WO2020253671A1 - 一种农杆菌介导的油菜黑胫病菌的遗传转化方法 - Google Patents

一种农杆菌介导的油菜黑胫病菌的遗传转化方法 Download PDF

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WO2020253671A1
WO2020253671A1 PCT/CN2020/096291 CN2020096291W WO2020253671A1 WO 2020253671 A1 WO2020253671 A1 WO 2020253671A1 CN 2020096291 W CN2020096291 W CN 2020096291W WO 2020253671 A1 WO2020253671 A1 WO 2020253671A1
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medium
agrobacterium
genetic transformation
transformation method
liquid
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French (fr)
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宋培玲
李子钦
史志丹
赵丽丽
燕孟娇
杨永青
皇甫海燕
皇甫九茹
贾晓清
郝丽芬
郭晨
朱春侠
张英
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内蒙古自治区农牧业科学院
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Publication of WO2020253671A1 publication Critical patent/WO2020253671A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation

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  • the invention belongs to the technical field of genetic transformation of plant pathogenic fungi, and in particular relates to a method for genetic transformation of black shank of rapeseed mediated by Agrobacterium.
  • Phoma stem canker is currently one of the important factors restricting the healthy development of rape industry in many countries.
  • L. maculans can cause the base of rape stems to rot and cause serious Loss of production.
  • the spreading trend and evolution law of rape black shank in various countries in the world show that L. biglobosa is gradually replaced by L. maculans.
  • 1999 the pathogenic bacterium L. biglobosa was isolated and obtained in my country for the first time. In recent years, the occurrence and damage of L.
  • the currently widely used fungal transformation methods include plasmid co-transformation, electric shock transformation, gene gun, restriction endonuclease-mediated transformation (REMI) and Agrobacterium-mediated transformation, but plasmid co-transformation, electric shock transformation, gene gun method, restriction Transformation methods such as sex endonuclease-mediated transformation are prone to problems such as unstable transformants and low transformation efficiency due to the complicated operation and poor reproducibility of the protoplast preparation and regeneration process.
  • the electrotransformation method, gene gun method, etc. can also use spores and mycelium as receptors, simplifying the operation steps, the transformation efficiency is low, and the genetic stability of the transformants is poor.
  • the purpose of the present invention is to provide an Agrobacterium-mediated genetic transformation method of Brassica napus with high transformation efficiency, low copy and genetic stability.
  • An Agrobacterium-mediated genetic transformation method of Black Shank of Brassica napus includes the following steps:
  • the plasmid pCHs-GFP includes hygromycin resistance gene, GFP green fluorescent protein gene and kana resistance gene;
  • step 1) There is no time sequence between step 1) and step 2).
  • the OD 600 of the bacterial liquid in step 1) is 0.6.
  • the concentration of acetosyringone in the IM liquid medium in step 1) is 180-220 ⁇ g/mL.
  • the LB liquid medium described in step 1) includes kanamycin and rifampicin.
  • the mixing volume ratio of the Agrobacterium liquid and the conidia suspension in step 3) is 1:1.
  • the concentration of the conidia suspension in step 2) is 1 ⁇ 10 6 /mL.
  • the co-cultivation temperature is 25° C.
  • the co-cultivation time is 4 days
  • the co-cultivation is performed under dark conditions.
  • the selection medium in step 3) is a PDA medium supplemented with cefotaxime sodium at a final concentration of 80-120 mg/mL and hygromycin B at a final concentration of 50-60 mg/mL.
  • the co-cultivation medium in step 3) is a solid CM medium.
  • the pH of the co-cultivation medium is 5.7-5.9.
  • the beneficial effects of the present invention uses the black shank conidia as the material, and the Agrobacterium broth and the conidia suspension are mixed and co-cultured to achieve genetic transformation of Brassica Phytophthora; the method of high conversion efficiency, low copy, genetically stable, every 106 to 161 120 conidia obtained transformants, five generations subcultured, hygromycin-specific PCR Detection and fluorescence detection showed that T-DNA was integrated into the black shank genome in the form of a single copy, and the transformant could be inherited stably.
  • Figure 1 shows the results of testing the stability of the transformed hygromycin resistance, from left to right: F1 to F5;
  • Figure 2 is the result of GFP PCR electrophoresis of the transformant, where Line M: is maker DL2000, Line 1-16 are transformants T1 ⁇ T16, and Line17 is wild strain NM-1;
  • Figure 3 shows the fluorescent expression results of transformants, where the left picture shows the transformant hyphae and the right picture shows the transformed sporozoites.
  • the present invention provides an Agrobacterium-mediated genetic transformation method of Black Shank of Brassica napus, comprising the following steps: 1) Agrobacterium containing plasmid pCHs-GFP is inoculated into LB liquid medium and cultivated until the OD 600 of the bacterial solution is After 0.5 ⁇ 0.7, transfer to CM liquid medium containing 150 ⁇ 250 ⁇ g/mL acetosyringone to resuspend to obtain Agrobacterium liquid; 2) Collect the conidia of Leptosphaeria biglobosa from rapeseed black shank and mix with sterile water to prepare concentration (0.5-9) ⁇ 10 6 /mL conidia suspension; 3) Mix the Agrobacterium liquid with the conidia suspension, co-culture for 3 ⁇ 4 days, then transfer to the screening medium for screening The transformant is obtained by culturing for 7-15 days; the plasmid pCHs-GFP includes the hygromycin resistance gene, the GFP green fluorescent protein gene and the kana resistance gene; there is no time
  • the Agrobacterium containing plasmid pCHs-GFP is inoculated into LB liquid medium and cultured until the OD 600 of the bacterial solution is 0.5-0.7, and then the bacterial cells are collected.
  • the plasmid pCHs-GFP includes hygromycin resistance gene, GFP green fluorescent protein gene and kana resistance gene; the Agrobacterium containing plasmid pCHs-GFP preferably LBA4404 Agrobacterium; the present invention
  • the source of the LBA4404 Agrobacterium is not particularly limited, and it can be obtained by using commercially available products in the field or by self-preparation; the LBA4404 Agrobacterium is more suitable for genetic transformation of black shank bacteria, with high transformation efficiency and stability.
  • the LB liquid medium preferably includes kanamycin and rifampicin; the concentration of kanamycin in the LB liquid medium is preferably 20-30 mg/mL, more preferably 25 mg/mL. mL; the concentration of rifampicin in the LB liquid medium is preferably 20-30 mg/mL, more preferably 25 mg/mL.
  • the present invention does not specifically limit the amount of inoculation of the Agrobacterium, as long as the amount of inoculation conventional in this field is used.
  • the OD 600 of the bacterial liquid is preferably 0.6; the OD 600 range of the bacterial liquid defined in the present invention can ensure successful transformation and will not affect the normal growth of the transformant; if the OD 600 is too low, the transformation cannot be achieved. Too high will affect the normal growth of transformants.
  • the present invention does not specifically limit the method for collecting bacterial cells, and the conventional method for collecting bacterial cells in the field may be used. After the bacteria are collected, the present invention preferably further includes a step of cleaning the bacteria; the cleaning of the bacteria is preferably carried out by using a CM liquid medium containing 150-250 ⁇ g/mL acetosyringone.
  • the collected bacteria are transferred into a CM liquid medium containing 150-250 ⁇ g/mL acetosyringone and resuspended to obtain Agrobacterium broth.
  • the concentration of acetosyringone in the CM liquid medium is preferably 180-220 ⁇ g/mL, more preferably 200 ⁇ g/mL; the acetosyringone in the CM liquid medium can be within the scope defined by the present invention. Ensure that the number of transformants obtained is large and can grow normally. If the concentration of acetosyringone increases, it will have a toxic effect on the strain, affect its normal growth, and reduce the number of transformants.
  • the OD 600 of the Agrobacterium liquid obtained after resuspension is preferably 0.6.
  • the CM liquid medium based on 1 L, preferably includes the following components: K 2 HPO 4 3.44g, KH 2 PO 4 1.45g, NaCl 0.15g, MgSO 4 ⁇ 7H 2 O 0.5g , (NH 4 ) 2SO 4 0.5g CaCl 2 ⁇ H 2 O 0.067g, FeSO 4 ⁇ 7H 2 O 0.0025g, Glucose 1.8g, MES 7.8g, glycerin 5mL and the remainder of distilled water.
  • the conidia of the black shank of rapeseed are collected and mixed with sterile water to prepare a conidia suspension with a concentration of (0.5-9) ⁇ 10 6 cells/mL.
  • the concentration of the conidia suspension is preferably (0.8-5) ⁇ 10 6 /mL, more preferably 1 ⁇ 10 6 /mL.
  • the conidia are preferably the conidia produced by culturing the rape black shank Leptosphaeria biglobosa for 10 to 14 days at 25°C, and more preferably the conidia produced by culturing for 12 days.
  • the black shank of rapeseed is preferably cultured on a spore-forming plate; the collection of the conidia preferably includes the following steps: adding sterile water to the spore-forming plate, standing for 8-12 minutes, Then the conidia were scraped off the sporulation plate.
  • the added amount of the sterile water is preferably 2 to 4 mL/spore-forming plate; the scraping of the conidia is preferably carried out by using a sterile glass slide; After separation, the scraping liquid is preferably filtered, and the filtrate is collected to obtain conidia; the filtering is preferably gauze filtration, and the gauze for filtering is preferably sterile gauze.
  • the Agrobacterium liquid and the conidia suspension are obtained, the Agrobacterium liquid and the conidia suspension are mixed, and after co-cultivation for 3 to 4 days, they are transferred to a screening medium for screening and culture
  • the transformants were obtained from 7 to 15 days.
  • the volume ratio of the mixing of the Agrobacterium liquid and the conidia suspension is preferably 1:1; the above ratio can ensure that the number of transformants obtained is moderate and easy to separate and purify; if the number of transformants obtained is too large If the hyphae of the transformant are stuck together, it is difficult to separate and purify.
  • the obtained mixed solution is thoroughly mixed after the mixing.
  • the method of the present invention is not particularly limited, and conventional shaking or mixing in the field may be used.
  • the co-cultivation temperature is preferably 25°C
  • the co-cultivation time is preferably 4 days
  • the co-cultivation is preferably performed under dark conditions.
  • the temperature of the co-cultivation defined by the present invention will not hinder the gene expression of VirB, VirD, and VirE, the infectivity of Agrobacterium is stable, and Agrobacterium grows well, and the number of transformants obtained is large;
  • the culturing time ensures that a sufficient number of transformants can be formed, and at the same time avoids the co-cultivation time being too long to produce too many false positive transformed colonies.
  • the co-cultivation medium is preferably CM solid medium
  • the CM solid medium based on 1 L, preferably includes the following components: K 2 HPO 4 3.44g, KH 2 PO 4 1.45g , NaCl 0.15g, MgSO 4 ⁇ 7H 2 O 0.5g, (NH 4 )2SO 4 0.5g CaCl 2 ⁇ H 2 O 0.067g, FeSO 4 ⁇ 7H 2 O 0.0025g, Glucose 1.8g, MES 7.8g, Glycerin 5ml , 15g agar powder and the remainder of distilled water; the pH value of the CM solid medium is preferably 5.7-5.9, more preferably 5.8.
  • the co-cultivation preferably coats the mixed liquid on a CM solid plate covered with glass fiber filter paper.
  • the coating amount of the mixed liquid is preferably 150-250 ⁇ L/CM A solid plate, more preferably a 200 ⁇ L/CM solid plate.
  • the amount of coating affects the number of transformants obtained. If the amount of bacterial liquid applied is small, the number of transformants obtained is relatively small. If the amount of bacterial liquid applied is large, the transformation obtained later The number of seeds is relatively large, but it takes 7-15 days for transformants to screen and culture, which will cause hyphae adhesion of transformants, which is not conducive to later separation and purification.
  • the product of the co-cultivation is transferred to a selection medium, and the transformant is obtained by 7-15 days of selection and culture.
  • the selection medium is preferably a PDA medium supplemented with cefotaxime sodium at a final concentration of 80 to 120 mg/mL and hygromycin B at a final concentration of 50 to 60 mg/mL, more preferably a final concentration of A PDA medium containing cefotaxime sodium at a concentration of 100 mg/mL and hygromycin B at a final concentration of 50 mg/mL.
  • the present invention preferably further includes an identification step of the transformant.
  • the identification of the transformant is preferably achieved by the following two methods: First, extract the DNA of the transformant, perform GFP-specific PCR amplification, and agarose gel electrophoresis to detect whether the amplified product is GFP-specific Sex band; if it is present, it is a correct positive transformant, and if it is not present, it is not a correct positive transformant. Second, picking of transformants As for observation under a fluorescence microscope, if it shows green fluorescence, it is a correct positive transformant, and if it does not show green fluorescence, it is not a correct positive transformant.
  • PDA medium 200g potato, 20g glucose, 15g agar powder, dilute to 1000mL with water, and autoclave at 121°C for 20min.
  • LB medium 5g yeast powder, 10g tryptone, 10g sodium chloride, 15g agar powder, add water to make up to 1000mL, autoclave at 121°C for 20min.
  • CM co-culture medium K 2 HPO 4 3.44g, KH 2 PO 4 1.45g, NaCl 0.15g, MgSO 4 ⁇ 7H 2 O 0.5g, (NH 4 )2SO 4 0.5g CaCl 2 ⁇ H 2 O 0.067g , FeSO 4 ⁇ 7H 2 O 0.0025g, Glucose 1.8g, MES 7.8g, glycerin 5mL, agar powder 15g, distilled water to dilute to 1000mL, pH 5.8.
  • IM medium Based on CM medium, add acetosyringone at a final concentration of 200 ⁇ g/mL to CM medium.
  • SM selection medium Based on PDA medium, add cefotaxime sodium at a final concentration of 100 mg/mL and hygromycin B at a final concentration of 50 mg/mL to the PDA medium.
  • the Agrobacterium strain LBA4404 containing the plasmid pCHs-GFP (the plasmid contains the hygromycin resistance gene, the GFP green fluorescent protein gene, and the kana resistance gene) contained 25 mg/mL kanamycin and 25 mg/mL rifampicin.
  • Cultured in LB liquid medium the culture temperature is 28°C, the rotation speed is 200rpm, and the culture time is 12h; when the culture reaches the OD 600 value of 0.6, transfer to IM liquid medium containing acetosyringone and resuspend to obtain Agrobacterium Bacteria.
  • the statistical method of transformants is to separate the colonies separately.
  • the ones that can grow normally on the hygromycin resistant medium are transformants, and a single colony is one transformant; 3 parallel replicates are set, and each replicate obtains an average of 161 transformations child.
  • the randomly selected transformants isolated from monospores were transferred to a PDA plate, and then transferred to a new PDA plate after 48 hours of culture. After five consecutive generations, the growth of the transformant was observed to determine its genetic stability. The results showed that after culturing on PDA medium for 5 generations, all the transformants on the PDA plate containing 50mg/mL hygromycin B could grow, but the wild-type black shank could not grow (Figure 1), indicating the transformation The transformants obtained by the method have high genetic stability.
  • test results showed that all the 16 transformant samples tested were able to amplify the GFP gene fragment with the same size as the plasmid, while the wild-type strain nm-1 failed to amplify a band with the same size as the target fragment ( Figure 2). It shows that the T-DNA of pCHs-GFP vector has been successfully introduced and integrated into the genomic DNA of Black Shank.
  • the present invention performs fluorescence detection on hygromycin-resistant transformants.
  • a sterile inoculation needle to pick up the fresh hyphae and conidia of the genetic transformants of Black Shank of Brassica napus, place them on a glass slide dripped with sterile water, lightly cover the cover slip to avoid air bubbles, and place it in inverted fluorescence Under a microscope (Nikon Eclipse Ti), the fluorescent protein in the hyphae and spores of the black shank transformant was observed at an excitation wavelength of 552 nm.
  • the results are shown in Fig. 3, that the hyphae and spores of the transformants all emit obvious green fluorescence, which proves that the black shank transformants identified by PCR screening are the correct positive transgenic strains.
  • PDA medium 200g potato, 20g glucose, 15g agar powder, dilute to 1000mL with water, and autoclave at 121°C for 20min.
  • LB medium 5g yeast powder, 10g tryptone, 10g sodium chloride, 15g agar powder, add water to make up to 1000mL, autoclave at 121°C for 20min.
  • CM co-culture medium K 2 HPO 4 3.44g, KH 2 PO 4 1.45g, NaCl 0.15g, MgSO 4 ⁇ 7H 2 O 0.5g, (NH 4 )2SO 4 0.5g CaCl 2 ⁇ H 2 O 0.067g , FeSO 4 ⁇ 7H 2 O 0.0025g, Glucose 1.8g, MES 7.8g, glycerin 5mL, agar powder 15g, distilled water to dilute to 1000mL, pH 5.8.
  • IM medium Based on CM medium, add acetosyringone at a final concentration of 200 ⁇ g/mL to CM medium.
  • SM selection medium Based on PDA medium, add cefotaxime sodium at a final concentration of 100 mg/mL and hygromycin B at a final concentration of 50 mg/mL to the PDA medium.
  • the Agrobacterium strain LBA4404 containing the plasmid pCHs-GFP (the plasmid contains the hygromycin resistance gene, the GFP green fluorescent protein gene, and the kana resistance gene) contained 25 mg/mL kanamycin and 25 mg/mL rifampicin.
  • LB liquid medium was cultured to an OD 600 value of 0.8, transferred to IM liquid medium containing acetosyringone and resuspended to obtain Agrobacterium broth.
  • the average number of transformants was calculated to be 36/10 6 conidia.
  • PDA medium 200g potato, 20g glucose, 15g agar powder, dilute to 1000mL with water, and autoclave at 121°C for 20min.
  • LB medium 5g yeast powder, 10g tryptone, 10g sodium chloride, 15g agar powder, add water to make up to 1000mL, autoclave at 121°C for 20min.
  • CM co-culture medium K 2 HPO 4 3.44g, KH 2 PO 4 1.45g, NaCl 0.15g, MgSO 4 ⁇ 7H 2 O 0.5g, (NH 4 )2SO 4 0.5g CaCl 2 ⁇ H 2 O 0.067g , FeSO 4 ⁇ 7H 2 O 0.0025g, Glucose 1.8g, MES 7.8g, glycerin 5mL, agar powder 15g, distilled water to dilute to 1000mL, pH 5.8.
  • IM medium Based on CM medium, add acetosyringone at a final concentration of 200 ⁇ g/mL to CM medium.
  • SM selection medium Based on PDA medium, add cefotaxime sodium at a final concentration of 100 mg/mL and hygromycin B at a final concentration of 50 mg/mL to the PDA medium.
  • the Agrobacterium strain LBA4404 containing the plasmid pCHs-GFP (the plasmid contains the hygromycin resistance gene, the GFP green fluorescent protein gene, and the kana resistance gene) contained 25 mg/mL kanamycin and 25 mg/mL rifampicin.
  • LB liquid medium was cultured to an OD 600 value of 0.6, then transferred to IM liquid medium containing acetosyringone and resuspended to obtain Agrobacterium broth.
  • the average number of transformants was calculated to be 6/10 6 conidia.
  • PDA medium 200g potato, 20g glucose, 15g agar powder, dilute to 1000mL with water, and autoclave at 121°C for 20min.
  • LB medium 5g yeast powder, 10g tryptone, 10g sodium chloride, 15g agar powder, add water to make up to 1000mL, autoclave at 121°C for 20min.
  • CM co-culture medium K 2 HPO 4 3.44g, KH 2 PO 4 1.45g, NaCl 0.15g, MgSO 4 ⁇ 7H 2 O 0.5g, (NH 4 )2SO 4 0.5g CaCl 2 ⁇ H 2 O 0.067g , FeSO 4 ⁇ 7H 2 O 0.0025g, Glucose 1.8g, MES 7.8g, glycerin 5mL, agar powder 15g, distilled water to dilute to 1000mL, pH 5.8.
  • IM medium Based on CM medium, add acetosyringone at a final concentration of 300 ⁇ g/mL to CM medium.
  • SM selection medium Based on PDA medium, add cefotaxime sodium at a final concentration of 100 mg/mL and hygromycin B at a final concentration of 50 mg/mL to the PDA medium.
  • the Agrobacterium strain LBA4404 containing the plasmid pCHs-GFP (the plasmid contains the hygromycin resistance gene, the GFP green fluorescent protein gene, and the kana resistance gene) contained 25 mg/mL kanamycin and 25 mg/mL rifampicin.
  • LB liquid medium was cultured to an OD 600 value of 0.6, then transferred to IM liquid medium containing acetosyringone and resuspended to obtain Agrobacterium broth.
  • the average number of transformants was calculated to be 17/10 6 conidia.
  • PDA medium 200g potato, 20g glucose, 15g agar powder, dilute to 1000mL with water, and autoclave at 121°C for 20min.
  • LB medium 5g yeast powder, 10g tryptone, 10g sodium chloride, 15g agar powder, add water to make up to 1000mL, autoclave at 121°C for 20min.
  • CM co-culture medium K 2 HPO 4 3.44g, KH 2 PO 4 1.45g, NaCl 0.15g, MgSO 4 ⁇ 7H 2 O 0.5g, (NH 4 )2SO 4 0.5g CaCl 2 ⁇ H 2 O 0.067g , FeSO 4 ⁇ 7H 2 O 0.0025g, Glucose 1.8g, MES 7.8g, glycerin 5mL, agar powder 15g, distilled water to dilute to 1000mL, pH 5.8.
  • IM medium Based on CM medium, add acetosyringone at a final concentration of 400 ⁇ g/mL to CM medium.
  • SM selection medium Based on PDA medium, add cefotaxime sodium at a final concentration of 100 mg/mL and hygromycin B at a final concentration of 50 mg/mL to the PDA medium.
  • the Agrobacterium strain LBA4404 containing the plasmid pCHs-GFP (the plasmid contains the hygromycin resistance gene, the GFP green fluorescent protein gene, and the kana resistance gene) contained 25 mg/mL kanamycin and 25 mg/mL rifampicin.
  • LB liquid medium was cultured to an OD 600 value of 0.6, then transferred to IM liquid medium containing acetosyringone and resuspended to obtain Agrobacterium broth.
  • the average number of transformants was calculated to be 7/10 6 conidia.

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Abstract

提供了一种农杆菌介导的油菜黑胫病菌的遗传转化方法，该方法包括以下步骤：1)将含质粒pCHs-GFP的农杆菌接种于LB液体培养基中培养，后转入含有150～250μg/mL乙酰丁香酮的CM液体培养基中重悬获得农杆菌菌液；2)收集油菜黑胫病菌的分生孢子与无菌水混合制备浓度为(0.5～9)×106个/mL的分生孢子悬浮液；3)将农杆菌菌液与分生孢子悬浮液混合，共培养3～4d后，转移至筛选培养基，筛选培养7～15d获得转化子。

Description

一种农杆菌介导的油菜黑胫病菌的遗传转化方法
本申请要求于2019年06月17日提交中国专利局、申请号为201910520818.9、发明名称为“一种农杆菌介导的油菜黑胫病菌的遗传转化方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于植物病原真菌遗传转化技术领域，尤其涉及一种农杆菌介导的油菜黑胫病菌的遗传转化方法。
背景技术
油菜黑胫病(Phoma stem canker)，目前是制约许多国家油菜产业健康发展的重要因素之一。研究显示，在世界上许多黑胫病流行的油菜产区，病原复合种Leptosphaeria maculans和Leptosphaeria biglobosa两者共同存在，单独或共同侵染寄主作物，L.maculans可引起油菜茎基部腐烂并造成严重的产量损失。油菜黑胫病在世界各国的扩散趋势及演化规律显示存在L.biglobosa被L.maculans逐渐替代的趋势。1999年，我国首次分离获得病原菌L.biglobosa，且近年来L.biglobosa在我国的发生危害呈逐年扩展蔓延趋势，随着L.biglobosa的扩展蔓延，我国极有可能出现L.maculans，即黑胫病对我国的油菜及十字花科作物安全生产构成了威胁。
为阻止、应对黑胫病给我国的油菜生产造成巨大的经济损失，我们必须明白黑胫病(L.biglobosa)与油菜的互作关系。荧光标记为研究病原微生物与寄主间的相互作用提供了更为直观的手段，利用这种方法能够进行长期的、实时的、直接的观测。而建立油菜黑胫病菌(L.biglobosa)的遗传转化体系，获得带有荧光标记阳性转化子是前提和基础。目前广泛采用的真菌转化方法有质粒共转化、电激转化、基因枪、限制性内切酶介导转化(REMI)和农杆菌介导转化，但质粒共转化、电激转化、基因枪法、限制性内切酶介导转化等转化方法因原生质体制备和再生过程操作繁琐、重复性差而易造成转化子不稳定、转化效率低等问题。虽然电转化法、基因枪法等也可以以孢子和菌丝体为受体，简化了操作步骤，但转化效率较低，且转化子遗传稳定性差。
发明内容
有鉴于此，本发明的目的在于提供一种转化效率高、低拷贝、遗传稳定的农杆菌介导的油菜黑胫病菌的遗传转化方法。
为了实现上述发明目的，本发明提供了以下技术方案：
一种农杆菌介导的油菜黑胫病菌的遗传转化方法，包括以下步骤：
1)将含质粒pCHs-GFP的农杆菌接种于LB液体培养基中培养至菌液的OD 600为0.5～0.7后，转入含有150～250μg/mL乙酰丁香酮的CM液体培养基中重悬获得农杆菌菌液；
2)收集油菜黑胫病菌Leptosphaeria biglobosa的分生孢子与无菌水混合制备浓度为(0.5～9)×10 6个/mL的分生孢子悬浮液；
3)将所述农杆菌菌液与分生孢子悬浮液混合，共培养3～4d后，转移至筛选培养基，筛选培养7～15d获得转化子；
所述质粒pCHs-GFP上包括潮霉素抗性基因、GFP绿色荧光蛋白基因和卡那抗性基因；
步骤1)和步骤2)之间无时间顺序限定。
优选的，步骤1)中所述菌液的OD 600为0.6。
优选的，步骤1)中所IM液体培养基中乙酰丁香酮的浓度为180～220μg/mL。
优选的，步骤1)所述的LB液体培养基中包括卡那霉素和利福平。
优选的，步骤3)中所述农杆菌菌液与分生孢子悬浮液混合的体积比为1:1。
优选的，步骤2)中所述分生孢子悬浮液的浓度为1×10 6个/mL。
优选的，步骤3)中所述共培养温度为25℃，所述共培养的时间为4d，所述共培养在黑暗条件下进行。
优选的，步骤3)中所述筛选培养基为添加终浓度为80～120mg/mL的头孢噻肟钠和终浓度为50～60mg/mL的潮霉素B的PDA培养基。
优选的，步骤3)中所述共培养的培养基为固体CM培养基。
优选的，所述共培养的培养基的pH值为5.7～5.9。
本发明的有益效果：本发明提供的农杆菌介导的油菜黑胫病菌的遗传转化方法，以黑胫病菌分生孢子为材料，将农杆菌菌液与分生孢子悬浮液混合共培养，实现对油菜黑胫病菌的遗传转化；所述方法转化效率高、低拷贝、遗传稳定，每10 6个分生孢子可获得120～161个转化子，5代继代培养、潮霉素特异性PCR检测及荧光检测显示，T-DNA以单拷贝的形式整合到黑胫病基因组中，转化子能够稳定遗传。
说明书附图
图1为转化子潮霉素抗性稳定性检测结果，从左至右分别为：F1～F5代；
图2为转化子的GFP PCR电泳结果图，其中Line M:为maker DL2000，Line 1～16为转化子T1～T16，Line17为野生菌株NM-1；
图3为转化子的荧光表达结果，其中左图为转化子菌丝，右图为转化子孢子。
具体实施方式
本发明提供了一种农杆菌介导的油菜黑胫病菌的遗传转化方法，包括以下步骤：1)将含质粒pCHs-GFP的农杆菌接种于LB液体培养基中培养至菌液的OD 600为0.5～0.7后，转入含有150～250μg/mL乙酰丁香酮的CM液体培养基中重悬获得农杆菌菌液；2)收集油菜黑胫病菌Leptosphaeria biglobosa的分生孢子与无菌水混合制备浓度为(0.5～9)×10 6个/mL的分生孢子悬浮液；3)将所述农杆菌菌液与分生孢子悬浮液混合，共培养3～4d后，转移至筛选培养基，筛选培养7～15d获得转化子；所述质粒pCHs-GFP上包括潮霉素抗性基因、GFP绿色荧光蛋白基因和卡那抗性基因；步骤1)和步骤2)之间无时间顺序限定。
在本发明中，将含质粒pCHs-GFP的农杆菌接种于LB液体培养基中培养至菌液的OD 600为0.5～0.7后，收集菌体。在本发明中，所述质粒pCHs-GFP上包括潮霉素抗性基因、GFP绿色荧光蛋白基因和卡那抗性基因；所述含质粒pCHs-GFP的农杆菌优选的LBA4404农杆菌；本发明对 所述LBA4404农杆菌的来源没有特殊限定，采用本领域市售产品或自行制备获得；所述LBA4404农杆菌更适于黑胫病菌的遗传转化，转化效率高且稳定。本发明中，优选的将质粒pCHs-GFP转入LBA4404农杆菌中获得含质粒pCHs-GFP的农杆菌；本发明对所述转入的方法没有特殊限定，采用本领域常规方法即可。在本发明中，所述LB液体培养基中优选的包括卡那霉素和利福平；所述LB液体培养基中卡那霉素的浓度优选为20～30mg/mL，更优选为25mg/mL；所述LB液体培养基中利福平的浓度优选为20～30mg/mL，更优选为25mg/mL。本发明对所述农杆菌的接种量没有特殊限定，采用本领域常规的接种量即可。在本发明中，所述菌液的OD 600优选为0.6；在本发明限定的菌液的OD 600范围能够保证转化成功，并且不会影响转化子的正常生长；OD 600过低无法实现转化，过高会影响转化子的正常生长。本发明对所述收集菌体的方法没有特殊限定，采用本领域常规的菌体收集方法即可。本发明在所述收集菌体后，优选的还包括清洗菌体的步骤；所述清洗菌体优选的采用含有150～250μg/mL乙酰丁香酮的CM液体培养基进行。
本发明将所述收集到的菌体转入含有150～250μg/mL乙酰丁香酮的CM液体培养基中重悬获得农杆菌菌液。在本发明中，所述CM液体培养基中乙酰丁香酮的浓度优选为180～220μg/mL，更优选为200μg/mL；所述CM液体培养基中乙酰丁香酮在本发明限定的范围内能够保证获得的转化子数量多，并且能够正常生长。若乙酰丁香酮的浓度增加，会对菌株产生毒害作用，影响其正常生长，降低转化子的数量。在本发明中，所述重悬后获得的农杆菌菌液的OD 600优选为0.6。在本发明中，所述CM液体培养基，以1L计，优选的包括以下组分：K 2HPO 43.44g、KH 2PO 4l.45g、NaCl 0.15g、MgSO 4·7H 2O 0.5g、(NH 4)2SO 4 0.5g CaCl 2·H 2O 0.067g、FeSO 4·7H 2O 0.0025g、Glucose 1.8g、MES 7.8g、甘油5mL和余量的蒸馏水。
在本发明中，收集油菜黑胫病菌的分生孢子与无菌水混合制备浓度为(0.5～9)×10 6个/mL的分生孢子悬浮液。在本发明中，所述分生孢子悬浮液的浓度优选为(0.8～5)×10 6个/mL，更优选为1×10 6个/mL。在本发 明中，所述分生孢子优选为25℃的光照培养油菜黑胫病菌Leptosphaeria biglobosa 10～14d产生的分生孢子，更优选为培养12d产生的分生孢子。在本发明中，所述油菜黑胫病菌优选的培养在产孢平板上；所述分生孢子的收集优选的包括以下步骤：向所述产孢平板添加无菌水，静置8～12min、然后将分生孢子刮离产孢平板。在本发明中，所述无菌水的添加量优选为2～4mL/产孢平板；所述分生孢子的刮离优选的采用无菌载玻片进行；本发明在所述分生孢子刮离后，优选的对刮离液进行过滤，收集滤液获得分生孢子；所述过滤优选为纱布过滤，所述过滤用纱布优选为无菌纱布。
本发明在获得所述农杆菌菌液和所述分生孢子悬浮液后，将所述农杆菌菌液与分生孢子悬浮液混合，共培养3～4d后，转移至筛选培养基，筛选培养7～15d获得转化子。在本发明中，所述农杆菌菌液与分生孢子悬浮液混合的体积比优选为1:1；上述比例能够确保获得的转化子数量适中，易于分离和纯化；若获得的转化子数量过多，会出现转化子菌丝粘连在一起的现象，分离和纯化困难。本发明在所述混合后将得到的混合液充分混匀，本发明对所述充分混匀的方法没有特殊限定，采用本领域常规的震荡混匀或搅拌混匀即可。在本发明中，所述共培养温度优选为25℃，所述共培养的时间优选为4d，所述共培养优选的在黑暗条件下进行。本发明所限定的共培养的温度不会使VirB和VirD、VirE的基因表达受阻，农杆菌的侵染能力稳定，并且农杆菌生长良好，获得转化子的数量较多；本发明所限定的共培养的时间在保证能形成足够数量的转化子的同时，又避免了共培养时间过长而产生过多的假阳性转化菌落。
本发明中，所述共培养的培养基优选为CM固体培养基，所述CM固体培养基，以1L计，优选的包括以下组分：K 2HPO 43.44g、KH 2PO 4l.45g、NaCl 0.15g、MgSO 4·7H 2O 0.5g、(NH 4)2SO 4 0.5g CaCl 2·H 2O 0.067g、FeSO 4·7H 2O 0.0025g、Glucose 1.8g，MES 7.8g，甘油5ml，琼脂粉15g和余量的蒸馏水；所述CM固体培养基的pH值优选为5.7～5.9，更优选为5.8。在本发明具体实施过程中，所述共培养优选的将混合液涂布于铺有玻璃纤维滤纸的CM固体平板，在本发明中，所述混合液的涂布量优选为150～250μL/CM固体平板，更优选为200μL/CM固体平板。在本发明中，涂布量的多少影响获得转化子数量的多少，如果涂布的菌液量较少， 则相对的获得转化子数量少，如果涂布的菌液量多，后期获得的转化子数量就较多，但转化子筛选培养需要7～15d，会造成转化子菌丝粘连，不利于后期分离纯化。
本发明在所述共培养结束后，将所述共培养的产物转移至筛选培养基，进行7～15d筛选培养获得转化子。在本发明具体实施过程中，优选的将带有共培养产物的所述玻璃纤维滤纸直接转移至筛选培养基上。在本发明中，所述筛选培养基优选为添加终浓度为80～120mg/mL的头孢噻肟钠和终浓度为50～60mg/mL的潮霉素B的PDA培养基，更优选为添加终浓度为100mg/mL的头孢噻肟钠和终浓度为50mg/mL的潮霉素B的PDA培养基。
本发明在获得所述转化子后，优选的还包括对所述转化子的鉴定步骤。在本发明中，所述转化子的鉴定优选的通过以下两种方法实现：第一，提取转化子的DNA，进行GFP特异性PCR扩增，琼脂糖凝胶电泳检测扩增产物是否存在GFP特异性条带；如果存在即为正确的阳性转化子，不存在则不是正确的阳性转化子。第二，挑取转化子至于荧光显微镜下观察，如果呈现绿色荧光即为正确的阳性转化子，不呈现绿色的荧光则不是正确的阳性转化子。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
本实施例中使用的培养基：
PDA培养基：马铃薯200g，葡萄糖20g，琼脂粉15g，加水定容至1000mL，121℃高压蒸汽灭菌20min。
LB培养基：酵母粉5g、胰蛋白胨10g、氯化钠10g、琼脂粉15g，加水定容至1000mL，121℃高压蒸汽灭菌20min。
CM共培养培养基：K 2HPO 43.44g、KH 2PO 4l.45g、NaCl 0.15g、MgSO 4·7H 2O 0.5g、(NH 4)2SO 4 0.5g CaCl 2·H 2O 0.067g、FeSO 4·7H 2O 0.0025g、Glucose 1.8g，MES 7.8g，甘油5mL，琼脂粉15g，蒸馏水定容至1000mL，pH值5.8。
IM培养基:以CM培养基为基础，在CM培养基中加入终浓度为200μg/mL的乙酰丁香酮。
SM筛选培养基：以PDA培养基为基础，在PDA培养基中加入终浓度为100mg/mL的头孢噻肟钠和终浓度为50mg/mL的潮霉素B。
方法步骤：
将含质粒pCHs-GFP(质粒上含有潮霉素抗性基因，GFP绿色荧光蛋白基因，卡那抗性基因)的农杆菌LBA4404菌株在含有25mg/mL卡那霉素和25mg/mL利福平的LB液体培养基中培养，培养温度为28℃，转速为200rpm，培养时间为12h；当培养至菌液OD 600值为0.6，转入含有乙酰丁香酮的IM液体培养基重悬获得农杆菌菌液。
25℃的光照培养油菜黑胫病菌Leptosphaeria biglobosa 12d产生分生孢子，在产孢平板上加3mL无菌水，静置10min，无菌载玻片轻刮平板表面待分生孢子充分释放后用无菌纱布过滤获得悬浮液，无菌水稀释至浓度为1×10 6个/mL的分生孢子悬浮液。
将上述配置好的农杆菌菌液和分生孢子悬浮液按1:1的体积比例混合，充分混匀后取200μL均匀涂布在铺有玻璃纤维滤纸的CM固体平板上25℃共培养4d，将玻璃纤维滤纸转移至SM筛选培养基上，培养10d，筛选获得转化子。
转化子的统计方式为将菌落单独分离出来，在潮霉素抗性培养基可正常生长的为转化子，一个单菌落为一个转化子；设置3个平行重复，每个重复平均获得161个转化子。
将随机挑选的单孢分离的转化子转接到PDA平板上，培养48h后转接到新的PDA平板上，连续转接五代后，观察转化子的生长情况确定其遗传稳定性。结果表明在PDA培养基上培养5代后，转至含50mg/mL潮霉素B的PDA平板上所有的转化子都能生长，而野生型黑胫病菌不能生长(图1)，说明该转化方法获得的转化子具有较高的遗传稳定性。
以野生型黑胫病菌NM-1和随机选取的16个转化子的基因组DNA为模板进行PCR扩增检测，
gfp基因扩增程序：
Figure PCTCN2020096291-appb-000001
Figure PCTCN2020096291-appb-000002
gfp基因扩增反应体系：
Figure PCTCN2020096291-appb-000003
检测结果表明所检测的16个转化子样品均能扩增出与质粒大小相同的GFP基因片段，而野生型菌株nm-1未能扩增出与目的片段大小一致的条带(图2)，表明pCHs-GFP载体的T-DNA已成功导入整合到黑胫病菌的基因组DNA上。
为了进一步确定pCHs-GFP基因已经成功地导入了野生型油菜黑胫病菌NM-1中并实现了表达，本发明对有潮霉素抗性的转化子进行了荧光检测。用无菌接种针挑取上述油菜黑胫病菌遗传转化子的新鲜菌丝和分生孢子，置于滴有无菌水的载玻片上，轻盖盖玻片，避免产生气泡，置于倒置荧光显微镜(Nikon Eclipse Ti)下，在552nm激发波长下观察黑胫病菌转化子的菌丝和孢子中的荧光蛋白。结果如图3所示，转化子的菌丝和孢子都发出明显的绿色荧光，证明本发明通过PCR筛选鉴定得到的黑胫病菌转化子为正确的阳性转基因菌株。
对比例1
本对比例中使用的培养基：
PDA培养基：马铃薯200g，葡萄糖20g，琼脂粉15g，加水定容至1000mL，121℃高压蒸汽灭菌20min。
LB培养基：酵母粉5g、胰蛋白胨10g、氯化钠10g、琼脂粉15g，加水定容至1000mL，121℃高压蒸汽灭菌20min。
CM共培养培养基：K 2HPO 43.44g、KH 2PO 4l.45g、NaCl 0.15g、MgSO 4·7H 2O 0.5g、(NH 4)2SO 4 0.5g CaCl 2·H 2O 0.067g、FeSO 4·7H 2O 0.0025g、Glucose 1.8g，MES 7.8g，甘油5mL，琼脂粉15g，蒸馏水定容至1000mL，pH值5.8。
IM培养基:以CM培养基为基础，在CM培养基中加入终浓度为200μg/mL的乙酰丁香酮。
SM筛选培养基：以PDA培养基为基础，在PDA培养基中加入终浓度为100mg/mL的头孢噻肟钠和终浓度为50mg/mL的潮霉素B。
方法步骤：
将含质粒pCHs-GFP(质粒上含有潮霉素抗性基因，GFP绿色荧光蛋白基因，卡那抗性基因)的农杆菌LBA4404菌株在含有25mg/mL卡那霉素和25mg/mL利福平的LB液体培养基中培养至OD 600值为0.8，转入含有乙酰丁香酮的IM液体培养基重悬获得农杆菌菌液。
25℃的光照培养油菜黑胫病菌Leptosphaeria biglobosa 12d产生分生孢子，在产孢平板上加3mL无菌水，静置10min，无菌载玻片轻刮平板表面待分生孢子充分释放后用无菌纱布过滤获得悬浮液，无菌水稀释至浓度为1×10 6个/mL的分生孢子悬浮液。
将上述配置好的农杆菌菌液和分生孢子悬浮液按1:1的体积比例混合，充分混匀后取200μL均匀涂布在铺有玻璃纤维滤纸的CM固体平板上25℃共培养4d，将玻璃纤维滤纸转移至SM筛选培养基上，培养10d，筛选获得转化子。
计算获得转化子的平均个数为36个/10 6分生孢子。
对比例2
本对比例中使用的培养基：
PDA培养基：马铃薯200g，葡萄糖20g，琼脂粉15g，加水定容至1000mL，121℃高压蒸汽灭菌20min。
LB培养基：酵母粉5g、胰蛋白胨10g、氯化钠10g、琼脂粉15g，加水定容至1000mL，121℃高压蒸汽灭菌20min。
CM共培养培养基：K 2HPO 43.44g、KH 2PO 4l.45g、NaCl 0.15g、MgSO 4·7H 2O 0.5g、(NH 4)2SO 4 0.5g CaCl 2·H 2O 0.067g、FeSO 4·7H 2O 0.0025g、Glucose 1.8g，MES 7.8g，甘油5mL，琼脂粉15g，蒸馏水定容至1000mL，pH值5.8。
IM培养基:以CM培养基为基础，在CM培养基中加入终浓度为200μg/mL的乙酰丁香酮。
SM筛选培养基：以PDA培养基为基础，在PDA培养基中加入终浓度为100mg/mL的头孢噻肟钠和终浓度为50mg/mL的潮霉素B。
方法步骤：
将含质粒pCHs-GFP(质粒上含有潮霉素抗性基因，GFP绿色荧光蛋白基因，卡那抗性基因)的农杆菌LBA4404菌株在含有25mg/mL卡那霉素和25mg/mL利福平的LB液体培养基中培养至OD 600值为0.6，转入含有乙酰丁香酮的IM液体培养基重悬获得农杆菌菌液。
25℃的光照培养油菜黑胫病菌Leptosphaeria biglobosa 12d产生分生孢子，在产孢平板上加3mL无菌水，静置10min，无菌载玻片轻刮平板表面待分生孢子充分释放后用无菌纱布过滤获得悬浮液，无菌水稀释至浓度为1×10 6个/mL的分生孢子悬浮液。
将上述配置好的农杆菌菌液和分生孢子悬浮液按1:1的体积比例混合，充分混匀后取200μL均匀涂布在铺有玻璃纤维滤纸的CM固体平板上25℃共培养5d，将玻璃纤维滤纸转移至SM筛选培养基上，培养10d，筛选获得转化子。
计算获得转化子的平均个数为6个/10 6分生孢子。
对比例3
本对比例中使用的培养基：
PDA培养基：马铃薯200g，葡萄糖20g，琼脂粉15g，加水定容至1000mL，121℃高压蒸汽灭菌20min。
LB培养基：酵母粉5g、胰蛋白胨10g、氯化钠10g、琼脂粉15g，加水定容至1000mL，121℃高压蒸汽灭菌20min。
CM共培养培养基：K 2HPO 43.44g、KH 2PO 4l.45g、NaCl 0.15g、MgSO 4·7H 2O 0.5g、(NH 4)2SO 4 0.5g CaCl 2·H 2O 0.067g、FeSO 4·7H 2O  0.0025g、Glucose 1.8g，MES 7.8g，甘油5mL，琼脂粉15g，蒸馏水定容至1000mL，pH值5.8。
IM培养基:以CM培养基为基础，在CM培养基中加入终浓度为300μg/mL的乙酰丁香酮。
SM筛选培养基：以PDA培养基为基础，在PDA培养基中加入终浓度为100mg/mL的头孢噻肟钠和终浓度为50mg/mL的潮霉素B。
方法步骤：
将含质粒pCHs-GFP(质粒上含有潮霉素抗性基因，GFP绿色荧光蛋白基因，卡那抗性基因)的农杆菌LBA4404菌株在含有25mg/mL卡那霉素和25mg/mL利福平的LB液体培养基中培养至OD 600值为0.6，转入含有乙酰丁香酮的IM液体培养基重悬获得农杆菌菌液。
25℃的光照培养油菜黑胫病菌Leptosphaeria biglobosa 12d产生分生孢子，在产孢平板上加3mL无菌水，静置10min，无菌载玻片轻刮平板表面待分生孢子充分释放后用无菌纱布过滤获得悬浮液，无菌水稀释至浓度为1×10 6个/mL的分生孢子悬浮液。
将上述配置好的农杆菌菌液和分生孢子悬浮液按1:1的体积比例混合，充分混匀后取200μL均匀涂布在铺有玻璃纤维滤纸的CM固体平板上25℃共培养4d，将玻璃纤维滤纸转移至SM筛选培养基上，培养10d，筛选获得转化子。
计算获得转化子的平均个数为17个/10 6分生孢子。
对比例4
本对比例中使用的培养基：
PDA培养基：马铃薯200g，葡萄糖20g，琼脂粉15g，加水定容至1000mL，121℃高压蒸汽灭菌20min。
LB培养基：酵母粉5g、胰蛋白胨10g、氯化钠10g、琼脂粉15g，加水定容至1000mL，121℃高压蒸汽灭菌20min。
CM共培养培养基：K 2HPO 43.44g、KH 2PO 4l.45g、NaCl 0.15g、MgSO 4·7H 2O 0.5g、(NH 4)2SO 4 0.5g CaCl 2·H 2O 0.067g、FeSO 4·7H 2O 0.0025g、Glucose 1.8g，MES 7.8g，甘油5mL，琼脂粉15g，蒸馏水定容至1000mL，pH值5.8。
IM培养基:以CM培养基为基础，在CM培养基中加入终浓度为400μg/mL的乙酰丁香酮。
SM筛选培养基：以PDA培养基为基础，在PDA培养基中加入终浓度为100mg/mL的头孢噻肟钠和终浓度为50mg/mL的潮霉素B。
方法步骤：
将含质粒pCHs-GFP(质粒上含有潮霉素抗性基因，GFP绿色荧光蛋白基因，卡那抗性基因)的农杆菌LBA4404菌株在含有25mg/mL卡那霉素和25mg/mL利福平的LB液体培养基中培养至OD 600值为0.6，转入含有乙酰丁香酮的IM液体培养基重悬获得农杆菌菌液。
25℃的光照培养油菜黑胫病菌Leptosphaeria biglobosa 12d产生分生孢子，在产孢平板上加3mL无菌水，静置10min，无菌载玻片轻刮平板表面待分生孢子充分释放后用无菌纱布过滤获得悬浮液，无菌水稀释至浓度为1×10 6个/mL的分生孢子悬浮液。
将上述配置好的农杆菌菌液和分生孢子悬浮液按1:1的体积比例混合，充分混匀后取200μL均匀涂布在铺有玻璃纤维滤纸的CM固体平板上25℃共培养4d，将玻璃纤维滤纸转移至SM筛选培养基上，培养10d，筛选获得转化子。
计算获得转化子的平均个数为7个/10 6分生孢子。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种农杆菌介导的油菜黑胫病菌的遗传转化方法，包括以下步骤：
    1)将含质粒pCHs-GFP的农杆菌接种于LB液体培养基中培养至菌液的OD 600为0.5～0.7后，转入含有150～250μg/mL乙酰丁香酮的CM液体培养基中重悬获得农杆菌菌液；
    2)收集油菜黑胫病菌Leptosphaeria biglobosa的分生孢子与无菌水混合制备浓度为(0.5～9)×10 6个/mL的分生孢子悬浮液；
    3)将所述农杆菌菌液与分生孢子悬浮液混合，共培养3～4d后，转移至筛选培养基，筛选培养7～15d获得转化子；
    所述质粒pCHs-GFP上包括潮霉素抗性基因、GFP绿色荧光蛋白基因和卡那抗性基因；
    步骤1)和步骤2)之间无时间顺序限定。
  2. 根据权利要求1所述的遗传转化方法，其特征在于，步骤1)中所述菌液的OD 600为0.6。
  3. 根据权利要求1或2所述的遗传转化方法，其特征在于，步骤1)中所述CM液体培养基中乙酰丁香酮的浓度为180～220μg/mL。
  4. 根据权利要求3所述的遗传转化方法，其特征在于，步骤1)所述的LB液体培养基中包括卡那霉素和利福平。
  5. 根据权利要求1所述的遗传转化方法，其特征在于，步骤3)中所述农杆菌菌液与分生孢子悬浮液混合的体积比为1:1。
  6. 根据权利要求1或5所述的遗传转化方法，其特征在于，步骤2)中所述分生孢子悬浮液的浓度为1×10 6个/mL。
  7. 根据权利要求1或5所述的遗传转化方法，其特征在于，步骤3)中所述共培养温度为25℃，所述共培养的时间为4d，所述共培养在黑暗条件下进行。
  8. 根据权利要求1所述的遗传转化方法，其特征在于，步骤3)中所 述筛选培养基为添加终浓度为80～120mg/mL的头孢噻肟钠和终浓度为50～60mg/mL的潮霉素B的PDA培养基。
  9. 根据权利要求1所述的遗传转化方法，其特征在于，步骤3)中所述共培养的培养基为固体CM培养基。
  10. 根据权利要求9所述的遗传转化方法，其特征在于，所述共培养的培养基的pH值为5.7～5.9。
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