枣黑斑病菌绿色荧光蛋白标记菌株的构建

Construction of a GFP-labeled Strain of Black Spot Pathogen of Jujube,Zizyphus Jujuba

以枣黑斑病菌和含有双元载体根癌农杆菌为材料,对分生孢子悬浮液的浓度、农杆菌液浓度、菌株共培养时间、共培养温度等条件进行优化,筛选出枣黑斑病菌遗传转化的最佳体系。以农杆菌介导枣黑斑病菌最佳的遗传转化体系为基础,进行GFP标记实验室保存枣黑斑病菌的菌落,继代培养后,通过观察菌落生长形态,生长速度以及致病力测定筛选获得遗传相对稳定、与野生枣黑斑病菌相比性状没有发生明显变化的荧光蛋白标记菌株。挑取单菌落于10 ml的LB液体培养基(含100μg·ml^(-1)卡那霉素)中,28℃震荡(180 r/min)培养48 h,吸取400μl培养液于20 ml含有200μmol·L-1的AS(乙酰丁香酮,acetosyringone)、100μg·ml^(-1)卡那霉素的AIM液体培养基中,28℃震荡(180 r/min)培养5~6 h共培养。将Alternaria alternata菌株所产的孢子洗下,制成浓度为1.0×106个/ml的悬浮液。分别取上述农杆菌液与孢子悬浮液各100μl均匀混合,涂布于含有200μmol·L-1AS和100μg·ml^(-1)卡那霉素的AIM平板上的硝酸纤维化膜(NC膜)表面,在25℃下黑暗共培养60 h,然后将NC膜转到含有潮霉素B的PDA平板上4~7天,获得约200个转化子,获得GFP标记的菌株。

The optimum conditions of conidial suspension concentration,Agrobacterium tumefaciens concentration,co-culture time and co-culture temperature were optimized,and the optimal system of genetic transformation of black spot fungus of jujube was screened used the material contained the jujube black spot pathogen and Agrobacterium tumefaciens.The Agrobacterium tumefaciens mediated the optimal genetic transformation system of black spot fungus of jujube,and GFP markers were carried out on the spores of black spot fungus preserved in laboratory.After subculture,genetic stability was obtained by colony morphology,growth rate and pathogenicity test,and the GFP marker strain was not significantly changed compared with wild type black spot fungus.LB liquid from a single colony in 10 ml was picked up,the culture medium（containing 100 μg·ml^（-1） kanamycin） was incubated at 28℃ for 48 h and then,400 μl culture medium was co-cultured for 5 to 6 hours in the AIM medium containing 20 ml which contained 200 μmol·L^（-1）（acetone acetosyringoneone 100 μg·ml^（-1） kanamycin） for 5 to 6 hours.The spores produced by Alternaria alternata were washed and the suspensions of 1.0×10~6/ml were prepared.Agrobacterium tumefaciens and spore suspensions were evenly mixed and coated on the surface of AIM plate containing 200 μmol·L^（-1） AS and 100 μg·mol-1 kanamycin,respectively.The membrane was co-cultured at 25 ℃ for 60 h.Then the NC membrane was transferred to the PDA plate containing hygromycin B for 4 to 7 days and about 200 transformants were obtained.：GFP labeled strains were obtained.