CN102618573A - Soybean efficient genetic transformation method - Google Patents

Abstract

The invention discloses a soybean efficient genetic transformation method, which includes the following steps: (1) guiding plasmids containing target deoxyribonucleic acid (DNA) into a soybean explant in a particle bombardment method; and (2) guiding the plasmids containing target DNA into the soybean explant finished with the step (1) in an agrobacterium mediated method, wherein the explant is an embryo point explant. The invention further discloses a method for preparing transgenic soybeans, which sequentially includes the following steps: (I) performing any transformation on germinated soybeans; and (II) sequentially performing co-culture, restoring culture, screening culture and rooting culture to obtain the transgenic soybeans. The methods can solve the problem of low soybean genetic transformation efficiency, provide powerful tools for researches of soybean molecular biology, genetic engineering breeding and the like, have the advantages of being convenient in material taking, short in period, simple, convenient and easy to achieve, and surely can lay a foundation for promoting development of soybean genetic engineering breeding.

Description

A kind of soybean high-efficiency genetic transforming method

Technical field

The present invention relates to a kind of soybean high-efficiency genetic transforming method.

Background technology

Soybean [Glycine max (L) Merr.] is important cash crop and oil crops, and its Study on Genetic Transformation receives scholars' extensive concern always, and main bottleneck problem is that transformation efficiency is low.

Existing soybean heredity method for transformation comprises agrobacterium-mediated transformation, electric shocking method, PEG conversion method, particle bombardment, pollen tube passage method etc., comparative maturity be the somatic embryo conversion method of particle gun mediation and agriculture bacillus mediated cotyledonary node conversion method.

Christou in 1988 etc. are applied to particle bombardment the genetic transformation of soybean callus tissue first, but do not obtain regeneration plant, and the same year, McCabe etc. utilized particle bombardment soybean transformation bud meristematic tissue, had obtained transfer-gen plant, but mostly had been mosaic.After this, the particle gun blast technique is mainly used in soybean immature embryo or body embryo suspension cell line, can stablize to obtain transfer-gen plant, but transformation efficiency is all lower, and has shortcomings such as regeneration plant cycle length and transfer-gen plant abortion.

First report such as Hinchee in 1998 example of agrobacterium tumefaciens (Agrobacterium tumfaciens) mediated method success soybean transformation; Many afterwards scholars adopt this method to carry out the soybean cotyledon node Study on Transformation; All obtained transfer-gen plant; But transformation efficiency is all lower, and mosaic is more.

Summary of the invention

The purpose of this invention is to provide a kind of soybean high-efficiency genetic transforming method.

The invention provides a kind of soybean high-efficiency genetic transforming method, in turn include the following steps:

The plasmid that (1) will contain target DNA through the particle gun blast technique imports the soybean explant;

(2) the said plasmid that contains target DNA is imported the soybean explant of completing steps (1) through agrobacterium-mediated transformation.

Said soybean explant can be embryo point explant, specifically can be soybean mature seed embryo point explant (soybean sprout seed embryo point explant).

The preparation method of said soybean mature seed embryo point explant in turn includes the following steps: the soaking at room temperature 24 hours in sterilized water of the ripe soybean seeds after (1) will sterilize; (2) under aseptic condition, remove kind of a skin, take off plumular axis together with the embryo point of just having sprouted; (3) hypocotyl is placed on the preparatory culture medium together with the embryo point of just having sprouted, the embryo point was cultivated 24 hours for 25 ℃-28 ℃ vertically upward, removed prophyll (leaf primordium is grown the back and formed true leaf) then, obtained explant (being soybean mature seed embryo point explant); Said pre-incubated illumination condition is illumination in 16 hours, intensity of illumination 150 μ mol m ^-2s ^-1, 8 hours dark.

Said preparatory culture medium is specially the MS-B that contains 3.5mg/L 6-benzyl aminopurine and 30g/L sucrose ₅Solid medium.

Said soybean specifically can be rich No. 28 soybean of K06-82 soybean or section.

In the said particle gun blast technique; The parameter of particle gun bombardment specifically can be: the bronze diameter is 1.0 μ m; Vacuum tightness 26～28 inches of mercury, helium pressure are 1100psi, and target distance is 6cm; The said plasmid that contains target DNA of 1 μ g is bombarded in every ware bombardment 2 times (bombardment back plate half-twist bombards for the second time more for the first time) at every turn.Specifically can adopt Bio-Rad PDS1000/He particle gun.

Said agrobacterium-mediated transformation specifically can be: with the soybean explant of completing steps (1) shaking culture 20 hours under 28 ℃ of dark conditions in reorganization Agrobacterium bacteria suspension; Said reorganization Agrobacterium is that the said plasmid that contains target DNA is imported the reorganization Agrobacterium that Agrobacterium obtains.Said Agrobacterium specifically can be Agrobacterium GV3101.Said reorganization Agrobacterium bacteria suspension specifically can be OD _600nmThe bacteria suspension of=0.45-0.5.Said reorganization Agrobacterium bacteria suspension specifically can prepare through following method: said reorganization Agrobacterium is resuspended in the substratum fourth, and the Agrobacterium bacteria suspension obtains recombinating.Said substratum fourth is the 1/2MS-B that contains 6mg/L6-benzyladenine, 200 μ M Syringylethanones, 30g/L sucrose and 10g/L glucose ₅Liquid nutrient medium.Syringylethanone can increase transfection efficiency.

More than arbitrary said method all can be used for preparing genetically engineered soybean.

Said soybean specifically can be rich No. 28 soybean of K06-82 soybean or section.

The present invention also protects a kind of method for preparing genetically engineered soybean, in turn includes the following steps:

(I) soybean that will set out carries out above arbitrary described genetic transformation;

(II) carry out common cultivation, recovery cultivation, screening and culturing and root culture successively, obtain genetically engineered soybean.

Said soybean specifically can be rich No. 28 soybean of K06-82 soybean or section.

Said cultivation altogether specifically can be and is being total on the culture medium, 22 ℃ of dark cultivations 4 days.Said culture medium altogether specifically can be and contains 6mg/L 6-benzyl aminopurine, 0.154g/L WR 34678,0.2g/L Na ₂S ₃O ₃, 1mg/L AgNO ₃, 1g/LL-halfcystine, 200 μ M Syringylethanones, 30g/L sucrose and 10g/L glucose 1/2MS-B ₅Solid medium.Syringylethanone can increase transfection efficiency.Said culture medium altogether can prevent brownization and improve transformation efficiency.

Said recovery is cultivated and specifically can be on recovery media, cultivates 6-7 days (illumination conditions: illumination in 16 hours, intensity of illumination 150 μ mol m for 25 ℃-28 ℃ ^-2s ^-1, 8 hours dark).Said recovery media specifically can be the 1/2MS-B that contains 300mg/L cephamycin and 30g/L sucrose ₅Solid medium.

Said screening and culturing can in turn include the following steps:

1. on the screening culture medium first, cultivate 14 days (illumination conditions: illumination in 16 hours, intensity of illumination 150 μ mol m for 25 ℃-28 ℃ ^-2s ^-1, 8 hours dark); Said screening culture medium first specifically can be the 1/2MS-B that contains 0.7mg/L Glyphosate 62 IPA Salt, 300mg/L cephamycin and 30g/L sucrose ₅Solid medium;

2. transfer to behind the subculture on the screening culture medium second, cultivate 14 days (illumination conditions: illumination in 16 hours, intensity of illumination 150 μ mol m for 25 ℃-28 ℃ ^-2s ^-1, 8 hours dark); Said screening culture medium second specifically can be the 1/2MS-B that contains 0.8mg/L Glyphosate 62 IPA Salt, 300mg/L cephamycin and 30g/L sucrose ₅Solid medium;

3. transfer on the screening culture medium third 25 ℃-28 ℃ cultivation (illumination conditions: illumination in 16 hours, intensity of illumination 150 μ mol m behind the subculture ^-2s ^-1, 8 hours dark) and be about 3cm (about 14 days) to bud; Said screening culture medium third specifically can be the 1/2MS-B that contains 0.9mg/L Glyphosate 62 IPA Salt, 300mg/L cephamycin and 30g/L sucrose ₅Solid medium.

Said root culture specifically can be 14-20 days (illumination conditions: illumination in 16 hours, intensity of illumination 150 μ mol m of 25 ℃-28 ℃ cultivations on root media ^-2s ^-1, 8 hours dark), open wide bottleneck then and practiced seedling 3-5 days; Said root media specifically can be the 1/2MS-B that contains 300mg/L cephamycin, 0.1mg/L Glyphosate 62 IPA Salt and 30g/L sucrose ₅Solid medium.

The invention provides the compound method for transformation of soybean embryo point of the common mediation of a kind of particle gun and Agrobacterium.In the method for the present invention; The plasmid that will contain goal gene earlier bombards through the apical meristem of particle gun to embryo point explant; This step can not only import some plasmids, and micropellet bombardment can cause numerous Wicresofts to hinder to soybean embryo point, is beneficial to Agrobacterium and infects.Wicresoft hinders can secrete a kind of solubility phenolic cpd, and this compound can be used as the inducement signal of agroinfection, activates the Agrobacterium increase and infects chance.Then, infect with the Agrobacterium that contains plasmid again, can more plasmids be imported in the receptor tissue again, so compound conversion can increase substantially transformation efficiency.Through cultivating altogether, recovering links such as cultivation, screening and culturing and root culture, can efficiently obtain transfer-gen plant again.Method provided by the invention can solve a undue acceptor gene type and the low difficult problem of transformation efficiency of relying in the long-term puzzlement soybean heredity conversion to a certain extent; The crops that are not merely other difficult conversions carry out genetic transformation provides reference, also for researchs such as soybean molecular biology and genetic engineering breedings strong instrument is provided.The present invention also has advantage such as draw materials conveniently, the cycle is short, simple and easy to do, will lay the foundation for the development of promotion soybean gene engineering breeding.

Description of drawings

Fig. 1 is the photo of each step in the whole flow process of compound genetic transformation of embodiment 1.

Fig. 2 is the result that PCR identifies the bar gene among the embodiment 1.

Fig. 3 is the result that PCR identifies the gus gene among the embodiment 1.

Fig. 4 is the painted result of GUS among the embodiment 1.

Fig. 5 identifies the proteic result of PAT with bar gene test test strip among the embodiment 1.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used experiment reagent among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.Transformation efficiency: transfer-gen plant quantity accounts for the particle gun bombardment and/or Agrobacterium is infected the total percentage of embryo point explant.K06-82 (being No. 2, section's beans) soybean: Anhui Tong Feng kind industry ltd.Rich No. 28 soybean of section: Anhui Tong Feng kind industry ltd.Binary vector pCAMBIA3301 (claiming the pCAMBIA3301 plasmid again): TRANSGEN company; Binary vector pCAMBIA3301 contains bar gene (coding PAT albumen) and gus gene (coding gus protein).Agrobacterium GV3101: available from TRANSGEN company.

1/2MS-B ₅The preparation method of liquid nutrient medium: get 1/2MS macroelement, MS trace element, MS molysite and B ₅Organic element, water-soluble and be settled to 1L.

1/2MS-B ₅Solid medium: at 1/2MS-B ₅On the basis of liquid nutrient medium, every liter is added 2g plant gel (available from Sigma, article No. P8169).

MS-B ₅The preparation method of solid medium: get MS macroelement, MS trace element, MS molysite and B ₅Organic element, water-soluble and be settled to 1L.

MS macroelement: saltpetre 1900mg/L, an ammonium nitrate 1650mg/L, potassium primary phosphate 170mg/L, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 370mg/L and Calcium dichloride dihydrate 440mg/L.

The 1/2MS macroelement: each component concentrations of MS macroelement all reduces by half.

MS trace element: potassiumiodide 0.83mg/L, boric acid 6.2mg/L, sal epsom 22.3mg/L, zinc sulfate 8.6mg/L, Sodium orthomolybdate 0.25mg/L, cupric sulfate pentahydrate 0.025mg/L and CoCL2 0.025mg/L.

MS molysite: EDTA Disodium 37.3mg/L and iron vitriol 27.8mg/L.

B ₅Organic element: vitamin (VITMAIN B1) 10mg/L, pyridoxine hydrochloride (Y factor) 1mg/L, nicotinic acid 1mg/L and inositol 100mg/L.

The compound genetic transformation of embodiment 1, K06-82 soybean

One, the acquisition of explant

1, sterilization

Get the full mature seed of K06-82 soybean that smooth surface does not have scab, in encloses container (containing chlorine), placed 16 hours.

2, soak

Get the soybean of completing steps 1, soaking at room temperature is 24 hours in sterilized water.

3, get the soybean of completing steps 2, on Bechtop, remove kind of a skin, hypocotyl is peeled off with two cotyledons together with the embryo point of just having sprouted, take off plumular axis together with the embryo point of just having sprouted.

4, the hypocotyl that step 3 is obtained is placed on the preparatory culture medium together with the embryo point of just having sprouted, and the embryo point is seen Figure 1A vertically upward, cultivates 24 hours (illumination conditions: illumination in 16 hours, intensity of illumination 150 μ mol m for 25 ℃ ^-2s ^-1, 8 hours dark), remove prophyll then, obtain explant.

Preparatory culture medium: the MS-B that contains 3.5mg/L 6-benzyl aminopurine (6-BA) and 30g/L sucrose ₅Solid medium.

Two, particle gun bombardment

1, the explant with completing steps one is placed on the new preparatory culture medium, and the intensive about 2.5cm circle of diameter of putting into is seen Figure 1B.

2, particle gun bombardment

Petridish with completing steps 1 on Bechtop carries out the particle gun bombardment, carries out according to Bio-Rad PDS1000/He particle gun working instructions.The parameter of particle gun bombardment: the bronze diameter is 1.0 μ m; Vacuum tightness 26～28 inches of mercury, helium pressure are 1100psi, and target distance is 6cm; 1 μ g pCAMBIA3301 plasmid is bombarded in every ware bombardment 2 times (bombardment back plate half-twist bombards for the second time more for the first time) at every turn.

Three, Agrobacterium is infected

1, the pCAMBIA3301 plasmid is imported Agrobacterium GV3101, obtain the Agrobacterium of recombinating.

2, the reorganization Agrobacterium with step 1 is resuspended in the substratum fourth, makes OD _600nm=0.45-0.5 is reorganization Agrobacterium bacteria suspension.

Substratum fourth: the 1/2MS-B that contains 6mg/L 6-benzyl aminopurine, 200 μ M Syringylethanones, 30g/L sucrose and 10g/L glucose ₅Liquid nutrient medium.

3, the explant of completing steps two is placed the reorganization Agrobacterium bacteria suspension (seeing Fig. 1 C) of step 2, shaking culture is 20 hours under 28 ℃ of dark conditions.

Four, cultivate altogether

Get the explant of completing steps three, blot bacterium liquid with aseptic filter paper, transfer to common culture medium then, 22 ℃ of dark cultivations 4 days are seen Fig. 1 D.

Be total to culture medium: contain 6mg/L 6-benzyl aminopurine, 0.154g/L WR 34678,0.2g/L Na ₂S ₃O ₃, 1mg/L AgNO ₃, 1g/L L-halfcystine, 200 μ M Syringylethanones, 30g/L sucrose and 10g/L glucose 1/2MS-B ₅Solid medium.

The GUS colored graph of the explant after cultivating is altogether seen Fig. 4 A, and the embryo point is shown as blueness, has promptly expressed gus protein.

Five, recover to cultivate

Get the explant of completing steps four, clean, transfer to then in the recovery media, cultivate 6-7 days (illumination conditions: illumination in 16 hours, intensity of illumination 150 μ mol m for 25 ℃-28 ℃ with sterilized water ^-2s ^-1, 8 hours dark), see Fig. 1 E.

Recovery media: the 1/2MS-B that contains 300mg/L cephamycin and 30g/L sucrose ₅Solid medium.

Six, screening and culturing

1, gets the explant of completing steps five, transfer to the screening culture medium first, cultivate 14 days (illumination conditions: illumination in 16 hours, intensity of illumination 150 μ mol m for 25 ℃-28 ℃ ^-2s ^-1, 8 hours dark), see Fig. 1 F.

Screening culture medium first: the 1/2MS-B that contains 0.7mg/L Glyphosate 62 IPA Salt, 300mg/L cephamycin and 30g/L sucrose ₅Solid medium.

2, with the explant subculture of completing steps 1, transfer to screening culture medium second, cultivate 14 days (illumination conditions: illumination in 16 hours, intensity of illumination 150 μ mol m for 25 ℃-28 ℃ ^-2s ^-1, 8 hours dark).

Screening culture medium second: the 1/2MS-B that contains 0.8mg/L Glyphosate 62 IPA Salt, 300mg/L cephamycin and 30g/L sucrose ₅Solid medium.

3,, transfer to the third, 25 ℃-28 ℃ cultivations of screening culture medium (illumination condition: illumination in 16 hours, intensity of illumination 150 μ mol m with the explant subculture of completing steps 2 ^-2s ^-1, 8 hours dark) and be about 3cm (about 14 days) to bud.

Screening culture medium third: the 1/2MS-B that contains 0.9mg/L Glyphosate 62 IPA Salt, 300mg/L cephamycin and 30g/L sucrose ₅Solid medium.

The GUS coloration result of bud is seen Fig. 4 B.Can observe and express gus protein.

Seven, root culture

Downcut the bud of step 6, transfer to root media (seeing Fig. 1 G), cultivate 14-20 days (illumination conditions: illumination in 16 hours, intensity of illumination 150 μ mol m for 25 ℃-28 ℃ ^-2s ^-1, 8 hours dark), see Fig. 1 H (root is long to enough healthy and strong), open wide bottleneck in the time of then and practice seedling 3-5 days (seeing Fig. 1 I).

Root media: the 1/2MS-B that contains 300mg/L cephamycin, 0.1mg/L Glyphosate 62 IPA Salt and 30g/L sucrose ₅Solid medium.

Eight, obtain transfer-gen plant

The seedling of completing steps seven is transferred to nutrition soil, normal cultured in the greenhouse (25 ℃-28 ℃ of temperature, natural lighting), 14 days photo of hot-house culture is seen Fig. 1 J.

Nine, the evaluation of transfer-gen plant

Get 14 days plant leaf of hot-house culture, identify as follows respectively:

1, extracts genomic dna, PCR is identified the bar gene with the primer that F1 and R1 form.

F1 (sequence 1 of sequence table): 5 '-GCACCATCGTCAACCACTAC-3 ';

R1 (sequence 2 of sequence table): 5 '-TGAAGTCCAGCTGCCAGAAAC-3 '.

The target sequence of F1 and R1 is the bar gene fragment of about 440bp.

Qualification result is seen Fig. 2.Among Fig. 2, M is Marker, and 1 is over against according to (binary vector pCAMBIA3301), and 2 are negative contrast (not carrying out the K06-82 soybean of genetic transformation), and 3 be blank (water), and 4 to 17 is to carry out the plant that obtains behind the genetic transformation); Arrow mark purpose fragment.

2, extract genomic dna, PCR is identified CaMV35S promotor and gus gene with the primer that F2 and R2 form.

F2：5’-AAGGAAGGTGGCTCCTACAA-3’；

R2：5’-AATATCTGCATCGGCGAACT-3’。

The target sequence of F2 and R2 is CaMV35S promotor and the gus gene fragment of about 669bp.

Qualification result is seen Fig. 3.Among Fig. 3, M is Marker, and 1 is over against according to (binary vector pCAMBIA3301), and 2 are negative contrast (not carrying out the K06-82 soybean of genetic transformation), and 3 be blank (water), and 4 to 17 is to carry out the plant that obtains behind the genetic transformation); Arrow mark purpose fragment.

3, GUS dyeing.

The result sees Fig. 4 C, shows blue spot, has promptly expressed gus protein.

4, extract total protein, identify PAT albumen with bar gene test test strip (available from EnviroLogix, article No. AS013LS), the result sees Fig. 5.Among Fig. 5,1 is over against according to (PAT albumen), and 2 are negative contrast (water), and 3 for not carrying out the K06-82 soybean of genetic transformation, 4 plant for carrying out obtaining behind the genetic transformation).

If plant step 1 to be measured and step 2 all are accredited as the positive, plant then to be measured is the successful transfer-gen plant of genetic transformation.Step 3 and step 4 are merely sampling verification, and positive judged result is all consistent with step 1 and step 2.

Carry out revision test three times, transformation efficiency is respectively up to 21.68%, 24.15% and 20.88%, and MV is 22.24% ± 1.70%.

Comparative Examples 1, particle gun blast technique transform Comparative Examples

Except middle hop is crossed step 3, other is all with embodiment 1.

The average conversion of three tests is 8.78% ± 1.22%.

Comparative Examples 2, agrobacterium-mediated transformation transform Comparative Examples

Except middle hop is crossed step 2, other is all with embodiment 1.

The average conversion of three tests is 7.94% ± 1.27%.

The genetic transformation of embodiment 2, rich No. 28 soybean of section

Replace the K06-82 soybean with rich No. 28 soybean of section, other is with embodiment 1.

The average conversion of three tests is 18.48% ± 1.50%.

Comparative Examples 3, particle gun blast technique transform Comparative Examples

Except middle hop is crossed step 3, other is all with embodiment 2.

The average conversion of three tests is 13.86% ± 1.31%.

Comparative Examples 4, agrobacterium-mediated transformation transform Comparative Examples

Except middle hop is crossed step 2, other is all with embodiment 2.

The average conversion of three tests is 9.78% ± 1.25%.

Claims (7)

1. soybean method for transformation in turn includes the following steps:

The plasmid that (1) will contain target DNA through the particle gun blast technique imports the soybean explant;

(2) the said plasmid that contains target DNA is imported the soybean explant of completing steps (1) through agrobacterium-mediated transformation.

2. the method for claim 1 is characterized in that: said explant is an embryo point explant, is specially the seed embryo point explant of sprouting.

3. according to claim 1 or claim 2 method, it is characterized in that: said soybean is rich No. 28 soybean of K06-82 soybean or section.

4. the application of arbitrary said method in the preparation genetically engineered soybean in the claim 1 to 3.

5. application as claimed in claim 4 is characterized in that: said soybean is rich No. 28 soybean of K06-82 soybean or section.

6. method for preparing genetically engineered soybean in turn includes the following steps:

(I) will set out soybean carry out claim 1 to 3 in arbitrary described conversion;

(II) carry out common cultivation, recovery cultivation, screening and culturing and root culture successively, obtain genetically engineered soybean.

7. method as claimed in claim 6 is characterized in that: said soybean is rich No. 28 soybean of K06-8 soybean 2 or section.

Images