有害疣孢霉Hypomyces perniciosus遗传转化体系的建立及其突变体库的构建  

Generation of Agrobacterium-mediated transformation system and construction of T-DNA insertional mutant library for Hypomyces perniciosus

作　　者：纪策 刘源[1] 李丹[1] Sossah Frederick Leo 杨阳[1] 付永平[1] 李玉[1] JI Ce;LIU Yuan;LI Dan;Sossah Frederick Leo;YANG Yang;FU Yong-Ping;LI Yu(Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi,College of Agronomy,Jilin Agricultural University,Changchun,Jilin 130118,China)

机构地区：[1]吉林农业大学食药用菌教育部工程研究中心,吉林长春130118

出　　处：《菌物学报》2018年第8期1027-1034,共8页Mycosystema

基　　金：公益性行业(农业)科研专项(201503137); 国家重点基础研究发展规划(973计划)(2014CB138305); 国家自然科学基金(31471926)

摘　　要：有害疣孢霉Hypomyces perniciosus是引起双孢蘑菇Agaricus bisporus湿泡病的病原真菌,目前其致病分子机理尚不清楚,而高效稳定的遗传转化体系和突变体库构建是挖掘和研究病原菌致病基因的基础和有效手段。因此,本实验以高致病力的有害疣孢霉菌株WH001为研究对象,采用冻融法将双元载体p BHt1转入农杆菌AGL-1中,建立并优化根癌农杆菌介导的遗传转化体系,并利用其构建T-DNA插入突变体库。结果表明有害疣孢霉菌株WH001的潮霉素(Hygromycin,Hyg)耐受浓度为250ng/L,当农杆菌侵染液浓度OD600=1,侵染时间为30min,乙酰丁香酮(Acetosyringone,AS)浓度为1.5mg/mL,共培养时间为3d时,转化体系效率最高。然后利用该优化体系构建有害疣孢霉的突变体库,通过PCR检测和形态学鉴定获得若干表型发生改变、稳定遗传的T-DNA插入突变体,与原菌种WH001相比,突变体在菌丝形态、生长速率、色素分泌和致病力等方面发生改变。本研究为进一步挖掘有害疣孢霉未知基因功能、解析生物学性状、探讨致病分子机制奠定基础。Wet bubble disease caused by Hypomyces perniciosus is a disease of mushroom Agaricus bisporus. The molecular pathogenesis mechanisms of the disease have not been investigated. Agrobacterium-mediated genetic transformation（ATMT） system is an efficient and stable method for fungal transformation and insertion-mutant library construction to determine the function of pathogenicity related genes. In this study, ATMT protocol for T-DNA insertional mutagenesis transformation of H. perniciosus was optimized. Agrobacterium tumefaciens AGL-1 strain containing the binary vector pBHt1 carrying hygromycin-resistance gene was used to generate H. perniciosus strain WH001 mutant library. Our results showed that the concentration of hygromycin sensitivity of H. perniciosus was 250 ng/L. The optimum T-DNA insertion mutation library systemfor Agrobacterium-mediated transformation of H. perniciosus was OD600=1 of the bacterial concentration, 30 min of infection duration, and 3 days of the co-cultivation period with the supplementation of 1.5 mg/mL of acetosyringone（AS） concentration. A large number and variety of mutants were acquired, and important phenotype included reduction in growth rate, loss of conidiation and loss of pathogenicity. This study shows that ATMT is a highly efficient method for studying the function of pathogenicity genes of H. perniciosus and understanding the molecular pathogenesis of wet bubble disease.

关 键 词：有害疣孢霉 农杆菌 转化 突变体库 表型变异 

分 类 号：S436.46[农业科学—农业昆虫与害虫防治]