CN102559746B - Method for obtaining transgenic wheat by impregnating young ears through agrobacterium tumefaciens - Google Patents
Method for obtaining transgenic wheat by impregnating young ears through agrobacterium tumefaciens Download PDFInfo
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Abstract
The invention relates to the field of plant transgenic engineering, in particular to a method for obtaining transgenic wheat by impregnating young ears through agrobacterium tumefaciens. The method for obtaining the transgenic wheat by impregnating the young ears through the agrobacterium tumefaciens comprises the following steps of: 1) culturing agrobacterium tumefaciens strains containing expression vectors which carry target genes, and suspending the strains by using an impregnating culture medium; and 2) selecting the wheat at a heading stage for transformation, removing underdeveloped small ears at the top ends and the tail ends of the young ears, shearing about 1/3 of the top ends of the wheat, and soaking the young ears of the wheat into the impregnating culture medium containing the agrobacterium tumefaciens. The method is easy and convenient to operate, saves manpower and material resources, and is high in transformation rate which can reach 26.3 to 82.3 percent; and the impregnated plants can normally grow and fruit, so the method has significance for theoretical research and genetic breeding practice on the aspect of wheat gene engineering.
Description
Technical field
The present invention relates to the transgenic plant genetic engineering field, particularly, relate to a kind of method that obtains transgenic wheat by infecting young ears with agrobacterium.
Background technology
Wheat is the annual or per nnial herb of Gramineae (Gramineae) Triticum (Tritucum), be extensively plant in the world, important food crop that ultimate production is only second to corn.The cultivation of new variety of wheat at present is still mainly by conventional breeding.Because the conventional hybridization breeding cycle is long, means are single, species hybridization is difficult, and efficient is lower, foresight is poor, cause that filial generation genetic background is narrow, the seed selection difficulty increases, make wheat breeding level difficulty that breakthrough raising be arranged, breeding is made slow progress.
The breed improvement that utilizes the modern genetic engineering technology to carry out wheat is a new direction of current wheat breeding.Its main means refer to utilize means such as biology and physical chemistry, with the plant transgenic technology of exogenous gene transfered plant cell with the acquisition transgenic plant.Since nineteen eighty-three Zambryski obtains in the world the first strain transfer-gen plant, the plant transgenic technology fast development, this also makes the gene engineering technique breeding become possibility.It can break the reproduction isolation, makes that the heredity interchange becomes possibility between different plant species, can utilize gene pool to create condition for widening plant, has remedied the deficiency of conventional hybridization breeding, has accelerated the development of Crop Genetic Breeding.
At present, nearly all crop has all been carried out transgenic technology research, and it is PEG method and electrization, ion-beam mediated method, pollen tube passage method, particle bombardment and the agrobacterium-mediated transformation etc. of acceptor that the method that has developed has with the protoplastis.Wherein, because it is simple, do not need expensive equipment with the vehicular method of Agrobacterium, cost is low, the transformation frequency height, and advantage such as copy number is few, and genetic expression stability is better more and more is subjected to the favor of researcher.Dicotyledons is the natural host of Agrobacterium, utilizes the mediation of Agrobacterium Ti-plasmids to change some foreign genes over to dicotyledons, and has set up the gene transfer system of multiple dicotyledons.But because monocotyledons is not the natural host of Agrobacterium, monocotyledonous explant regeneration ability is low in addition, competent cell lacks and genetic transformation instability etc., has limited the application of this method on gramineous crop.Researchdevelopment to wheat transgenic in recent years is very fast, has also obtained certain achievement, but compares with cereal crop such as paddy rice, corn and dicotyledons, still has bigger gap.Wheat is compared the special difficult point of carrying out transgenic breeding and is mainly shown a kind of method of gene introduction efficiently of shortage with other crops.Secondly, protoplastis is cultivated and plant regeneration is only limited to indivedual genotype, and the genetic stability of regeneration plant it be unclear that.And wheat itself is allohexaploid, and its genome is bigger and complicated, and it is reticent with to modify phenomenon serious to import foreign gene.
Good explant material during children's fringe transforms as wheat genetic, its callus induction rate and induce quality all to be better than rataria.Yet the genetic transformation of young fringe also need pass through tissue culture, and this just inevitably will be through carrying out the process that tissue culture obtains regeneration plant owing to single transformant.For fear of this process, this method is just arisen at the historic moment in conjunction with the live body conversion.
Agriculture bacillus mediated infecting young ears method for transformation is a kind of easy, quick, efficient, the good reproducibility that gets up of developed recently, the non-tissue culture transgenic method that stability is high.Its biggest advantage is directly to obtain the seed that transforms, tissue culture and succeeding transfer culture have been avoided, got rid of in the tissue culture and given the correct expression of goal gene and totally unfavorable genetic background that molecule genetics research brings because of somatic variation, the new way of transgenosis is provided for some agrotypes that are difficult for setting up hereditary regeneration system simultaneously.
What road first-class (2003) splashes into Agrobacterium among the wheat Xiao Hua transforms, and carries out molecular level by PCR and Southern hybridization and identifies that finally obtaining transformed plant efficient is 2.0%～3.2%.The method that Zale etc. (2009) soak young fringe with Agrobacterium has successfully transformed North America hard red spring wheat Crocus, and can the stable expression of exogenous gene.Transformation efficiency is 0.44%～6.8%.(2011) are carried out Agrobacterium with reference to the method for (2009) such as Zale to a plurality of current leading winter wheat varieties such as northern China winter wheat district's polling 987, section's farmings 199 and are infected conversion and all obtain to transform seed or plant yet leaf is made the country prosperous etc.; the method that (2009) such as Zale are described may and be not suitable for the genetic transformation to the current leading winter wheat variety of China, need carry out technique improvement according to the characteristic of China's wheat.And, utilize the transformation efficiency of agriculture bacillus mediated young fringe method acquisition transgenic wheat also unsatisfactory at present, and used kind mainly is spring wheat.Therefore, press for method at a kind of stable, efficient, the easy acquisition transgenic wheat of the current leading wheat breed of China
Summary of the invention
The purpose of this invention is to provide a kind of method that obtains transgenic wheat by infecting young ears with agrobacterium.
According to the method by infecting young ears with agrobacterium acquisition transgenic wheat of the present invention, may further comprise the steps:
1) cultivate the agrobacterium strains that comprises the expression vector that is loaded with goal gene, with dip-dye substratum suspension thalline,
Wherein, arrive OD with YEB substratum incubated overnight
600Be 1.5～2.0, centrifugal, use behind the precipitation thalline and contaminate substratum suspension thalline to OD
600=0.6～0.8, described YEB substratum is yeast extract 1g/L, extractum carnis 5g/L, Tryptones 5g/L, sucrose 5g/L, MgSO47H
2O 0.5g/L, pH7.0;
2) wheat at heading stage of choosing greenhouse or field planting transforms, remove young fringe top and terminal embryotic small ear, cut off wheat growth vertex of a cone end about 1/3, wheat children tassel is immersed 1～2min in the dip-dye substratum that contains Agrobacterium, contaminate the back and preserve moisture with the glassine paper bagging, prevent the plant intermolecular hybrid simultaneously.
Select young fringe to be directly to obtain the seed that transforms as transformation receptor, the small ear top is cut off about 1/3 can make bacterium liquid fully contact with gynoecium among the young fringe Xiao Hua, raising changes into power, remains with enough lepicena simultaneously and has guaranteed that also seed can not come off.
According to the method by infecting young ears with agrobacterium acquisition transgenic wheat of the present invention, wherein, described agrobacterium strains is EHA105 or GV3101.
According to the method by infecting young ears with agrobacterium acquisition transgenic wheat of the present invention, wherein, described carrier is pBI121.
According to the method that obtains transgenic wheat by infecting young ears with agrobacterium of the present invention, wherein, described dip-dye substratum is for being minimum medium with MS, add MES buffer 0.5mM, Syringylethanone (AS) 200 μ M and tensio-active agent Silwet L-77 0.04%～0.06%, pH5.8～6.0.
According to the method by infecting young ears with agrobacterium acquisition transgenic wheat of the present invention, wherein, described wheat is winter wheat variety Ji wheat 38 or all wheats 16.
According to the present inventionly obtain the method for transgenic wheat by infecting young ears with agrobacterium, wherein, described heading stage wheat be the wheat growth awl part expose and the wheat children tassel when from sheath, extracting out fully as yet.
The insertion that studies show that T-DNA with the Agrobacterium-mediated Transformation Arabidopis thaliana is that a kind of allos is inserted, this also just show conversion may occur in flower development late period and embryo reach full growth finish before, and at the conversion aspect of Arabidopis thaliana, acquired genetic transformation plant mostly before blooming 5-10d carry out During Agrobacterium.Through test of many times, we select the wheat children tassel at heading stage, carry out Agrobacterium-mediated Transformation when the existing part of its growing tip is exposed and do not extracted out from sheath fully as yet, have not only obtained transfer-gen plant, and have increased substantially transformation efficiency.
The present invention's During Agrobacterium wheat children tassel, successfully foreign gene is changed in the winter wheat, do not need through genetic transformation processes such as protoplastis cultivation, cell cultures, tissue culture and plant regenerations, thereby the cycle of having avoided other method is long, shortcomings such as the somatic variation possibility is big, callus embryo regenerative power difference.The present invention is simple to operation, use manpower and material resources sparingly, and transformation efficiency height, can reach 26.3%～82.3%, the plant after the dip-dye can normal growth, solid, and this theoretical investigation and genetic breeding practice for wheat cdna engineering aspect has great importance.
Description of drawings
Fig. 1 is the T-DNA zone of plasmid pBI121-OsDREB2.2.
Fig. 2 is the wheat before contaminating.
Fig. 3 is the ripe fringe portion of the transfer-gen plant that obtains.
Fig. 4 is OsDREB2.2 transfer-gen plant T
0For the PCR detected result.1, dna molecular amount mark; 16, positive plasmid contrast pBI121-OsDREB2.2; 17, negative empty carrier contrast pBI121; 15, do not infect wild-type wheat breed plant; 14, PCR water; 2-13, Agrobacterium-mediated Transformation T
0For the seed plant.The positive purpose band of arrow indication.
Fig. 5 is OsDREB2.2 transfer-gen plant T
1For the PCR detected result.1, dna molecular amount mark; 14, positive plasmid contrast pBI121-OsDREB2.2; 15, negative empty carrier contrast pBI121; 13, do not infect wild-type wheat breed plant; 12, PCR water; 2,7-11 is for respectively from different T
0T for seed
1For plant; 3-4,5-6 is for respectively from same T
0Different T for seed
1For plant.The positive purpose band of arrow indication.
Embodiment
1. the preparation of vegetable material
Plant in the laboratory Ji wheat 38, all wheats 16 wheat breeds or the land for growing field crops, normal management is to beginning heading.
2. the conversion of Agrobacterium
1) (contain corresponding microbiotic: Rif30mg/ml), 28 ℃ of shaking culture are spent the night in the LB of 10ml liquid nutrient medium for difference picking agrobacterium tumefaciens EHA105 and GV3101 mono-clonal.
2) get incubated overnight bacterium liquid 2ml and be inoculated in 50ml LB (the containing Rif30mg/ml) liquid nutrient medium, 28 ℃ of shaking culture are to OD
600Be 0.6～0.7.
3) bacterium liquid branch is filled in the 10ml pipe of 4 sterilizations ice bath 10min.Then in 4 ℃, 5000rpm, centrifugal 5min.Remove supernatant, precipitate with the ice-cold CaCl of 200 μ l 20mM
2Resuspended, 4 ℃, 5000rpm, centrifugal 5min.Precipitate with the ice-cold CaCl of 200 μ l 20mM
2Resuspended again, be filled in the 1.5ml centrifuge tube of 8 precoolings by the volume integral of every pipe 100 μ l.
4) (EHA105, GV3101) mixing behind the liquid nitrogen flash freezer 2min, place 37 ℃ of water-baths, incubation 5min immediately with 100 μ l Agrobacterium competent cells respectively to get recombinant plasmid (pBI121-OsDREB2.2) 5 μ l.
Wherein, OsDREB2.2 (laboratory name), GenBank Acession No.AY064403 (Oryza sativa DREB1mRNA) has resisting abiotic environment stress function.
5) add 1ml and do not contain antibiotic LB liquid nutrient medium, 28 ℃, 150rpm cultivates 3h.The centrifugal 30s of 4000rpm removes most of supernatant, stays about 100ul bacterium liquid, and is resuspended.
6) 100 μ l bacterium liquid all are applied to LB flat board (30mg/ml Rif, 100mg/ml Kan), are inverted for 28 ℃ and cultivate.Grow single bacterium colony behind 2～3d.
3. the evaluation of positive colony
The single bacterium colony that grows on the picking flat board is inoculated in the LB liquid nutrient medium (30mg/ml Rif, 100mg/ml Kan), and 28 ℃ of shaking culture are spent the night; Extracting plasmid DNA in a small amount, is that template is carried out the pcr amplification evaluation with the plasmid DNA.According to CaMV 35S promoter sequences Design upstream primer 35S on the pBI121 carrier, according to gene order design downstream primer Os2.2-R, amplified fragments 496bp.The positive colony of identifying is protected bacterium in-70 ℃ of refrigerators.The primer sequence is 35S:5 '-GACGCACAATCCCACTATCC-3 ' (SEQ ID NO.1); Os2.2-R:5 '-GGGACCAT ACATTGCCCTTGCC-3 ' (SEQ ID NO.2).
4. Agrobacterium is cultivated
Contaminate wheat children tassel and from-70 ℃ of refrigerators, took out the Agrobacterium that contains goal gene in preceding 3 days, rule in containing on the corresponding antibiotic LB solid medium, activation culture 2 days, the picking mono-clonal adds 2ml and contains 28 ℃ of shaken overnight cultivations the corresponding antibiotic YEB liquid nutrient medium from substratum then, adds an amount of YEB liquid nutrient medium and continues to cultivate 6～8h in second day.The YEB substratum is yeast extract 1g/L, extractum carnis 5g/L, Tryptones 5g/L, sucrose 5g/L, MgSO47H
2O 0.5g/L sterilizes behind the adjusting pH7.0.
5. the genetic transformation of wheat
As the Agrobacterium OD that cultivates
600When reaching 1.5 left and right sides, the centrifugal 15min of 4500rpm collects thalline, abandons supernatant, with contaminating the resuspended precipitation of substratum, makes last OD
600Reach 0.6～0.8.Wherein, described dip-dye substratum adds MES buffer 0.5mM, Syringylethanone 200 μ M and tensio-active agent Silwet L-77 0.04%～0.06%, pH5.8～6.0 for being minimum medium with MS.
Bloomed preceding 4～7 days at wheat, remove young fringe top and terminal embryotic small ear, it is about 1/3 to cut off wheat growth vertex of a cone end, and the wheat children tassel of having pruned (Fig. 2) is immersed 1～2min in the Agrobacterium bacterium liquid, uses the cellophane bags bagging then.
6. the wheat normal management after transforming, the results seed.Get blade extraction DNA after the plantation and carry out PCR detection acquisition transfer-gen plant, detected result such as Fig. 4, transfer-gen plant such as Fig. 3.
7. s-generation seed is detected, whether the checking goal gene genetic stability.
With embodiment 1 described method, carry out During Agrobacterium in wheat children tassel different steps at heading stage, the result is as shown in table 2.As known from Table 2, do not contaminate before wheat children tassel goes out sheath, wheat heteroplasia can not be set seeds; Contaminate when young fringe partly goes out sheath, wheat is grown normal, sets seeds, and can obtain transfer-gen plant.
Table 1T
0For the transfer-gen plant detected result
Table 2 dip-dye is compared period
Annotate: * expression is not set seeds;
√ represents to set seeds, and positive seedling is arranged.
Claims (5)
1. the method by infecting young ears with agrobacterium acquisition transgenic wheat is characterized in that, may further comprise the steps:
1) cultivates the agrobacterium strains that comprises the expression vector that is loaded with goal gene, with contaminating substratum suspension thalline;
2) choosing the heading stage wheat transforms, remove young fringe top and terminal embryotic small ear, cut off wheat small ear top 1/3, wheat children tassel immersed contain in the dip-dye substratum of Agrobacterium, described heading stage wheat be the wheat growth awl part expose and the wheat children tassel when from sheath, extracting out fully as yet.
2. according to the described method by infecting young ears with agrobacterium acquisition transgenic wheat of claim 1, it is characterized in that described agrobacterium strains is EHA105 or GV3101.
3. according to the described method by infecting young ears with agrobacterium acquisition transgenic wheat of claim 1, it is characterized in that described expression vector is pBI121.
4. according to the described method by infecting young ears with agrobacterium acquisition transgenic wheat of claim 1, it is characterized in that described wheat is winter wheat variety Ji wheat 38 or all wheats 16.
5. according to the described method that obtains transgenic wheat by infecting young ears with agrobacterium of claim 1, it is characterized in that, described dip-dye substratum is for being minimum medium with MS, add MES damping fluid 0.5mM, Syringylethanone 200 μ M and tensio-active agent Silwet L-770.04%～0.06%, pH5.8～6.0.
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| CN102229946A (en) * | 2011-05-30 | 2011-11-02 | 山东农业大学 | Improved transforming method of wheat young ears mediated by agrobacterium tumefaciens and application thereof |
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Non-Patent Citations (6)
| Title |
|---|
| Evidence for stable transformation of wheat by floral dip in Agrobacterium tumefaciens;Janice M. Zale;《Plant Cell Rep》;20091231;903-913 * |
| Janice M. Zale.Evidence for stable transformation of wheat by floral dip in Agrobacterium tumefaciens.《Plant Cell Rep》.2009,903-913. |
| 叶兴国.小麦转基因方法及其评述.《遗传》.2011,422-430. |
| 小麦组织培养再生系统及农杆菌介导的转基因技术研究;朱晋云;《植物遗传资源学报》;20081231;第9卷(第1期);84-89 * |
| 小麦转基因方法及其评述;叶兴国;《遗传》;20110531;422-430 * |
| 朱晋云.小麦组织培养再生系统及农杆菌介导的转基因技术研究.《植物遗传资源学报》.2008,第9卷(第1期),84-89. |
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