粉红粘帚菌的ATMT体系构建及定殖甘草条件优化  

Construction of ATMT System for Clonostachys rosea and Its Conditional Optimization of Colonization into Glycyrrhiza uralensis Seeds

作　　者：史启今 王潇晗 任广喜[1] 姜丹[1] 刘春生[1] SHI Qijin;WANG Xiaohan;REN Guangxi;JIANG Dan;LIU Chunsheng(School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 102488,China)

机构地区：[1]北京中医药大学中药学院,北京102488

出　　处：《中国实验方剂学杂志》2023年第4期126-133,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82173921);北京中医药大学重点攻关项目(2020-JYB-ZDGG-037)。

摘　　要：目的:建立并优化根癌农杆菌介导的甘草内生真菌粉红粘帚菌的遗传转化体系,优选粉红粘帚菌定殖甘草的条件,为生物菌肥的开发及优质甘草的育种奠定基础。方法:分别从乙酰丁香酮浓度、共培养时间和受体真菌分生孢子浓度3个方面对根癌农杆菌介导的遗传转化(ATMT)条件进行优化并优选转化子。采用正交试验,以共培养温度、共培养时间和孢子浓度为考察因素,定殖率为指标,优化粉红粘帚菌定殖甘草的条件。结果:粉红粘帚菌最适转化条件为共培养时间60 h、孢子浓度1×10^(7) cfu·mL^(-1)、乙酰丁香酮浓度150μmol·L^(-1),此条件下的转化效率为每1×10^(7)个受体真菌孢子可获得135个转化子。通过标记基因绿色荧光蛋白(GFP)的克隆和β-葡萄糖苷酸酶(GUS)染色结果检测转化的准确性与稳定性。粉红粘帚菌通过浸种法定殖甘草的最佳条件为共培养温度25℃、共培养时间36 h、孢子浓度1×10^(6) cfu·mL^(-1),此条件下定殖率71.11%。结论:本研究成功建立了稳定高效的粉红粘帚菌ATMT体系及其定殖甘草的技术体系,可为甘草生物菌肥的开发奠定基础。Objective: Agrobacterium tumefaciens-mediated transformation(ATMT) of Clonostachys rosea, an endophytic fungus of Glycyrrhiza uralensis seeds, was established and optimized, and orthogonal test was designed to optimize the colonization conditions of C. rosea for G. uralensis seeds, so as to lay foundation for the development of biofertilizer and the breeding of high-quality G. uralensis. Method: The conditions of ATMT were optimized from three aspects, including the concentration of acetosyringone, co-culture time and the concentration of conidia of recipient fungi. Then, high-quality transformants were selected. Orthogonal test was used to optimize the colonization conditions by taking co-culture temperature, co-culture time and spore concentration as factors and colonization rate as index. Result: When spore concentration was 1×10^(7) cfu·mL^(-1), acetosyringone concentration was 150 μmol·L^(-1) and the co-culture time was 60 h, the transformation efficiency of C. rosea was the highest, which was 135 transformants per 1×10^(7) recipient fungal spores. The accuracy and stability of the transformations were tested by cloning the marker gene green fluorescent protein(GFP) and β-glucuronidase(GUS) staining. When co-culture temperature was 25 ℃, co-culture time was 36 h and the spore concentration was 1×10^(6) cfu·mL^(-1), the colonizing rate for C. rosea back dyeing into G. uralensis seeds by seed soaking method was the highest, which was 71.11%. Conclusion: This study successfully establishes stable and efficient technical systems not only of ATMT in C. rosea, but also of colonization of the transformants into G. uralensis seeds, which can lay a foundation for the development of biofertilizer of G. uralensis.

关 键 词：粉红粘帚菌 根癌农杆菌介导的遗传转化(ATMT) 绿色荧光蛋白 β-葡萄糖苷酸酶(GUS)染色 甘草 转化子 正交试验 

分 类 号：R22[医药卫生—中医基础理论] R931[医药卫生—中医学] R28[生物学—微生物学] Q939.1