheat breeds; Simple to operate, can obtain transfer-gen plant without the step of callus induction and plant regeneration; Transformation period is short, only needs 11-14 days from infecting to the transgenic wheat seedling that obtains to have root, stem and leaf.The present invention has crucial meaning for the genetic improvement of wheat breed, the new germ plasm of acquisition transgenic wheat.

Description of drawings

Fig. 1 is T ₀PCR for the transgenic wheat plant detects.Wherein, 1 is molecular weight standard DL2000 (being followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom), and 2-4 is respectively blank, positive control, negative control, and 5-9 is respectively the T of each kind ₀For the agricultural 7742-24 in transfer-gen plant east, Hite-17, imperial wheat 9814-41, imperial wheat 26-5, imperial spoke wheat 10-32.

Fig. 2 is T ₀Southern hybridization for the transgenic wheat plant detects.Wherein, 1-3 is respectively positive control, negative control, molecular weight standard pBR328/BamH I+Bg I+Hinf I (being followed successively by 4907bp, 2176bp, 1766bp, 1230bp, 1033bp and 653bp from top to bottom); 4-8 is respectively the T of each kind ₀For the transgenic positive individual plant: eastern agricultural 7742-24, Hite-17, imperial wheat 9814-41, imperial wheat 26-5 and imperial spoke wheat 10-32.

Embodiment

Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.

Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.

Wheat breed used in the present invention, plasmid and Agrobacterium rhizogenes are following in detail:

East farming 7742: Li Xinling, Xu Xiangling, Zhang Nannan. the wheat descendant inheritting that changes chitinase gene is analyzed and the disease resistance evaluation. and Plant Physiology Communications, 2008,44 (1): 75-78, the public can obtain from Harbin Teachers' Univ..

Hite: Li Xinling, Qu Min, Xu Xiangling, Li Ji face the research of the plasmid-mediated chitinase gene transformed wheat of .Ri. and plant research, 2006,26 (3): 297-301, the public can obtain from Harbin Teachers' Univ..

Dragon wheat 9814: Li Xinling, Qu Min, Yan Yuqin, Xu Xiangling. influence the research of wheat mature embryo cultivation and plant regeneration factor. plant research, 2005,25 (1): 49-52, the public can obtain from Harbin Teachers' Univ..

Dragon wheat 26: Li Xinling, Qu Min, Yan Yuqin, Xu Xiangling. influence the research of wheat mature embryo cultivation and plant regeneration factor. plant research, 2005,25 (1): 49-52, the public can obtain from Harbin Teachers' Univ..

Dragon spoke wheat 10: Xing Quanhua, Wang Guangjin, Shi Jinfeng, Wang Yueguang; Li Zhongjie, beam Fengshan, Jin Demin, Wang Bin. β-1; The structure of 3-glucanase gene efficient expression vector reaches the conversion to wheat. and Acta Genetica Sinica, 2003,30 (8): 717-722, the public can obtain from Harbin Teachers' Univ..

Plasmid pMLH7133-Chi: Li Xinling, Qu Min, Xu Xiangling, Li Ji face the research of the plasmid-mediated chitinase gene transformed wheat of .Ri. and plant research, 2006,26 (3): 297-301, the public can obtain from Harbin Teachers' Univ..

Agrobacterium rhizogenes R1000 (Agrobacterium rhizogenes strain R1000): Li Xinling, Qu Min, Xu Xiangling; Li Ji faces the research of the plasmid-mediated chitinase gene transformed wheat of .Ri. plant research; 2006,26 (3): 297-301, the public can obtain from Harbin Teachers' Univ..

The prescription such as the table 1 of basic medium MS substratum used in the present invention:

Table 1.MS culture medium prescription

YEB liquid nutrient medium: Carnis Bovis seu Bubali cream 5g/L, yeast powder 1g/L, sucrose 5g/L, peptone 5g/L, MgSO ₄494.94mg/L, pH7.2.

YEB solid medium: add agar 10g/L in the above-mentioned YEB liquid nutrient medium.

Embodiment 1, utilize Agrobacterium rhizogenes that wheat is carried out chitinase gene to transform

One, the structure that contains the reorganization Agrobacterium rhizogenes of target DNA

The recombinant vectors that is used for transforming agrobacterium rhizogenes is pMLH7133-Chi, and the goal gene on this carrier is chitinase gene (Chi), and the label screening gene on this carrier is hygromix phosphotransferase (HPT) gene.

Change recombinant vectors pMLH7133-Chi over to reorganization Agrobacterium rhizogenes R1000/pMLH7133-Chi that Agrobacterium rhizogenes R1000 (Agrobacterium rhizogenes strain R1000) obtains containing chitinase gene.Concrete grammar is following: the single bacterium colony of (1) picking Agrobacterium rhizogenes R1000 from the YEB solid plate that contains the 50mg/L Streptomycin sulphate, and be inoculated in and contain in the 10ml YEB liquid nutrient medium test tube of (containing the 50mg/L Streptomycin sulphate), 28 ℃ of shaking culture are spent the night; (2) get 400 μ l bacterium liquid and be inoculated in 40ml and contain in the YEB liquid nutrient medium of 50mg/L Streptomycin sulphate, cultivate 4-6h to logarithmic phase (OD ₆₀₀=0.5); (3) ice bath 30min gets 1.5ml bacterium liquid and joins in the aseptic centrifuge tube, and the centrifugal 5min of 13000rpm abandons supernatant; (4) deposition is with 800 μ l 150mmol/L CaCl ₂Again suspend, 4 ℃, the centrifugal 5min of 3000rpm abandons supernatant; (5) deposition is with 800 μ l 20mmol/L CaCl ₂Again suspend, divide by every pipe 200 μ l to install in the aseptic centrifuge tube of 1.5ml; (6) in 200 μ l Agrobacterium competent cells, add 1 μ g recombinant vectors pMLH7133-Chi, place 30min on ice; (7) freezing 2min in the liquid nitrogen places 37 ℃ of water-bath 2min immediately after the taking-up; (8) add 800 μ l YEB liquid nutrient mediums, 28 ℃ of shaking culture 1-2h; (9) get 200 μ l and coat on the YEB solid medium that contains 50mg/L kantlex, 50mg/L Streptomycin sulphate, replace DNA to establish a negative contrast with sterilized water simultaneously, put in 28 ℃ of incubators; (10) single bacterium colony of the above-mentioned cultivation acquisition of picking, shaking culture in 28 ℃ of liquid YEB substratum is extracted DNA, carries out pcr amplification and identifies reorganization Agrobacterium rhizogenes R1000/pMLH7133-Chi, and the method for pcr amplification is identical with following step 3.

Two, the reorganization Agrobacterium rhizogenes transformed wheat that contains target DNA

Rataria with wheat east farming 7742, Hite, imperial wheat 9814, imperial wheat 26, imperial spoke wheat 10 is an explant respectively; The reorganization Agrobacterium rhizogenes R1000/pMLH7133-Chi that utilization contains chitinase gene (Chi) changes chitinase gene in the wheat cell over to; Agrobacterium rhizogenes R1000 not contain plasmid pMLH7133-Chi is converted into contrast, and method for transformation is following:

1, explant obtains and cultivates in advance: take away and spend 13-15 days the wheat immature seed in back; Under the aseptic condition, earlier with 70% ethanol disinfection 30s, 0.1%HgCl is used in aseptic washing 3 times again ₂Sterilization 12min, aseptic washing 5 times; From immature seed, separate then and long be the rataria of 1.5mm-2.0mm, be inoculated on the preparatory culture medium, 25 ℃, secretly cultivated 24 hours;

Preparatory culture medium: contain 1.0g/L caseinhydrolysate and 2mg/L 2, the MS substratum of 4-D, pH5.8.

2, infect cultivation together: will be soaked in OD through the pre-incubated rataria of step 1 ₆₀₀Be 0.6 the 5min in the liquid that infects that contains reorganization Agrobacterium rhizogenes R1000/pMLH7133-Chi, during be respectively the ultrasonication 20s of 27kHz and 80w with frequency and power, be inoculated on the common culture medium 25 ℃, secretly cultivated 3 days then;

The preparation method who infects liquid is following: get the reorganization Agrobacterium rhizogenes R1000/pMLH7133-Chi bacterium liquid that step 1 obtains; Streak inoculation is on the YEB solid medium that contains 50mg/L Streptomycin sulphate and 50mg/L Totomycin; 28 ℃ of dark cultivations 1 day, picking list bacterium colony is inoculated in 5mL and contains in the YEB liquid nutrient medium of 50mg/L Streptomycin sulphate and 50mg/L Totomycin; 28 ℃, dark, 200rpm shaken overnight cultivation; Again this 5mL nutrient solution all is added to 100ml and contains enlarged culturing in the YEB liquid nutrient medium of 150 μ mol/L Syringylethanones, 50mg/L Streptomycin sulphate and 50mg/L Totomycin, when treating that Agrobacterium grows into logarithmic phase, room temperature, the centrifugal 5min of 4000rpm collect thalline; Be resuspended in the MS substratum that contains 150 μ mol/L Syringylethanones, transfer OD ₆₀₀Value is 0.6;

Be total to culture medium: contain the MS substratum of 150 μ mol/L Syringylethanones and 1g/L WR 34678, pH5.2.

3, long seedling: the wheat immature embryo that will cultivate altogether through step 2 is with sterile water wash 2-3 time; Be inoculated in and contain on the antibiotic long seedling substratum; 25 ℃, illumination every day 16 hours (intensity of illumination 4000Lux), dark 8 hours, cultivated 7-10 days, acquisition has the transgenic wheat seedling of root, stem and leaf;

Long seedling substratum: contain the MS substratum of 200mg/L cephamycin and 100mg/L Totomycin, pH5.8.

4, transplant strong sprout: the transgenic wheat seedling replanting that step 3 is obtained in the little alms bowl that contains vermiculite, water every day 1/10 do not contain agar MS substratum and regularly moisturizing, hot-house culture obtains T ₀For the transgenic wheat plant.

The rataria of each article grow wheat is after the Agrobacterium rhizogenes R1000 that does not contain plasmid pMLH7133-Chi infects, and all dead after the screening of step 3, no seedling obtains.The T that infects rataria number, acquisition of each article grow wheat ₀Statistics for transgenic wheat plant number is seen table 2.

Three, the PCR of transgenic wheat detects

1, pcr template: each T that the embodiment 1 that extracts with the SDS method obtains ₀For total DNA of transgenic wheat plant leaf, with the wheat breed that does not carry out Agrobacterium-mediated Transformation of SDS method extraction eastern agricultural 7742 total DNA of plant leaf (negative control) and plasmid pMLH7133-Chi (positive control).

2, PCR primer: two terminal sequences of chitinase gene: 5 '-GCA GTG TGG AAG GCA AGC AG-3 ' (sequence 1 in the sequence table) and 5 '-CTG AGA GGT GAC AAG GTC AG-3 ' (sequence 2 in the sequence table).

3, PCR program: the PCR response procedures is: 94 ℃ of 5min, 95 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 60s, 30 circulations, 72 ℃ of 5min again.

4, result: the purpose clip size of pcr amplification is 1100bp; 1% agarose gel electrophoresis detects the PCR product of each sample template, obtains east agricultural 7742 transgenic positive plant 5 strains altogether, 6 strains of Hite positive plant; Dragon wheat 9814 is 2 strains, and imperial wheat 26 is 3 strains, and imperial spoke wheat 10 is 3 strains; Average conversion is 1.02%, and the result is shown in Fig. 1 and table 2.

The detailed process that above-mentioned SDS method is extracted DNA is following: the about 100mg of regeneration plant spire is got in (1), puts into the 1.5ml centrifuge tube, and liquid nitrogen or silica sand grind; (2) add extraction damping fluid 600 μ l, mixing, room temperature reaction 30min; (3) 4 ℃, the centrifugal 5min of 13000rpm gets supernatant; (4) add isopyknic phenol chloroform (phenol: chloroform=1: 1), put upside down 3-5min gently; (5) 4 ℃, the centrifugal 5min of 13000rpm carefully draws supernatant; (6) add isopyknic chloroform, put upside down 3-5min gently; (7) 4 ℃, the centrifugal 5min of 13000rpm, water is with equal-volume Virahol or 2 times of volume absolute ethyl alcohol alcohol precipitations, and slowly mixing is put more than the 10min for 4 ℃.This step can spend the night; (8) 4 ℃, the centrifugal 10min of 13000rpm abandons supernatant, with 70% washing with alcohol deposition and drain; (9) add 30-50 μ l TE dissolution precipitation, add 37 ℃ of temperature of 1 μ l RNase A and bathe 30min; (10) get 5-10 μ l DNA electrophoresis detection.

Extract damping fluid: 200mmol/L Tris-HCl, 250mmol/L NaCl, 25mmol/L EDTA, 0.5%SDS, pH7.5.

TE：10mmol/L(pH8.0)Tris-HCl，1mmol/L(pH8.0)EDTA。

Transformation efficiency=(PCR positive plant number/infect rataria number) * 100%.

Table 2.T ₀Pcr amplification result for transgenic wheat

Genotype	The rataria number that infects	T ₀For transgenic wheat plant number	PCR positive plant number	Transformation efficiency (%)
					East farming 7742	494	159	?5	1.01
Hite	408	153	?6	1.47
					Dragon wheat 9814	251	95	?2	0.79
Dragon wheat 26	290	82	?3	1.03

Dragon spoke wheat 10	411	126	3	0.73
					Add up to	1854	615	19	1.02

Four, the Southern of transgenic wheat hybridization

1, the preparation of probe: use digoxigenin labeled detection kit (Roche; Cat.No.11745832910) synthetic hybridization probe; Method is following: (with plasmid pMLH7133-Chi is template to add 1 μ g chitinase gene DNA; The pcr amplification product that obtains according to the method for step 3) in the 0.2ml centrifuge tube, uses ddH ₂O replenishes and makes final volume to 16 μ l; Heating 10min makes the DNA sex change also fast in cooled on ice in boiling water bath; Add 4 μ l Mix Dig-High Prime, mixing is also of short duration centrifugal, places 1h for 37 ℃; EDTA (pH=8.0) or the 65 ℃ heating 10min that add 2 μ l 0.2M are with termination reaction, and-20 ℃ frozen subsequent use.

2, Southern hybridization: because there are the single endonuclease digestion site in restriction enzyme BamHI and SacI on the T-DNA of plasmid pMLH7133-Chi; And these two restriction enzyme sites are positioned at the both sides of chitinase gene (1.1kb), and this experiment is cut 19 T that step 1 obtains with restriction enzyme BamHI and SacI while enzyme ₀For the total DNA of transgenic wheat plant leaf, plasmid pMLH7133-Chi (positive control), the total DNA of plant leaf (negative control) that do not carry out the wheat breed east farming 7742 of Agrobacterium-mediated Transformation; (molecular cloning experiment guide (third edition), Science Press 2005, p495-499) carry out Southern hybridization and detect according to the method for Sa nurse Brooker.

3, result: as shown in Figure 2.19 T that PCR is positive ₀The hybrid belt about 1 1.1kb all occurred for the transgenic wheat plant, and the negative control plant there is not any hybrid belt.

Five, the chitinase activity of transgenic wheat is measured

With disease-resistant relevant chitinase mainly is endochitinase, and the chitinase of entrained bean chitinase gene coding also is an endochitinase among the plasmid pMLH7133-Chi.Adopt the GS method to measure 8 strain T respectively ₀For the chitinase activity of transgenic wheat plant (transformant) blade, be contrast with the wheat plant (contrast strain) that does not carry out Agrobacterium-mediated Transformation respectively, measuring method is following:

1, the extraction of chitinase and processing

1. take by weighing T respectively ₀For the blade 2g of transgenic wheat plant with the contrast strain, put into the mortar of precooling, and add 6ml 0.1M sodium citrate buffer solution (pH5.0) and a little silica sand, grind to form homogenate in the ice bath; 2. 4 ℃, the centrifugal 15min of 12000rpm, supernatant is crude enzyme liquid; 3. get the 1ml crude enzyme liquid, add 1ml 0.5% chitosan (pH6.0) and 2ml 0.2M sodium-acetate (pH5.2), mixing.Do blank with the chitosan and the sodium-acetate mixed solution that do not contain crude enzyme liquid simultaneously; 4. 37 ℃ of water-bath 2h, 4 ℃, the centrifugal 5min of 12000rpm, it is subsequent use to get supernatant.

0.5% chitosan: soak 4h with 5% Glacial acetic acid min. 99.5 in advance, after abundant dissolving, transfer pH6.0 with NaOH.

2, circumscribed chitinase activity is measured

1. get supernatant that 1ml step 1 obtains in test tube, add 1ml H ₂O and 1ml methyl ethyl diketone reagent shake up; 2. seal the test tube mouth with glass sphere, heat 20min in the boiling water, be cooled to room temperature then; 3. add 5ml absolute ethyl alcohol and 1ml 1% paradimethy laminobenzaldehyde (DMAB reagent), complement to TV 10ml with absolute ethyl alcohol again, fully mixing; 4. 65 ℃ of-70 ℃ of water-bath 10min quicken CO ₂Discharge, be cooled to room temperature then, the 530nm place measures the solution light absorption value.Water replaces the crude enzyme liquid of each sample to do blank.

Methyl ethyl diketone reagent: 1ml heavily steams methyl ethyl diketone and is dissolved in 50ml 0.5M Na ₂CO ₃In the solution, preparation before using.

DMAB reagent: 0.8g is right-and the dimethylamino benzaldehyde is dissolved in the 30ml ethanol, adds the 30ml concentrated hydrochloric acid, and it is faint yellow that solution is.

3, total chitinase activity is measured

1. get supernatant that 1ml step 1 obtains in test tube, add 100 μ l 1M phosphoric acid buffers (pH7.1) and 70 μ l, 3% Snailase, shake up; 2. 37 ℃ of water-bath 2h, adding water to TV is 2ml; 3. 65 ℃ of-70 ℃ of water-bath 10min quicken CO ₂Discharge, be cooled to room temperature, the 530nm place measures the solution light absorption value.Water replaces the crude enzyme liquid of each sample to do blank.

4, the endochitinase vigor calculates

(X represents OD according to typical curve equation: Y=329.82X-3.3249 ₅₃₀Value, Y represents GS amount (μ g), linear correlation coefficient r=0.9923), the total chitinase activity of each sample that mensuration is obtained and the OD of circumscribed chitinase activity ₅₃₀Value is converted into GS output respectively, produces 1 μ g GS and is designated as an enzyme activity unit (U) per hour to decompose the colloid chitosan again.

According to formula: endochitinase vigor (U)=total chitinase activity (U)-circumscribed chitinase activity (U);

5, result: see table 3.The result shows, the T of different varieties ₀Endochitinase vigor for the transgenic wheat plant all is higher than the contrast strain significantly, and is more than 2 times of strain of contrast at least, and the chitinase gene that external source is described is at T ₀For expressing in the transgenic wheat.

The endochitinase vigor of table 3. transgenic wheat

Claims

1. cultivate the method for transgenic wheat, change target DNA over to wheat cell, it is characterized in that, said method comprising the steps of through agriculture bacillus mediated:

2) will pass through the said wheat immature embryo direct inoculation that step 1) cultivates altogether and in long seedling substratum, cultivate, promptly obtain having root, the transgenic wheat seedling of stem and leaf.

2. method according to claim 1 is characterized in that: before the said reorganization Agrobacterium that will contain target DNA of step 1) was inoculated in wheat immature embryo, said wheat immature embryo also passed through 12-24 hour preparatory cultivation.

3. method according to claim 1 and 2 is characterized in that: said Agrobacterium is an Agrobacterium rhizogenes.

4. according to arbitrary described method among the claim 1-3, it is characterized in that: said wheat immature embryo is 13-15 days the said wheat immature embryo in back of blooming.

5. according to arbitrary described method among the claim 1-4; It is characterized in that: the said reorganization Agrobacterium that will contain target DNA of step 1) is inoculated in wheat immature embryo; Carry out according to the method that comprises the steps: said wheat immature embryo was soaked in the bacterium liquid of said reorganization Agrobacterium 5～10 minutes, as 5 minutes; The OD of said reorganization Agrobacterium bacterium liquid ₆₀₀Be 0.5～0.6, as 0.6.

6. according to arbitrary described method among the claim 1-5, it is characterized in that: in said immersion, contain the said bacterium liquid 20～30 seconds of said wheat immature embryo, as 20 seconds with ultrasonication.

7. according to arbitrary described method among the claim 1-6, it is characterized in that: the said time of cultivating altogether of step 1) was 2-3 days, as 3 days.

8. according to arbitrary described method among the claim 1-7, it is characterized in that: said wheat is a common wheat.

9. according to arbitrary described method among the claim 1-8, it is characterized in that: step 2) selective pressure of screening transformant arranged in the described long seedling substratum.

10. according to arbitrary described method among the claim 1-9, it is characterized in that: do not comprise the step that forms callus in the said method.