CN110195078B - Genetic transformation method of agrobacterium-mediated rape black shank - Google Patents
Genetic transformation method of agrobacterium-mediated rape black shank Download PDFInfo
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Abstract
The invention provides a genetic transformation method of agrobacterium-mediated rape black shank bacteria, belonging to the technical field of genetic transformation of plant pathogenic fungiThe method comprises the following steps of 1) inoculating agrobacterium containing plasmid pCHs-GFP into an LB liquid culture medium for culture, transferring the agrobacterium into a CM liquid culture medium containing 150-250 mu g/mL acetosyringone for resuspension to obtain agrobacterium tumefaciens bacterial liquid, 2) collecting conidia of the rape phytophthora parasitica and mixing the conidia with sterile water to prepare the bacillus subtilis with the concentration of (0.5-9) × 106Conidium suspension of seed/mL; 3) mixing the agrobacterium liquid and the conidium suspension, transferring to a screening culture medium after co-culturing for 3-4 d, and screening and culturing for 7-15 d to obtain a transformant; the method has high transformation efficiency, low copy and stable heredity.
Description
Technical Field
The invention belongs to the technical field of plant pathogenic fungi genetic transformation, and particularly relates to an agrobacterium-mediated genetic transformation method of rape phytophthora parasitica.
Background
Rape black shank (Phoma stem hanger) is one of the important factors which restrict the healthy development of rape industry in many countries at present. Studies have shown that in many black shank endemic rape producing regions of the world, both the pathogenic complex Leptosphaeramains and Leptosphaeria biglobosa co-exist, infecting host crops individually or together, L.maculans can cause rot at the base of the rape stem and cause severe yield loss. The diffusion trend and the evolution rule of the rape black shank in various countries in the world show that the trend that L.biglobosa is gradually replaced by L.maculans exists. In 1999, the pathogenic bacteria L.biglobosa is obtained by first separation in China, in recent years, the harm of L.biglobosa in China is in the trend of year-by-year expansion and spread, and along with the expansion and spread of L.biglobosa, L.maculans is likely to appear in China, namely, the black shank threatens the safe production of rape and cruciferous crops in China.
In order to prevent and deal with black shank and cause huge economic loss to rape production in China, the interaction relationship between the black shank (L. biglobosa) and rape must be understood. The fluorescent marker provides a more intuitive means for researching the interaction between the pathogenic microorganism and the host, and the method can be used for carrying out long-term, real-time and direct observation. And the establishment of a genetic transformation system of the rape phytophthora parasitica (L.biglobosa) and the obtainment of a positive transformant with a fluorescent marker are the premise and the basis. The currently widely adopted fungal transformation methods include plasmid cotransformation, electric stimulation transformation, gene gun, restriction enzyme mediated transformation (REMI) and agrobacterium mediated transformation, but the transformation methods such as plasmid cotransformation, electric stimulation transformation, gene gun method, restriction enzyme mediated transformation and the like have the problems of easy operation of protoplast preparation and regeneration processes, poor repeatability, easy generation of transformant instability, low transformation efficiency and the like. Although the electrotransformation method, the particle gun method and the like can also take spores and mycelia as receptors, so that the operation steps are simplified, the transformation efficiency is low, and the genetic stability of transformants is poor.
Disclosure of Invention
In view of the above, the present invention aims to provide an agrobacterium-mediated genetic transformation method of leptosphaeria maculans, which has high transformation efficiency, low copy number and stable heredity.
In order to achieve the above purpose, the invention provides the following technical scheme:
an agrobacterium-mediated genetic transformation method for rape phytophthora parasitica, which comprises the following steps:
1) inoculating agrobacterium containing plasmid pCHs-GFP into LB liquid culture medium to culture to OD of bacterial liquid600Transferring the bacillus subtilis to a CM liquid culture medium containing 150-250 mu g/mL acetosyringone after the bacillus subtilis content is 0.5-0.7, and re-suspending to obtain agrobacterium liquid;
2) collecting conidia of Leptosphaeria biglobosa of rape, mixing the conidia with sterile water to prepare × 10 (0.5-9) with the concentration6Conidium suspension of seed/mL;
3) mixing the agrobacterium liquid and the conidium suspension, transferring to a screening culture medium after co-culturing for 3-4 d, and screening and culturing for 7-15 d to obtain a transformant;
the plasmid pCHs-GFP comprises a hygromycin resistance gene, a GFP green fluorescent protein gene and a kanamycin resistance gene;
no time sequence is defined between the step 1) and the step 2).
Preferably, OD of the bacterial liquid in the step 1)600Is 0.6.
Preferably, the concentration of the acetosyringone in the IM liquid culture medium in the step 1) is 180-220 mu g/mL.
Preferably, the LB liquid medium described in step 1) includes kanamycin and rifampicin.
Preferably, the volume ratio of the mixture of the agrobacterium liquid and the conidium suspension in the step 3) is 1: 1.
Preferably, the concentration of the conidia suspension in the step 2) is 1 × 106one/mL.
Preferably, the co-cultivation temperature in step 3) is 25 ℃, the co-cultivation time is 4d, and the co-cultivation is performed under dark conditions.
Preferably, the screening culture medium in the step 3) is a PDA culture medium added with cefotaxime sodium with the final concentration of 80-120 mg/mL and hygromycin B with the final concentration of 50-60 mg/mL.
Preferably, the co-cultured medium in step 3) is a solid CM medium.
Preferably, the pH value of the co-culture medium is 5.7-5.9.
The invention has the beneficial effects that: the agrobacterium-mediated genetic transformation method for rape black shank bacteria provided by the invention takes the conidia of the black shank bacteria as materials, and the agrobacterium liquid and the conidia suspension are mixed and cultured together to realize a genetic transformation system for the rape black shank bacteria; the method has high transformation efficiency, low copy number, and stable heredity, and each 10 times6120-161 transformants can be obtained from each conidium, 5-generation subculture, hygromycin specificity PCR detection and fluorescence detection show that T-DNA is integrated into the black shank genome in a single copy mode, and the transformants can be stably inherited.
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FIG. 1 shows the results of stability testing of resistance of transformed ascomycin from left to right: F1-F5 generation;
FIG. 2 is a GFPPCR electrophoresis result chart of transformants, wherein LineM is makerDL2000, Line 1-16 are transformants T1-T16, and Line17 is wild strain NM-1;
FIG. 3 shows the results of fluorescence expression of transformants, wherein the left panels show the hyphae and the right panels show the spores of the transformants.
Detailed Description
The invention provides an agrobacterium-mediated genetic transformation method for rape phytophthora parasitica, which comprises the following steps: 1) inoculating agrobacterium containing plasmid pCHs-GFP into LB liquid culture medium to culture to OD of bacterial liquid600After 0.5-0.7, transferring the mixture into a CM liquid culture medium containing 150-250 mu g/mL acetosyringone for resuspension to obtain an agrobacterium tumefaciens bacterial solution, 2) collecting conidia of Leptosphaeria biglobosa of the Leptosphaeria brassicae, and mixing the conidia with sterile water to prepare × 10 with the concentration of (0.5-9)6Conidium suspension of seed/mL; 3) mixing the agrobacterium liquid and the conidium suspension, transferring to a screening culture medium after co-culturing for 3-4 d, and screening and culturing for 7-15 d to obtain a transformant; the plasmid pCHs-GFP comprises hygromycinA resistance gene, a GFP green fluorescent protein gene and a kanamycin resistance gene; no time sequence is defined between the step 1) and the step 2).
In the invention, agrobacterium containing plasmid pCHs-GFP is inoculated in LB liquid culture medium and cultured to OD of bacterial liquid600And collecting the thalli after the number of the thalli is 0.5-0.7. In the invention, the plasmid pCHs-GFP comprises a hygromycin resistance gene, a GFP green fluorescent protein gene and a kanamycin resistance gene; the agrobacterium preferably LBA4404 agrobacterium containing the plasmid pCHs-GFP; the source of the LBA4404 agrobacterium is not particularly limited, and the LBA4404 agrobacterium is obtained by adopting a product sold in the field or self-preparation; the LBA4404 agrobacterium is more suitable for genetic transformation of the Heiphilus fuliginosus, and the transformation efficiency is high and stable. In the present invention, the plasmid pCHs-GFP is preferably transferred into Agrobacterium LBA4404 to obtain Agrobacterium containing the plasmid pCHs-GFP; the method for transferring the gene is not particularly limited, and the conventional method in the field can be adopted. In the present invention, the LB liquid medium preferably includes kanamycin and rifampicin; the concentration of kanamycin in the LB liquid culture medium is preferably 20-30 mg/mL, and more preferably 25 mg/mL; the concentration of rifampicin in the LB liquid culture medium is preferably 20-30 mg/mL, and more preferably 25 mg/mL. The inoculation amount of the agrobacterium is not particularly limited, and the conventional inoculation amount in the field can be adopted. In the present invention, OD of the bacterial liquid600Preferably 0.6; OD of bacterial suspension defined in the present invention600The range can ensure the successful transformation without influencing the normal growth of transformants; OD600Too low a transformation cannot be achieved, and too high a transformation affects the normal growth of transformants. The method for collecting the cells in the present invention is not particularly limited, and a conventional method for collecting cells in the art may be used. After the collection of the cells, the present invention preferably further comprises a step of washing the cells; the cleaning of the thalli is preferably carried out by adopting a CM liquid culture medium containing 150-250 mu g/mL acetosyringone.
The collected thalli are transferred into a CM liquid culture medium containing 150-250 mu g/mL acetosyringone for resuspension to obtain agrobacterium tumefaciens bacterial liquid. In the invention, the concentration of acetosyringone in the CM liquid mediumPreferably 180-220 mug/mL, more preferably 200 mug/mL; the acetosyringone in the CM liquid medium can ensure that the obtained transformants have a large number and can normally grow in the range defined by the invention. If the concentration of the acetosyringone is increased, the acetosyringone can produce toxic action on the strains, influence the normal growth of the strains and reduce the number of transformants. In the invention, OD of the agrobacterium liquid obtained after the heavy suspension600Preferably 0.6. In the present invention, the CM liquid medium, in 1L, preferably includes the following components: k2HPO43.44g、KH2PO4l.45g、NaCl0.15g、MgSO4·7H2O0.5g、(NH4)2SO40.5gCaCl2·H2O0.067g、FeSO4·7H2O0.0025g, Glucose1.8g, MES7.8g, 5mL of glycerin, and the balance of distilled water.
In the invention, conidia of the rape phytophthora parasitica are collected and mixed with sterile water to prepare the mixture with the concentration of (0.5-9) × 106The concentration of the conidium suspension is preferably (0.8-5) × 106one/mL, more preferably 1 × 106one/mL. In the invention, the conidia are preferably conidia generated by culturing Leptosphaeria biglobosa 10-14 d of rape under illumination at 25 ℃, and are more preferably conidia generated by culturing 12 d. In the invention, the rape phytophthora parasitica is preferably cultured on a sporulation plate; the collecting of conidia preferably comprises the steps of: and adding sterile water into the spore production plate, standing for 8-12 min, and scraping conidia off the spore production plate. In the invention, the addition amount of the sterile water is preferably 2-4 mL/spore production plate; the conidium scraping is preferably performed by using a sterile glass slide; after conidia are scraped, preferably filtering a scraping liquid, and collecting filtrate to obtain conidia; the filtration is preferably gauze filtration, and the gauze for filtration is preferably sterile gauze.
After the agrobacterium liquid and the conidium suspension are obtained, the agrobacterium liquid and the conidium suspension are mixed, are transferred to a screening culture medium after being cultured for 3-4 days, and are screened and cultured for 7-15 days to obtain a transformant. In the invention, the volume ratio of the mixture of the agrobacterium liquid and the conidium suspension is preferably 1: 1; the proportion can ensure that the obtained transformants have moderate quantity and are easy to separate and purify; if the number of transformants obtained is too large, the transformant mycelia may stick together, and isolation and purification may be difficult. The mixed solution obtained after the mixing is fully and uniformly mixed, the fully and uniformly mixing method is not particularly limited, and the shaking uniformly mixing or stirring uniformly mixing which is conventional in the field is adopted. In the present invention, the co-cultivation temperature is preferably 25 ℃ and the co-cultivation time is preferably 4d, and the co-cultivation is preferably performed in the dark. The co-culture temperature limited by the invention can not hinder the gene expression of VirB, VirD and VirE, the infection capacity of the agrobacterium is stable, the agrobacterium grows well, and the number of obtained transformants is large; the co-culture time defined by the invention ensures that enough transformants can be formed, and simultaneously avoids the phenomenon that too many false positive transformed colonies are generated due to too long co-culture time.
In the present invention, the co-culture medium is preferably a CM solid medium, and the CM solid medium preferably includes the following components in 1L: k2HPO43.44g、KH2PO4l.45g、NaCl0.15g、MgSO4·7H2O0.5g、(NH4)2SO40.5gCaCl2·H2O0.067g、FeSO4·7H2O0.0025g, Glucose1.8g, MES7.8g, 5ml of glycerol, 15g of agar powder and the balance of distilled water; the pH value of the CM solid medium is preferably 5.7-5.9, and more preferably 5.8. In the practice of the present invention, the co-culture is preferably performed by applying the mixture to a CM solid plate coated with glass fiber filter paper, and in the present invention, the amount of the mixture applied is preferably 150 to 250. mu.L/CM solid plate, and more preferably 200. mu.L/CM solid plate. In the invention, the amount of the applied bacterium liquid affects the amount of the obtained transformant, if the amount of the applied bacterium liquid is less, the amount of the obtained transformant is relatively less, and if the amount of the applied bacterium liquid is more, the amount of the transformant obtained at the later stage is relatively lessHowever, the screening and culturing of transformants need 7-15 days, which causes the hypha of the transformants to adhere and is not beneficial to the later separation and purification.
After the co-culture is finished, transferring the co-cultured product to a screening culture medium, and carrying out screening culture for 7-15 days to obtain a transformant. In the practice of the invention, it is preferred that the glass fiber filter paper with the co-cultured product be transferred directly to the screening medium. In the invention, the screening culture medium is preferably a PDA culture medium added with cefotaxime sodium with the final concentration of 80-120 mg/mL and hygromycin B with the final concentration of 50-60 mg/mL, and is more preferably a PDA culture medium added with cefotaxime sodium with the final concentration of 100mg/mL and hygromycin B with the final concentration of 50 mg/mL.
After obtaining the transformant, the present invention preferably further comprises a step of identifying the transformant. In the present invention, the identification of the transformant is preferably achieved by the following two methods: firstly, extracting DNA of a transformant, carrying out GFP specific PCR amplification, and detecting whether an amplification product has a GFP specific band or not by agarose gel electrophoresis; if present, a positive transformant is correct, and if not, a positive transformant is not correct. Secondly, selecting transformants to be observed under a fluorescence microscope, and determining the transformants to be correct positive if the transformants show green fluorescence, or determining the transformants to be not correct positive if the transformants do not show green fluorescence.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The medium used in this example:
PDA culture medium: 200g of potato, 20g of glucose and 15g of agar powder, adding water to a constant volume of 1000mL, and sterilizing with high-pressure steam at 121 ℃ for 20 min.
LB culture medium: 5g of yeast powder, 10g of tryptone, 10g of sodium chloride and 15g of agar powder, adding water to a constant volume of 1000mL, and sterilizing for 20min by high-pressure steam at 121 ℃.
CM co-culture medium: k2HPO43.44g、KH2PO4l.45g、NaCl0.15g、MgSO4·7H2O0.5g、(NH4)2SO40.5gCaCl2·H2O0.067g、FeSO4·7H2O0.0025g, Glucose1.8g, MES7.8g, 5mL of glycerol, 15g of agar powder and distilled water to reach the constant volume of 1000mL and the pH value of 5.8.
IM culture medium, taking CM culture medium as a base, adding acetosyringone with the final concentration of 200 mug/mL into the CM culture medium.
SM screening culture medium: a final concentration of 100mg/mL cefotaxime sodium and a final concentration of 50mg/mL hygromycin B were added to PDA medium based on PDA medium.
The method comprises the following steps:
culturing Agrobacterium LBA4404 strain containing plasmid pCHs-GFP (hygromycin resistance gene, GFP green fluorescent protein gene and kana resistance gene on the plasmid) in LB liquid culture medium containing 25mg/mL kanamycin and 25mg/mL rifampicin at 28 deg.C and 200rpm for 12 h; when the culture is carried out until the bacterial liquid OD is reached600The value is 0.6, transferring the strain into an IM liquid culture medium containing acetosyringone, and re-suspending to obtain an agrobacterium liquid.
Culturing Leptosphaeria biglobosa12d at 25 deg.C under illumination to generate conidia, adding 3mL sterile water on the conidia-producing plate, standing for 10min, scraping the surface of the plate with sterile glass slide to obtain suspension after conidia are fully released, and filtering with sterile gauze to obtain suspension, wherein the concentration of the suspension is 1 × 106Conidia suspension per mL.
Mixing the prepared agrobacterium liquid and conidium suspension according to the volume ratio of 1:1, uniformly mixing, uniformly coating 200 mu L of the mixture on a CM solid plate paved with glass fiber filter paper for co-culture at 25 ℃ for 4d, transferring the glass fiber filter paper to an SM screening medium, culturing for 10d, and screening to obtain a transformant.
The statistical mode of the transformant is to separate out the colony alone, can be the transformant in the normal growth of hygromycin resistance culture medium, a single colony is a transformant; 3 parallel replicates were set up and an average of 161 transformants was obtained for each replicate.
And transferring the randomly selected single-spore separated transformant to a PDA (personal digital assistant) plate, culturing for 48h, transferring to a new PDA plate, continuously transferring for five generations, and observing the growth condition of the transformant to determine the genetic stability of the transformant. The results show that all transformants transferred to PDA plates containing 50mg/mL hygromycin B can grow after 5 generations of culture on PDA medium, while wild type phytophthora parasitica can not grow (FIG. 1), which indicates that the transformants obtained by the transformation method have higher genetic stability.
Carrying out PCR amplification detection by taking wild type phytophthora parasitica NM-1 and randomly selected genome DNA of 16 transformants as templates,
gfp gene amplification program:
gfp gene amplification reaction system:
the detection result shows that 16 transformant samples detected can amplify GFP gene fragments with the same size as the plasmid, while the wild strain nm-1 cannot amplify a band with the same size as the target fragment (figure 2), which shows that the T-DNA of the pCHs-GFP vector is successfully introduced and integrated into the genome DNA of the phytophthora parasitica.
In order to further confirm that the pCHs-GFP gene has been successfully introduced into the wild type rape phytophthora parasitica NM-1 and realizes the expression, the invention carries out fluorescence detection on a transformant with hygromycin resistance. And (3) selecting fresh hyphae and conidia of the rape black shank genetic transformant by using a sterile inoculating needle, placing the fresh hyphae and the conidia on a glass slide dropped with sterile water, slightly covering a cover glass to avoid generating bubbles, placing the glass slide under an inverted fluorescence microscope (Nikon eclipseti), and observing fluorescent proteins in the hyphae and the conidia of the black shank genetic transformant under the excitation wavelength of 552 nm. The result is shown in fig. 3, hypha and spore of the transformant emit obvious green fluorescence, and the result proves that the phytophthora parasitica transformant obtained by PCR screening and identification is a correct positive transgenic strain.
Comparative example 1
The culture medium used in this comparative example:
PDA culture medium: 200g of potato, 20g of glucose and 15g of agar powder, adding water to a constant volume of 1000mL, and sterilizing with high-pressure steam at 121 ℃ for 20 min.
LB culture medium: 5g of yeast powder, 10g of tryptone, 10g of sodium chloride and 15g of agar powder, adding water to a constant volume of 1000mL, and sterilizing for 20min by high-pressure steam at 121 ℃.
CM co-culture medium: k2HPO43.44g、KH2PO4l.45g、NaCl0.15g、MgSO4·7H2O0.5g、(NH4)2SO40.5gCaCl2·H2O0.067g、FeSO4·7H2O0.0025g, Glucose1.8g, MES7.8g, 5mL of glycerol, 15g of agar powder and distilled water to reach the constant volume of 1000mL and the pH value of 5.8.
IM culture medium, taking CM culture medium as a base, adding acetosyringone with the final concentration of 200 mug/mL into the CM culture medium.
SM screening culture medium: a final concentration of 100mg/mL cefotaxime sodium and a final concentration of 50mg/mL hygromycin B were added to PDA medium based on PDA medium.
The method comprises the following steps:
agrobacterium LBA4404 strain containing plasmid pCHs-GFP (hygromycin resistance gene, GFP green fluorescent protein gene, kanamycin resistance gene on the plasmid) was cultured to OD in LB liquid medium containing 25mg/mL kanamycin and 25mg/mL rifampicin600The value is 0.8, transferring the strain into an IM liquid culture medium containing acetosyringone, and re-suspending to obtain an agrobacterium liquid.
Culturing Leptosphaeria biglobosa12d to produce conidia at 25 deg.C, adding 3mL sterile water on the conidia-producing plate, standing for 10min, scraping the surface of the plate with sterile glass slide to obtain suspension, filtering with sterile gauze to obtain suspension, and diluting with sterile water to concentration of 1 × 106Conidia suspension per mL.
Mixing the prepared agrobacterium liquid and conidium suspension according to the volume ratio of 1:1, uniformly mixing, uniformly coating 200 mu L of the mixture on a CM solid plate paved with glass fiber filter paper for co-culture at 25 ℃ for 4d, transferring the glass fiber filter paper to an SM screening medium, culturing for 10d, and screening to obtain a transformant.
The average number of transformants obtained was calculated to be 36/106Conidia.
Comparative example 2
The culture medium used in this comparative example:
PDA culture medium: 200g of potato, 20g of glucose and 15g of agar powder, adding water to a constant volume of 1000mL, and sterilizing with high-pressure steam at 121 ℃ for 20 min.
LB culture medium: 5g of yeast powder, 10g of tryptone, 10g of sodium chloride and 15g of agar powder, adding water to a constant volume of 1000mL, and sterilizing for 20min by high-pressure steam at 121 ℃.
CM co-culture medium: k2HPO43.44g、KH2PO4l.45g、NaCl0.15g、MgSO4·7H2O0.5g、(NH4)2SO40.5gCaCl2·H2O0.067g、FeSO4·7H2O0.0025g, Glucose1.8g, MES7.8g, 5mL of glycerol, 15g of agar powder and distilled water to reach the constant volume of 1000mL and the pH value of 5.8.
IM culture medium, taking CM culture medium as a base, adding acetosyringone with the final concentration of 200 mug/mL into the CM culture medium.
SM screening culture medium: a final concentration of 100mg/mL cefotaxime sodium and a final concentration of 50mg/mL hygromycin B were added to PDA medium based on PDA medium.
The method comprises the following steps:
agrobacterium LBA4404 strain containing plasmid pCHs-GFP (hygromycin resistance gene, GFP green fluorescent protein gene, kanamycin resistance gene on the plasmid) was cultured to OD in LB liquid medium containing 25mg/mL kanamycin and 25mg/mL rifampicin600The value is 0.6, transferring the strain into an IM liquid culture medium containing acetosyringone, and re-suspending to obtain an agrobacterium liquid.
Culturing Leptosphaeria biglobosa12d to produce conidia at 25 deg.C, adding 3mL sterile water on the conidia-producing plate, standing for 10min, and scraping the surface of the plate with sterile glass slide until the conidia are fully releasedFiltering with sterile gauze to obtain suspension, and diluting with sterile water to obtain a solution with a concentration of 1 × 106Conidia suspension per mL.
Mixing the prepared agrobacterium liquid and conidium suspension according to the volume ratio of 1:1, uniformly mixing, uniformly coating 200 mu L of the mixture on a CM solid plate paved with glass fiber filter paper for co-culture at 25 ℃ for 5d, transferring the glass fiber filter paper to an SM screening medium, culturing for 10d, and screening to obtain a transformant.
The average number of transformants obtained was calculated to be 6/106Conidia.
Comparative example 3
The culture medium used in this comparative example:
PDA culture medium: 200g of potato, 20g of glucose and 15g of agar powder, adding water to a constant volume of 1000mL, and sterilizing with high-pressure steam at 121 ℃ for 20 min.
LB culture medium: 5g of yeast powder, 10g of tryptone, 10g of sodium chloride and 15g of agar powder, adding water to a constant volume of 1000mL, and sterilizing for 20min by high-pressure steam at 121 ℃.
CM co-culture medium: k2HPO43.44g、KH2PO4l.45g、NaCl 0.15g、MgSO4·7H2O0.5g、(NH4)2SO40.5gCaCl2·H2O0.067g、FeSO4·7H20.0025g of O, 1.8g of Glucose, 7.8g of MES, 5mL of glycerol, 15g of agar powder and distilled water to reach the constant volume of 1000mL and the pH value of 5.8.
IM culture medium, taking CM culture medium as a base, adding acetosyringone with the final concentration of 300 mug/mL into the CM culture medium.
SM screening culture medium: a final concentration of 100mg/mL cefotaxime sodium and a final concentration of 50mg/mL hygromycin B were added to PDA medium based on PDA medium.
The method comprises the following steps:
agrobacterium LBA4404 strain containing plasmid pCHs-GFP (hygromycin resistance gene, GFP green fluorescent protein gene, kanamycin resistance gene on the plasmid) was cultured to OD in LB liquid medium containing 25mg/mL kanamycin and 25mg/mL rifampicin600A value of 0.6, transferred to a composition containing acetobutylAnd (4) carrying out heavy suspension on the IM liquid culture medium of the ketonic to obtain an agrobacterium liquid.
Culturing Leptosphaeria biglobosa12d to produce conidia at 25 deg.C, adding 3mL sterile water on the conidia-producing plate, standing for 10min, scraping the surface of the plate with sterile glass slide to obtain suspension, filtering with sterile gauze to obtain suspension, and diluting with sterile water to concentration of 1 × 106Conidia suspension per mL.
Mixing the prepared agrobacterium liquid and conidium suspension according to the volume ratio of 1:1, uniformly mixing, uniformly coating 200 mu L of the mixture on a CM solid plate paved with glass fiber filter paper for co-culture at 25 ℃ for 4d, transferring the glass fiber filter paper to an SM screening medium, culturing for 10d, and screening to obtain a transformant.
The average number of transformants obtained was calculated to be 17/106Conidia.
Comparative example 4
The culture medium used in this comparative example:
PDA culture medium: 200g of potato, 20g of glucose and 15g of agar powder, adding water to a constant volume of 1000mL, and sterilizing with high-pressure steam at 121 ℃ for 20 min.
LB culture medium: 5g of yeast powder, 10g of tryptone, 10g of sodium chloride and 15g of agar powder, adding water to a constant volume of 1000mL, and sterilizing for 20min by high-pressure steam at 121 ℃.
CM co-culture medium: k2HPO43.44g、KH2PO4l.45g、NaCl 0.15g、MgSO4·7H2O 0.5g、(NH4)2SO40.5gCaCl2·H2O 0.067g、FeSO4·7H20.0025g of O, 1.8g of Glucose, 7.8g of MES, 5mL of glycerol, 15g of agar powder and distilled water to reach the constant volume of 1000mL and the pH value of 5.8.
IM culture medium, taking CM culture medium as a base, adding acetosyringone with a final concentration of 400 μ g/mL into the CM culture medium.
SM screening culture medium: a final concentration of 100mg/mL cefotaxime sodium and a final concentration of 50mg/mL hygromycin B were added to PDA medium based on PDA medium.
The method comprises the following steps:
agrobacterium LBA4404 strain containing plasmid pCHs-GFP (hygromycin resistance gene, GFP green fluorescent protein gene, kanamycin resistance gene on the plasmid) was cultured to OD in LB liquid medium containing 25mg/mL kanamycin and 25mg/mL rifampicin600The value is 0.6, transferring the strain into an IM liquid culture medium containing acetosyringone, and re-suspending to obtain an agrobacterium liquid.
Culturing Leptosphaeria biglobosa12d to produce conidia at 25 deg.C, adding 3mL sterile water on the conidia-producing plate, standing for 10min, scraping the surface of the plate with sterile glass slide to obtain suspension, filtering with sterile gauze to obtain suspension, and diluting with sterile water to concentration of 1 × 106Conidia suspension per mL.
Mixing the prepared agrobacterium liquid and conidium suspension according to the volume ratio of 1:1, uniformly mixing, uniformly coating 200 mu L of the mixture on a CM solid plate paved with glass fiber filter paper for co-culture at 25 ℃ for 4d, transferring the glass fiber filter paper to an SM screening medium, culturing for 10d, and screening to obtain a transformant.
The average number of transformants obtained was calculated to be 7/106Conidia.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (6)
1. An agrobacterium-mediated genetic transformation method for rape phytophthora parasitica, which comprises the following steps:
1) inoculating agrobacterium containing plasmid pCHs-GFP into LB liquid culture medium to culture to OD of bacterial liquid600After the concentration is 0.6, transferring the bacillus subtilis into a CM liquid culture medium containing 180-220 mu g/mL acetosyringone for resuspension to obtain agrobacterium liquid;
2) collecting conidia of Leptosphaeria biglobosa of Brassica napus, mixing with sterile water to obtain a mixture with a concentration of 1 × 106Conidium suspension of seed/mL;
3) mixing the agrobacterium liquid and the conidium suspension, transferring to a screening culture medium after co-culture, and screening and culturing for 7-15 d to obtain a transformant;
the plasmid pCHs-GFP comprises a hygromycin resistance gene, a GFP green fluorescent protein gene and a kanamycin resistance gene;
the co-culture temperature in the step 3) is 25 ℃, the co-culture time is 4d, and the co-culture is carried out under the dark condition;
no time sequence is defined between the step 1) and the step 2).
2. The genetic transformation method according to claim 1, wherein the LB liquid medium of step 1) comprises kanamycin and rifampicin.
3. The genetic transformation method according to claim 1, wherein the volume ratio of the mixture of the agrobacterium liquid and the conidium suspension in step 3) is 1: 1.
4. The genetic transformation method according to claim 1, wherein the selection medium in step 3) is 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.
5. The genetic transformation method according to claim 1, wherein the co-culture medium in step 3) is a solid CM medium.
6. The genetic transformation method according to claim 5, wherein the pH of the co-cultured medium is 5.7 to 5.9.
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