CN101016568B - Method of screening transgene barley strain without antibiotic mediated by agrobacterium - Google Patents
Method of screening transgene barley strain without antibiotic mediated by agrobacterium Download PDFInfo
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Abstract
The invention discloses a method to screen and culture barley transfer-gene plant with non-antibiotic of agricillin dielectric conductance in plant transfer-gene technical domain, which comprises the following steps: leading reported gene into receptor barley cell through agricillin dielectric conductance; utilizing mannose to replace antibiotic as chosen agent; screening the reverting receptor cell; adjusting the genetic reforming process of barley through phosphomannose isomerase; further-screening transfer-gene barley with chlorophenol red colouration method and PCR method; getting the end product. The barley cultivating strain conversion rate can reach 12% with agricillin dielectric conductance and can reach 100% with chlorophenol red colouration method.
Description
Technical field
The invention belongs to the plant transgenic technology field, be specifically related to the method for utilizing agriculture bacillus mediated no antibiosis rope screening to cultivate the barley transfer-gen plant.The invention still further relates to PMI (phosphomannose isomerase)/seminose screen body and tie up to the application in the barley genetic transformation, and the application of dichlorophenol sulfonphthalein (CPR) development process in differentiating the barley transgenic plant.
Background technology
Barley is one of the most ancient in the world crop, is to list in wheat, corn and paddy rice the 4th important cereal crop afterwards, mainly medicine industry raw material and the protective foods that is used as feed, grain, brewing industry raw material and causes concern in recent years.Barley also is one of model plant of genetics research widespread use.
Since Tingay in 1997 etc. transformed the barley success with agrobacterium-mediated transformation first, agriculture bacillus mediated method for transformation became in recent years the most frequently used a kind of method (Tingay S, McElroy D in the barley genetic transformation, Kalla R, Fieg S, Wang M, Thornton S﹠amp; Brettell R.Agrobacterium tumefaciens-mediated barley transformation.J Plant, 1997,11:1369-1376; McCormac A C, WuH, Bao M, Wang Y, Xu R, Elliott M C﹠amp; Chen D F.The use of visual marker genes as cell-specific reporters ofAgrobacterium-mediated T-DNA delivery to wheat (Triticum aestivum L.) and Barley (Hordeum vulgare L.) .Euphytica, 1998,99:17-25; Matthews P R, Wang M B, Waterhouse P M, Thornton S, Fieg S J, Gubler F, JacobsenJ V.Marker gene elimination from transgenic barley, using co-transformation with adjacent ' twinT-DNAs ' on a standard Agrobacterium transformation vector.Mol Breeding, 2001,7:195-202; Murray F, BrettellR, Matthews P, Bishop D, Jacobsen J.Comparison of Agrobacterium-mediated transformation of four barleycultivars using the GFP and GUS reporter genes.Plant Cell Rep, 2004,22:397-402; Wu H, McCormac A C, ElliottM C﹠amp; Chen D F.Agrobacterium-mediated stable transformation of cell suspension cultures of barley (Hordeumvulgare L.) .Plant Cell Tiss.Org.Cult, 1998,54:161-171; Trifonova A, Madsen S, Olesen A.Agrobacterium-mediated transgene delivery and integration into barley under a range of in vitro culture conditions.Plant Sci, 2001,161:871-880; Fang Y D, Akula C, Altpeter F.Agrobacterium-mediated barley (Hordeum vulgareL.) transformation using green fluorescent protein as a visual marker and sequence analysis of the T-DNA::barleygenomic DNA junctions.J Plant Physiol, 2002,159:1131-1138; Travella S, Ross S M, Harden J, Everett C, Snape J W, Harwood W A.A comparison of transgenic barley lines produced by particle bombardment andAgrobacterium-mediated techniques.Plant Cell Rep, 2005,23:780-789; Kumlehn J, Serazetdinova L, Hensel G, Becker D, Loerz H.Genetic transformation of barley (Hordeum vulgare L.) via infection of androgenetic pollencultures with Agrobacterium tumefaciens.Plant Biotechnol J, 2006,4:251-261; Or the like).The method of employings such as Tingay is to bombard callus earlier with particle gun, will have the acceptor of the callus of tiny wound as Agrobacterium-mediated Transformation again.Though it has advantages such as the low copy of transgenosis, inheritance stability, has also strengthened the experiment difficulty and has improved experimental cost.Simultaneously, in the agrobacterium mediation converted of barley, the outstanding problem that exists is subjected to the genotype restriction exactly at present, transformation frequency is not high and selection markers is single.In real work, must improve the defective and the deficiency of prior art system.The present invention starts with from callus culture and Transformation Program, and tie up to application in the barley genetic transformation by PMI/ seminose screen body, further overcoming callus influences the problem of breaking up owing to screen subculture for a long time in conversion process, thereby sets up a kind of barley genetic transforming method of efficient agrobacterium mediation.
PMI/ seminose screening system is cheap to human body and environmental safety, screening reagent, do not influence the growing of transformed plant, the screening efficiency height.Dichlorophenol sulfonphthalein (CPR) development process is to utilize plant to carry out seminose metabolism meeting to make substratum acidifying principle.PH indicator dichlorophenol sulfonphthalein takes on a red color when pH6.0, is transformed into yellow along with pH descends.This method is the active most popular method that detects of PMI, on transgenic wheat, corn and sweet orange detect, all have application (Boscariol R L, Almeida WA B, Derbyshire M T V C,
Filho F A A, Mendes B M J.Theuse of the PMI/mannose selection system to recover transgenic sweet orange plants (Citrus sinensis L.Osbeck) .Plant Cell Rep, 2003,22:122-128; Wright M, Dawson J, Dunder E.Efficient biolistic transformation of maize (Zea mays L.) and wheat (Triticum aestivum L.) using the phosphomannose isomerase gene, pmi, as the selectablemarker.Plant Cell Rep, 2001,20:429-436; Gao Z, Xie X, Yan L, Muthukrishnan S, Liang G H.Agrobacteriumtumefaciens-mediated sorghum ransformation using a mannose selection system.Plant Biotechnol J, 2005,3:591-599; Degenhardt J, Poppe A, Montag J, Szankowski I.The use of the phosphomannose isomerase/mannoseselection system to recover transgenic apple plants.Plant Cell Rep, 2006,25:1149-1156; Lucca P, Ye X D, Potrykus I.Effective selection and regeneration of transgenic rice plants with mannose as selective agent.MolBreeding, 2001,7:43-49; Or the like), also Shang Weiyou reports in the genetic transformation of barley.Compare with the GUS tissue staining, the dichlorophenol sulfonphthalein development process need not expensive reagent, and disturbance reponse is few, reliable results.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, develop a kind of method of antibiotic-free screening cultivation barley transfer-gen plant of efficient agrobacterium mediation, utilize PMI (phosphomannose isomerase)/seminose screening system and dichlorophenol sulfonphthalein (CPR) in genetic transformation of barley and the application in the discriminating barley transgenic plant, to improve the efficient of barley gene transformation.
The present invention is achieved through the following technical solutions:
1, design of primers and vector construction:
According to the dna sequence dna that Genebank announces, design the special primer of a pair of intestinal bacteria manA gene, the numbering and the structure of this primer are as follows:
1)PMIP1：5’-ATGGATCCATGCAAAAACTCATTAACTCA-3’；
PMIP2：’-AAGGATCCTTACAGCTTGTTGTAAACACG-3’。
2) pcr amplification manA gene from the bacillus coli gene group, the order-checking back is used for vector construction as selectable marker gene.The gene of pUC18-manA will be cloned into, cut with the BamHI enzyme, from middle carrier pMBL-3 and pBAR (being so kind as to give), cut with HindIII and BamHI-HinIII enzyme respectively simultaneously and obtain corn ubiquitin promoter and correlated series by German Ma Pu doctor Guo Yan of institute, clone on the carrier that is connected to based on pUC8, make up and formed the pMBL-5 plant expression vector.Its concrete structure route is seen Fig. 2.The plant expression vector that builds is transformed agrobacterium tumefaciens.
2, utilize the barley genetic transformation of agriculture bacillus mediated antibiotic-free screening, it may further comprise the steps:
1), callus induces
With wide about 0.5mm～1.5mm, the translucent barley rataria of color is as explant.Earlier the prematurity barley seed was sterilized 30 seconds with 70% ethanol, aseptic water washing 1 time, again with 0.1% mercuric chloride sterilization 8 minutes, aseptic water washing 3 times, every all over 3 minutes.Strip described explant, the explant scultellum upwards is inoculated on the inducing culture, under the condition of 25 ℃ of dark, cultivate, induce callus.
2), the pre-cultivation of callus
Picking is eugonic from the callus that step 1) obtains is inoculated on the pre-culture medium, cultivates 5 hours under the condition of 25 ℃ of dark;
3), the dip-dye of callus
To change in the Agrobacterium suspension culture base through pre-incubated callus, and place vibrate on the shaking table (100 rev/mins) to contaminate 30 minutes;
4), callus and Agrobacterium are cultivated altogether
Callus is pressed from both sides out from Agrobacterium suspension culture base, place on the aseptic filter paper to dry up a little, be seeded to common culture medium, under 25 ℃ of dark conditions, cultivated 2 days;
5), the recovery of callus is cultivated
Callus is changed in the sterilized water that contains the 500mg/L cephamycin, place vibrate on the shaking table (100 rev/mins) to soak 30 minutes, on aseptic filter paper, dry up a little, be inoculated into the last 25 ℃ of dark culturing of recovery media (with the purpose that reaches antibacterial and recover to cultivate) at last 7 days;
6), the screening and culturing of callus
Antibacterial good callus is inoculated into screening culture medium, 3 weeks of screening and culturing under 25 ℃ of dark conditions;
7), differentiation of calli
Select the callus that screens new growth in back and not brownization death and be inoculated into division culture medium, 25 ℃, 2000Lux, illumination cultivation is to differentiating green bud point;
8), the screening again of regeneration plant
Green bud point or seedling that differentiation is produced insert the strong seedling culture base that contains seminose, and 25 ℃, 2000Lux, illumination cultivation obtains candidate's transformed plant to transplanting.
3, utilize dichlorophenol sulfonphthalein liquid to detect substratum and detect transfer-gen plant phosphomannose isomerase (PMI) activity, concrete steps are:
Choose and state up-to-date longer a slice leaf on the candidate's transformed plant stem that obtains, the blade tip section of the about 0.3cm of clip * 0.5cm area size immerses dichlorophenol sulfonphthalein liquid with the leaf section and detects in the substratum, observes the substratum colour-change after 25 ℃ of lucifuges are cultivated 4 days.According to the identical leaf section that requires the unconverted plant of clip, immerse dichlorophenol sulfonphthalein liquid respectively and detect in substratum and the dichlorophenol sulfonphthalein liquid control medium simultaneously, cultivate 4 days as contrast in 25 ℃ of lucifuges.
In the present invention, following substratum is vital, all kinds of nutrient media componentses and fill a prescription as follows:
1, inducing culture (numbering: MS2):
1900mg/L KNO
3, 1650mg/L NH
4NO
3, 170mg/L KH
2PO
4, 370mg/L MgSO
47H
2O, 440mg/L CaCl
22H
2O, 27.8mg/L FeSO
47H
2O, 37.3mg/L Na
2EDTA, 0.83mg/L KI, 6.2mg/L H
3BO
3, 22.3mg/L MnSO
44H
2O, 8.6mg/LZnSO
47H
2O, 0.25mg/L Na
2MoO
42H
2O, 1.25mg/L CuSO
45H
2O, 0.025mg/L CoCl
26H
2O, 2mg/L glycine, 1mg/L V
B1, 0.5mg/L V
B6, 0.5mg/L V
B5, the 690mg/L proline(Pro), the 250mg/L inositol, the 1000mg/L caseinhydrolysate, 2mg/L2,4-D, 30g/L maltose, 6g/L agar keeps the skin wet to 1L pH5.8.
2, pre-culture medium (numbering: MS3):
1900mg/L KNO
3, 1650mg/L NH
4NO
3, 170mg/L KH
2PO
4, 370mg/L MgSO
47H
2O, 440mg/L CaCl
22H
2O, 27.8mg/L FeSO
47H
2O, 37.3mg/L Na
2EDTA, 0.83mg/L KI, 6.2mg/L H
3BO
3, 22.3mg/L MnSO
44H
2O, 8.6mg/LZnSO
47H
2O, 0.25mg/L Na
2MoO
42H
2O, 1.25mg/L CuSO
45H
2O, 0.025mg/L CoCl
26H
2O, 2mg/L glycine, 1mg/L V
B1, 0.5mg/L V
B6, 0.5mg/L V
B5, the 690mg/L proline(Pro), the 250mg/L inositol, the 1000mg/L caseinhydrolysate, 2mg/L2,4-D, 0.4mol/L N.F,USP MANNITOL, 30g/L maltose, 6g/L agar keeps the skin wet to 1L pH5.8
3, the fluid suspension culture base during During Agrobacterium (numbering: MS11):
1900mg/L KNO
3, 1650mg/L NH
4NO
3, 170mg/L KH
2PO
4, 370mg/L MgSO
47H
2O, 440mg/L CaCl
22H
2O, 27.8mg/L FeSO
47H2O, 37.3mg/L Na
2EDTA, 0.83mg/L KI, 6.2mg/L H
3BO
3, 22.3mg/L MnSO
44H
2O, 8.6mg/LZnSO
47H
2O, 0.25mg/L Na
2MoO
42H
2O, 1.25mg/L CuSO
45H
2O, 0.025mg/L CoCl
26H
2O, 2mg/L glycine, 1mg/L V
B1, 0.5mg/L V
B6, 0.5mg/L V
B5, the 690mg/L proline(Pro), the 250mg/L inositol, the 1000mg/L caseinhydrolysate, 2mg/L2,4-D, 100 μ mol/L Syringylethanones, 30g/L maltose keeps the skin wet to 1L pH5.8
4, altogether culture medium (numbering: MS4):
1900mg/L KNO
3, 1650mg/L NH
4NO
3, 170mg/L KH
2PO
4, 370mg/L MgSO
47H
2O, 440mg/L CaCl
22H
2O, 27.8mg/L FeSO
47H
2O, 37.3mg/L Na
2EDTA, 0.83mg/L KI, 6.2mg/L H
3BO
3, 22.3mg/L MnSO
44H
2O, 8.6mg/LZnSO
47H
2O, 0.25mg/L Na
2MoO
42H
2O, 1.25mg/L CuSO
45H
2O, 0.025mg/L CoCl
26H
2O, 2mg/L glycine, 1mg/L V
B1,0.5mg/L V
B6, 0.5mg/L V
B5, the 690mg/L proline(Pro), the 250mg/L inositol, the 1000mg/L caseinhydrolysate, 2mg/L2,4-D, 100 μ mol/L Syringylethanones, 30g/L maltose, 6g/L agar keeps the skin wet to 1L pH5.8.
5, recovery media (numbering: MS5):
1900mg/L KNO
3, 1650mg/L NH
4NO
3, 170mg/L KH
2PO
4, 370mg/L MgSO
47H
2O, 440mg/L CaCl
22H
2O, 27.8mg/L FeSO
47H
2O, 37.3mg/L Na
2EDTA, 0.83mg/L KI, 6.2mg/L H
3BO
3, 22.3mg/L MnSO
44H
2O, 8.6mg/LZnSO
47H
2O, 0.25mg/L Na
2MoO
42H
2O, 1.25mg/L CuSO
45H
2O, 0.025mg/L CoCl
26H
2O, 2mg/L glycine, 1mg/L V
B1, 0.5mg/L V
B6, 0.5mg/L V
B5, the 690mg/L proline(Pro), the 250mg/L inositol, the 1000mg/L caseinhydrolysate, 2mg/L2,4-D, the 500mg/L cephamycin, 30g/L maltose, 6g/L agar keeps the skin wet to 1L pH5.8
6, screening culture medium (numbering: MS6):
1900mg/L KNO
3, 1650mg/L NH
4NO
3, 170mg/L KH
2PO
4, 370mg/L MgSO
47H
2O, 440mg/L CaCl
22H
2O, 27.8mg/L FeSO
47H
2O, 37.3mg/L Na
2EDTA, 0.83mg/L KI, 6.2mg/L H
3BO
3, 22.3mg/L MnSO
44H
2O, 8.6mg/LZnSO
47H
2O, 0.25mg/L Na
2MoO
42H
2O, 1.25mg/L CuSO
45H
2O, 0.025mg/L CoCl
26H
2O, 2mg/L glycine, 1mg/L V
B1, 0.5mg/L V
B6, 0.5mg/L V
B5, the 690mg/L proline(Pro), the 250mg/L inositol, the 1000mg/L caseinhydrolysate, 2mg/L2,4-D, the 500mg/L cephamycin, 10g/L maltose, the 20g/L seminose, 6g/L agar keeps the skin wet to 1L pH5.8.
7, division culture medium (numbering: MS7):
1900mg/L KNO
3, 1650mg/L NH
4NO
3, 170mg/L KH
2PO
4, 370mg/L MgSO
47H
2O, 440mg/L CaCl
22H
2O, 27.8mg/L FeSO
47H
2O, 37.3mg/L Na
2EDTA, 0.83mg/L KI, 6.2mg/L H
3BO
3, 22.3mg/L MnSO
44H
2O, 86mg/LZnSO
47H
2O, 0.25mg/L Na
2MoO
42H
2O, 1.25mg/L CuSO
45H
2O, 0.025mg/L CoCl
26H
2O, 2mg/L glycine, 1mg/L V
B1, 0.5mg/L V
B6, 0.5mg/L V
B5, the 690mg/L proline(Pro), the 250mg/L inositol, the 1000mg/L caseinhydrolysate, the 300mg/L cephamycin, 30g/L maltose, 6g/L agar keeps the skin wet to 1L pH5.8.
8, root media (numbering: MS8):
950mg/L KNO
3, 825mg/L NH
4NO
3, 85mg/L KH
2PO
4, 185mg/L MgSO
47H
2O, 220mg/L CaCl
22H
2O, 13.9mg/LFeSO
47H
2O, 18.65mg/L Na
2EDTA, 0.415mg/L KI, 3.1mg/L H
3BO
3, 11.15mg/L MnSO
44H
2O, 4.3mg/LZnSO
47H
2O, 0.125mg/L Na
2MoO
42H
2O, 0.625mg/L CuSO
45H
2O, 0.0125mg/L CoCl
26H
2O, 2mg/L glycine, 0.5mg/L V
B1, 0.5mg/L V
B6, 0.5mg/L V
B5, the 690mg/L proline(Pro), the 250mg/L inositol, the 1000mg/L caseinhydrolysate, 1mg/L NAA, 10g/L maltose, the 20g/L seminose, the 200mg/L cephamycin, 6g/L agar keeps the skin wet to 1L pH5.8.
9, dichlorophenol sulfonphthalein liquid detect substratum (numbering: MS9):
1900mg/L KNO
3, 1650mg/L NH
4NO
3, 170mg/L KH
2PO
4, 370mg/L MgSO
47H
2O, 440mg/L CaCl
22H
2O, 27.8mg/L FeSO
47H
2O, 37.3mg/L Na
2EDTA, 0.83mg/L KI, 6.2mg/L H
3BO
3, 22.3mg/L MnSO
44H
2O, 8.6mg/LZnSO
47H
2O, 0.25mg/L Na
2MoO
42H
2O, 0.025mg/L CuSO
45H
2O, 0.025mg/L CoCl
26H
2O, 2mg/L glycine, 100mg/L inositol, 0.1mg/L V
B1, 0.5mg/L V
B6, 0.5mg/L V
B5, the 15g/L seminose, 100mg/L dichlorophenol sulfonphthalein keeps the skin wet to 1L pH6.0.
10, dichlorophenol sulfonphthalein liquid control medium (numbering: MS10):
1900mg/L KNO
3, 1650mg/L NH
4NO
3, 170mg/L KH
2PO
4, 370mg/L MgSO
47H
2O, 440mg/L CaCl
22H
2O, 27.8mg/L FeSO
47H
2O, 37.3mg/L Na
2EDTA, 0.83mg/L KI, 6.2mg/L H
3BO
3, 22.3mg/L MnSO
44H
2O, 8.6mg/LZnSO
47H
2O, 0.25mg/L Na
2MoO
42H
2O, 0.025mg/L CuSO
45H
2O, 0.025mg/L CoCl
26H
2O, 2mg/L glycine 100mg/L inositol, 0.1mg/LV
B1, 0.5mg/L V
B6, 0.5mg/LV
B5, 15g/L sucrose, 100mg/L dichlorophenol sulfonphthalein keeps the skin wet to 1L pH6.0
The high pressure steam sterilization of above-mentioned substratum carries out according to the ordinary method of report.
The invention has the beneficial effects as follows:
The present invention is with three barley varieties producing extensive popularizing planting (precocious No. 3, Hubei Province barley No. 7 and spend 30, kind from: be test materials Hubei Prov. Acdemy of Agricutural Sciences), with reference to the barley conversion method for agrobacterium delivered, and obtain on the barley seminose tolerance studies result's who has obtained in conjunction with the applicant the basis.Generally all utilize microbiotic to screen in the agriculture bacillus mediated conversion of the barley of having reported, adopt pattern kind Golden Promise as acceptor.Golden Promise is precious wine brewing barley variety, and the work of barley genetic transformation at present concentrates on the research to Golden Promise, but its planting cost is high, in not extensively plantation of China.Simultaneously, (Tingay S, McElroyD, Kalla R, Fieg S, Wang M, Thornton S﹠amp such as Tingay; Brettell R.Agrobacterium tumefaciens-mediated barleytransformation.J Plant, 1997,11:1369-1376; ) the bombardment of classical conversion method for agrobacterium applying gene rifle as supplementary means, improved experimental cost, antibiotic screening has strengthened the mortality ratio of callus, transformation efficiency only 4.2%.The present invention has overcome the deficiencies in the prior art, has broken the genotypic restriction of barley genetic transformation, utilizes seminose to be selective agent, has developed a kind of conversion method for agrobacterium and dichlorophenol sulfonphthalein detection method of simple and effective.The barley Cultivar transformation efficiency of agriculture bacillus mediated genetic transformation that the present invention adopts is up to 12% (seeing Table 1), efficient with dichlorophenol sulfonphthalein development process screening transformed plant reaches 100%, promptly all are through the transgenic plant of dichlorophenol sulfonphthalein development process test positive, and pcr analysis is all positive.Dichlorophenol sulfonphthalein development process qualification result has confirmed that the gene that is incorporated in the Plant Genome not only expresses, and expressed proteins mass-energy forms the biological enzyme with function.Therefore the dichlorophenol sulfonphthalein development process is a kind of transgenosis Function Identification method.Method of the present invention is simple, and is easy and simple to handle, and reliable results is with a wide range of applications.
The pattern kind main technical details that barley variety that table 1 the present invention transforms and bibliographical information method transform relatively
Illustrate: a reference: Trifonova A, Madsen S, Olesen A.Agrobacterium-mediated transgene delivery and integrationinto barley under a range of in vitro culture conditions.Plant Sci, 2001,161:871-880.
B: the present invention divides multiple batches of conversion, precocious No. 3 cotransformations of kind 916 ratarias, No. 7 cotransformations of kind Hubei Province barley 418 ratarias, kind has been spent 30 cotransformations 920 ratarias.
Description of drawings
Fig. 1 is a techniqueflow chart of the present invention.
Fig. 2 is a plant expression vector pMBL-5 physical build-up collection of illustrative plates among the present invention.
Fig. 3 is the transformed plant that utilizes agriculture bacillus mediated antibiotic-free screening method for transformation to obtain among the present invention.The upper right corner is blocky single fringe among the figure.
Fig. 4 is the CPR detected result of transformed plant among the present invention.Mark A1 and A2 are the dichlorophenol sulfonphthalein control medium among the figure, and A2-C4 is that dichlorophenol sulfonphthalein detects substratum, and A2 and A4 are the blades of unconverted plant, and B1-C4 is the blade of different transformed plants.
Fig. 5 is the PCR result of transformed plant among the present invention.Mark M is a molecular weight marker among the figure, and 1-8 is a transgene barley, 9 positive contrast pMBL-5, and 10 is non-conversion barley strain contrast, 11 is blank.
Fig. 6 is seminose is spent 30 callus of induce, subculture, differentiation to barley variety influence.
Embodiment
Embodiment 1: the barley gene transformation of agriculture bacillus mediated antibiotic-free
1, callus induces
With wide about 0.5mm～1.5mm, the translucent barley rataria of color (kind precocious No. 3-from Hubei Prov. Acdemy of Agricutural Sciences) is as explant.Earlier the prematurity barley seed was sterilized 30 seconds with 70% ethanol, aseptic water washing 1 time, again with 0.1% mercuric chloride sterilization 8 minutes, aseptic water washing 3 times, every all over 3 minutes.Strip described explant, the explant scultellum is inoculated on the inducing culture (referring to " summary of the invention " part) up, under the condition of 25 ℃ of dark, cultivate, induce callus.
2, the pre-cultivation of callus
Picking is eugonic from the callus that the first step obtains is inoculated on the pre-culture medium (referring to " summary of the invention " part), cultivates 5 hours under the condition of 25 ℃ of dark;
3, the dip-dye of callus
In the fluid suspension culture base (referring to " summary of the invention " part), place vibrate on the shaking table (100 rev/mins) to contaminate 30 minutes in the time of will changing During Agrobacterium over to through pre-incubated callus:
4, callus and Agrobacterium are cultivated altogether
Callus is pressed from both sides out from Agrobacterium suspension culture base, place on the aseptic filter paper to dry up a little, be seeded to common culture medium (referring to " summary of the invention " part), under 25 ℃ of dark conditions, cultivated 2 days;
5, the recovery of callus is cultivated
Callus is changed in the sterilized water that contains the 500mg/L cephamycin, place on the shaking table vibration (100 rev/mins) to soak 30 minutes, on aseptic filter paper, dry up a little, be inoculated on the recovery media 25 ℃ of dark culturing at last 7 days;
6, the screening and culturing of callus
Antibacterial good callus is inoculated into screening culture medium (referring to " summary of the invention " part), 3 weeks of screening and culturing under 25 ℃ of dark conditions;
7, differentiation of calli
Select the callus that screens new growth in back and not brownization death and be inoculated into division culture medium (referring to " summary of the invention " part), 25 ℃, 2000Lux, illumination cultivation is to differentiating green bud point;
8, the screening again of regeneration plant
Green bud point or seedling that differentiation is produced insert the root media (referring to " summary of the invention " part) that contains seminose, and 25 ℃, 2000Lux, illumination cultivation obtains transformed plant to transplanting.Its invention effect as shown in Figure 3.
Embodiment 2: the dichlorophenol sulfonphthalein development process (CPR) of transformed plant detects
Select up-to-date longer a slice leaf on the transformed plant stem of embodiment 1, the blade tip section of the about 0.3cm of clip * 0.5cm area size, the leaf section is immersed dichlorophenol sulfonphthalein liquid detect in the substratum (composition of this substratum is seen the preparation part of dichlorophenol sulfonphthalein liquid detection substratum in " summary of the invention "), after 25 ℃ of lucifuges are cultivated 4 days, observe the substratum colour-change.Simultaneously according to the identical leaf section that requires the unconverted plant of clip, immersing dichlorophenol sulfonphthalein liquid respectively detects in substratum and the dichlorophenol sulfonphthalein liquid control medium (referring to the preparation part of " summary of the invention " dichlorophenol sulfonphthalein liquid control medium), the concrete operations step is cultivated 4 days as contrast referring to " summary of the invention " part in 25 ℃ of lucifuges.If its colour changed into yellow explanation of observing dichlorophenol sulfonphthalein liquid detection substratum detects the activity of phosphomannose isomerase (PMI) enzyme,, the activity that detects in the rotaring gene plant blade less than the PMI enzyme is described if this substratum still keeps red or becomes purple.Under the control medium condition that with sucrose is carbon source,, illustrate that the blade of the transfer-gen plant of being cultivated has utilized sucrose if the color of control medium becomes yellow.The result of present embodiment shows in the 8 tested strain transgene barley strains the positive transformed plant of 6 strains is arranged, and specificity of the present invention has been described.The invention effect of present embodiment as shown in Figure 4.
Embodiment 3: the PCR to transformed plant detects
Get the blade of the barley transgenosis transformed plant about 0.1g, grind into powder in liquid nitrogen is with conventional CTAB method (REFERENCE TO RELATED people's number of patent application is 200610019482.0 disclosed methods) the total DNA of extracting barley.Design a pair of Auele Specific Primer Primerl (forward, 5 '-GTTTCTTTTGTCGATGCTCACCC-3 ') and Primer2 (oppositely, 5 '-TCCGGCTTGTGGTTAGGATC-3 ') and carry out pcr amplification.In 25ul PCR reaction solution, contain the 300ng template DNA, 2.5ul PCR damping fluid, 1.5ul 25mM MgCl
2, 2ul 1.25mMdNTPs, 0.5ul Primer1 (10pmol/ul), 0.5ul Primer2 (10pmol/ul), 1U Taq polysaccharase.The PCR reaction conditions is: 94 ℃ of 1min of 95 ℃ of 5min, 58 ℃ of 50sec, 72 ℃ of 90sec, 35 circulations; Last 72 ℃ of 5min, 10 ℃ of 10min.After reaction is finished, get 18ul PCR product electrophoresis on 1.2% sepharose.Gel imaging system medium ultraviolet lamp is taken a picture down, judges the integration situation of foreign gene according to PCR product segment.The specific band size that produces is 480bp, and judging according to the banding pattern of PCR product has the positive transformed plant of 6 strains in the 8 tested strain transformed plants, and the invention effect as shown in Figure 5.
Embodiment 4: seminose is to the test of barley callus induction, subculture and differentiation influence
In order to determine with effective working concentration of seminose as selective agent, present embodiment is an explant with the mature embryo of barley Cultivar (kind serves as that flower 30-is from Hubei Prov. Acdemy of Agricutural Sciences), 50 explants of every processing, compared of the influence of different seminose gradient concentrations, obtained result as shown in Figure 6 callus of induce, subculture and the differentiation of barley mature embryo.Fig. 6 shows: on callus inducing medium, when mannose concentration in the substratum is 10g/L or 15g/L, the callus induction rate of barley mature embryo reduces by 50%, and when mannose concentration increased to 20g/L or 25g/L, barley mature embryo callus of induce and growth were suppressed fully; In addition, callus is the highest to the tolerance of seminose in the differentiation culture stage.So the present invention determines to use the 20g/L seminose as selective agent in the callus growth stage (being subculture).Continue to use the 20g/L seminose as the selective agent of taking root into seedling in the stage of taking root, all can reach ideal and induce effect.
Claims (1)
1. the method for an agriculture bacillus mediated screening transgene barley strain without antibiotic, its step comprises design of primers, pcr amplification, vector construction, genetic transformation, Screening and Identification transgenic plant, it is characterized in that, by agriculture bacillus mediated the manA gene is imported in the acceptor barley, utilize seminose to substitute microbiotic as selective agent, recipient cell after transforming is screened, be tested and appraised the active and PCR detection of phosphomannose isomerase, screening obtains transfer-gen plant, and its preparation process is as follows:
1) designs a pair of special primer: PMIP1:5 '-ATGGATCCATGCAAAAACTCATTAACTCA-3 ' and PMIP2:5 '-AAGGATCCTTACAGCTTGTTGTAAACACG-3 ', amplification intestinal bacteria manA gene, with this gene is that selectable marker gene makes up plant expression vector pMBL-5, transforms agrobacterium tumefaciens;
2) on callus inducing medium, induce the barley rataria, obtain callus;
3) with step 2) callus be transferred on the pre-culture medium and cultivated 5 hours;
4) callus and the Agrobacterium of step 3) are cultivated altogether, the callus after training altogether is transferred on recovery, screening, differentiation and the root media successively cultivates, screening obtains the candidate transfer-gen plant;
5) detect the candidate transfer-gen plant with the dichlorophenol sulfonphthalein development process, and the candidate's transfer-gen plant that is screened is transplanted into seedling;
6) further with the transfer-gen plant after the PCR evaluation transplanting, obtain transgenic plant;
Wherein:
Step 2) described callus inducing medium is by following formulation:
Callus inducing medium: KNO
31900mg/L, NH
4NO
31650mg/L, KH
2PO
4170mg/L, MgSO
47H
2O370mg/L, CaCl
22H
2O 440mg/L, FeSO
47H
2O 27.8mg/L, Na
2EDTA 37.3mg/L, KI 0.83mg/L, H
3BO
3 
22.3mg/L, ZnSO
47H
2O 8.6mg/L, Na
2MoO
42H
2O 0.25mg/L, CuSO
45H
2O1.25mg/L, CoCl
26H
2O 0.025mg/L, glycine 2mg/L, V
B11mg/L, V
B60.5mg/, V
B50.5mg/L, proline(Pro) 690mg/L, inositol 250mg/L, caseinhydrolysate 1000mg/L, 2,4-D 2mg/L, maltose 30g/L, agar 6g/L keeps the skin wet to 1L pH5.8;
The described substratum of step 3) is pressed following formulation:
Pre-culture medium: with the callus inducing medium is minimum medium, additional N.F,USP MANNITOL 72.8g/L, pH5.8;
The described substratum of step 4) is pressed following formulation:
Be total to culture medium: with the callus inducing medium is minimum medium, additional Syringylethanone 100 μ mol/L, pH5.8;
Recovery media: with the callus inducing medium is minimum medium, additional header p0-357 500mg/L, pH5.8;
Screening culture medium: with the callus inducing medium is minimum medium, and wherein maltose 30g/L is revised as 10g/L, additional seminose 20g/L, pH5.8;
Division culture medium: with the callus inducing medium is minimum medium, and additional header p0-357 300mg/L removes 2 simultaneously, 4-D 2mg/L, pH5.8;
Root media: KNO
3950mg/L, NH
4NO
3825mg/L, KH
2PO
485mg/L, MgSO
47H
2O 185mg/L, CaCl
22H
2O220mg/L, FeSO
47H
2O 13.9mg/L, Na
2EDTA 18.65mg/L, KI 0.415mg/L, H
3BO
33.1mg/L, MnSO
44H
2O 11.15mg/L, ZnSO
47H
2O 4.3mg/L, Na
2MoO
42H
2O 0.125mg/L, CuSO
45H
2O 0.625mg/L, CoCl
26H
2O 0.0125mg/L, glycine 2mg/L, V
B10.5mg/L, V
B60.5mg/L, V
B50.5mg/L, proline(Pro) 690mg/L, inositol 250mg/L, caseinhydrolysate 1000mg/L, NAA 1mg/L, maltose 10g/L, seminose 20g/L, cephamycin 200mg/L, agar 6g/L keeps the skin wet to 1L pH5.8;
The described method of step 5) is as follows:
1. prepare dichlorophenol sulfonphthalein and detect substratum;
2. step detection substratum branch 1. is filled in the Tissue Culture Plate;
3. rotaring gene plant blade is immersed and detect in the substratum 25 ℃ of dark cultivations 4 days;
4. identify the special color reaction of transgenic plant;
Wherein the 1. described dichlorophenol sulfonphthalein of step detects substratum according to following formulation:
KNO
31900mg/L, NH
4NO
31650mg/L, KH
2PO
4170mg/L, MgSO
47H
2O
370mg/L, CaCl
22H
2O440mg/L, FeSO
47H
2O 27.8mg/L, Na
2EDTA37.3mg/L, KI6.2mg/L, H
3BO
30.83mg/L, MnSO
44H
2O, 22.3mg/L, ZnSO
47H
2O 8.6mg/L, Na
2MoO
42H
2O 0.25mg/L, CuSO
45H
2O 0.025mg/L, CoCl
26H
2O 0.025mg/L, glycine 2mg/L, inositol 100mg/L, V
B10.1mg/L, V
B60.5mg/L, V
B50.5mg/L, seminose 15g/L, dichlorophenol sulfonphthalein 100mg/L keeps the skin wet to 1L pH6.0;
The described method of step 6) is as follows:
Adopt a pair of Auele Specific Primer Primer1 forward, 5 '-GTTTCTTTTGTCGATGCTCACCC-3 ' and Primer2 are reverse, 5 ' TCCGGCTTGTGGTTAGGATC-3 ' pcr amplification manA gene specific dna fragmentation, and this fragment length is 480bp.
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|---|---|---|---|---|
| CN108070603A (en) * | 2017-12-28 | 2018-05-25 | 河南健特生物科技集团有限公司 | A kind of transgenic method for improving oil and using peony seeds oil content |
Families Citing this family (3)
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| CN102304544B (en) * | 2011-09-01 | 2013-03-20 | 华中农业大学 | Agrobacterium tumefaciens-mediated barley stem apex transformation method |
| CN103667167A (en) * | 2012-05-02 | 2014-03-26 | 上海凯赛生物技术研发中心有限公司 | Stabilized recombinant expression plasmid vector in Hafnia alvei and application thereof |
| CN113396821A (en) * | 2021-06-15 | 2021-09-17 | 西北农林科技大学 | Tissue culture method for agrobacterium-mediated transformation of lettuce |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1657630A (en) * | 2005-03-25 | 2005-08-24 | 三峡大学 | Process for constructing transgenosis cucumber using mannoheptose as screening marker |
-
2007
- 2007-01-26 CN CN200710051390A patent/CN101016568B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1657630A (en) * | 2005-03-25 | 2005-08-24 | 三峡大学 | Process for constructing transgenosis cucumber using mannoheptose as screening marker |
Non-Patent Citations (6)
| Title |
|---|
| Lucca P ,Ye X D ,Potrykus I.Effective selection and regeneration of transgenicriceplantswith mannose as selective agent.Mol Breed7 1.2001,7(1),43 - 49. |
| Lucca P,Ye X D,Potrykus I.Effective selection and regeneration of transgenicriceplantswith mannose as selective agent.Mol Breed7 1.2001,7(1),43- 49. * |
| 杨莉 等.PMI/甘露糖筛选体系在植物转基因中的应用.林业科学41 3.2005,41(3),137-141. |
| 杨莉 等.PMI/甘露糖筛选体系在植物转基因中的应用.林业科学41 3.2005,41(3),137-141. * |
| 郭晓琳 等.大麦成熟胚愈伤组织的诱导和植株再生的研究.植物遗传资源学报6 4.2005,6(4),418-422. |
| 郭晓琳 等.大麦成熟胚愈伤组织的诱导和植株再生的研究.植物遗传资源学报6 4.2005,6(4),418-422. * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108070603A (en) * | 2017-12-28 | 2018-05-25 | 河南健特生物科技集团有限公司 | A kind of transgenic method for improving oil and using peony seeds oil content |
| CN108070603B (en) * | 2017-12-28 | 2021-06-08 | 洛阳健特药业有限公司 | Transgenic method for improving oil content of oil peony seeds |
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