大丽轮枝菌微菌核发育相关基因VdPKS的功能分析  

Functional analysis of VdPKS related to microsclerotia development of Verticilliumdahliae

作　　者：姚传飞 张昕[2] 邓晟[2] 林玲[2] 戴亦军[1] YAO Chuan-fei;ZHANG Xin;DENG Sheng;LIN Ling;DAI Yi-jun(College of Life Science,Nanjing Normal University,Nanjing 210046,China;Institute of Plant Protection,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China)

机构地区：[1]南京师范大学生命科学学院,江苏南京210046 [2]江苏省农业科学院植物保护研究所,江苏南京210014

出　　处：《江苏农业学报》2019年第6期1299-1307,共9页Jiangsu Journal of Agricultural Sciences

基　　金：国家公益性行业(农业)科研专项(201503109);国家棉花产业技术体系项目(CARS-18-16);国家重点研发计划项目(2017YFD0201900)

摘　　要：为阐明与大丽轮枝菌微菌核发育相关的聚酮合成酶基因VdPKS的功能,在前期获得VdPKS被单拷贝T-DNA插入的微菌核发育异常突变体2H3的基础上,通过基因敲除和回补技术,经分子鉴定和表型验证,获得了2株VdPKS基因敲除突变体和3株回补突变体。研究发现野生型菌株孢子在接种到PDA培养基上培养的第6 d开始积累黑色素,而此时VdPKS基因的表达量也最高。敲除突变体虽然没有形成黑色素,却也观察到微菌核的初始结构,表明VdPKS基因不是微菌核形成的必须基因,仅参与微菌核发育后期黑色素的形成。致病力测定结果表明VdPKS基因与菌株的致病力无关。杀菌剂嘧菌酯和咯菌腈对敲除突变体菌丝生长的抑制率相对于野生型菌株和回补突变体有显著提高,表明VdPKS基因与大丽轮枝菌的抗逆性相关。A single T-DNA insertion mutant 2H3 that showed abnormal microsclerotia development was selected from the T-DNA insertion mutant library of a Verticilliumdahliae sclerotium-type strain V08DF1.The inserted gene in the mutant 2H3 was VdPKS which encoded a conidial yellow pigment biosynthesis polyketide synthetase.Based on these results,the experiments of gene knockout and genetic complement were carried out in order to confirm the function of the gene VdPKS.Two deletion mutants△VdPKS1,△VdPKS2 and three complementary mutants 2H3-PKS6,2H3-PKS8,2H3-PKS9 were obtained through molecular and colonial morphological identification.After six days cultured on the PDA medium,the spores of the wildtype strain V08DF1 began to accumulate melanin of microsclerotia,and the VdPKS gene expression level detected by qRT-PCR was also the highest.The initial structure of the microsclerotia was also observed in the△VdPKS1 and△VdPKS2 although they did not form melanin.These results showed that the VdPKS gene was involved in the formation of microsclerotia melanin,but it was not an essential gene for microsclerotia formation.The results of the pathogenicity assay indicated that the VdPKS gene had nothing to do with the pathogenicity.The inhibition rate of azoxystrobin and fludioxonil on the mycelial growth of△VdPKS1 and△VdPKS2 was significantly higher than that of the wildtype strain and complementary mutants.The results showed that the VdPKS gene was related to the stress resistance of Verticilliumdahliae.

关 键 词：大丽轮枝菌 微菌核 聚酮合成酶 黑色素 致病力 

分 类 号：S432.1[农业科学—植物病理学]