枣黑斑病菌绿色荧光蛋白标记菌株的构建
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摘要
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.
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.
引文
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[7]张小飞,李晓,崔丽娜,等.轮枝镰孢菌的绿色荧光蛋白基因标记[J].西南农业学报,2014,27(06):2374-2376.
[8]袁鹰,李启云,郝文媛,等.农杆菌介导玉米遗传转化影响因子的研究[J].分子植物育种,2006(02):228-232.
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[10]贾娜娜.梨腐烂病菌GFP标记及其在梨组织中侵染的显微观察[D].华中农业大学,2015.
[2]董宁,冯宏祖,王兰,等.南疆骏枣黑斑病症状表现及病原菌鉴定[J].植物保护学报,2016,43(06):922-927.
[3]李夏鸣,郭黄萍,胡增丽.枣黑斑病研究[J].山西农业科学,2009,37(11):37-40.
[4]张俊华,刘烨,韩雨桐,等.农杆菌介导稻瘟病菌绿色荧光蛋白(GFP)遗传转化研究[J].东北农业大学学报,2014,45(11):1-7.
[5]顾雪迎,施文骁,王洪凯,等.链格孢菌苹果致病型的ATMT转化体系的建立[J].中国农业科学,2016,49(10):1885-1891.
[6]陈天子,袁洪波,杨郁文,等.农杆菌介导转化大丽轮枝菌的体系优化[J].棉花学报,2011,23(06):507-514.
[7]张小飞,李晓,崔丽娜,等.轮枝镰孢菌的绿色荧光蛋白基因标记[J].西南农业学报,2014,27(06):2374-2376.
[8]袁鹰,李启云,郝文媛,等.农杆菌介导玉米遗传转化影响因子的研究[J].分子植物育种,2006(02):228-232.
[9]徐后娟,徐荣燕,李多川.链格孢ATMT转化体系的构建及弱毒突变株验证[J].基因组学与应用生物学,2009,28(06):1056-1062.
[10]贾娜娜.梨腐烂病菌GFP标记及其在梨组织中侵染的显微观察[D].华中农业大学,2015.