大丽轮枝菌VdKeR基因的克隆及功能分析  

Cloning and Functional Analysis of VdKeR in Verticillium dahliae

作　　者：陈睿 李彪 王媛[1] 黄家风[1] CHEN Rui;LI Biao;WANG Yuan;HUANG Jia-Feng(College of Agriculture/Key Laboratory of Oasis Agricultural Pest Management and Plant Protection Resources Utilization,Xinjiang Uygur Autonomous Region,Shihezi University,Shihezi 832003,China)

机构地区：[1]石河子大学农学院/新疆绿洲农业病虫害治理与植保资源利用重点实验室,石河子832003

出　　处：《农业生物技术学报》2021年第11期2212-2222,共11页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(32060590,31760497)。

摘　　要：由大丽轮枝菌(Verticillium dahliae)引起的棉花黄萎病(cotton verticillium wilt)是威胁我国棉花(Gossypium hirsutum)生产的一种重要土传真菌维管束病害。本研究从棉花黄萎病菌强致病力菌株V592的基因组中克隆得到大丽轮枝菌的一个假定蛋白基因全长;根据同源重组原理和农杆菌(Agrobacterium tumefaciens)介导的遗传转化获得该基因的敲除突变体及其互补菌株,并对突变体表型、致病力进行了分析,利用逆转录荧光定量PCR(reverse transcription qPCR,RT-qPCR)对其他4个致病相关基因的表达情况进行了研究。结果显示,该基因全长1439 bp,编码蛋白含有Kelch重复结构域和1个半乳糖氧化酶结构域,Kelch重复形成6叶螺旋桨结构,因编码蛋白含有Kelch重复(Kelch repeat,KeR),将该基因命名为VdKeR(GenBank No.MZ855770)。与野生型菌株V592和互补菌株相比,VdKeR基因敲除导致棉花黄萎病菌在马铃薯葡萄糖琼脂(potato dextrose agar,PDA)培养基上的菌落生长速率降低、气生菌丝增多、产孢量减少、对棉花的致病力下降;RT-qPCR分析结果表明,VdKeR基因敲除后,c AMP依赖的蛋白激酶PKA的C亚基基因(C subunit gene of cAMP-dependent protein kinase A,VdPKAC1)、G蛋白β亚基基因(G proteinβsubunit gene,VGB)、类Nep1蛋白(necrosis-and ethylene-inducing-proteins(Nep1)-like protein,NLP)家族基因VdNLP1和VdNLP2的表达量降低。本研究表明,VdKeR基因与棉花黄萎病菌生长发育和致病性有关,并影响其他致病相关基因的表达。研究结果为深入研究大丽轮枝菌致病机制提供了基础资料。Cotton verticillium wilt caused by Verticillium dahliae is an important soil-borne fungal vascular disease threatening cotton production in China.In previous studies,the transcriptional expression of a gene encoding hypothetical protein in the virulent defoliating V.dahliae V592 strain isolated from cotton(Gossypium hirsutum)was dramatically down-regulated when V592 strain was cultured with cotton roots.To determine the function of the gene,the full length of the gene was cloned from the genome of V592 strain.The target gene knockout plasmid was constructed based on homologous recombination,and its complementation plasmid containing the full length of the target gene encoding region was also constructed,then to generate the target knockout mutant and its complementary strain by Agrobacterium-mediated transformation.The growth phenotype characteristics and pathogenicity to cotton of the gene knockout mutant were assayed.The transcriptional expression of 4 genes involved in virulence in the target gene knockout mutants were measured by reverse transcription qPCR(RT-qPCR).The results showed that the target gene full length was determined to be 1439 bp and the deduced protein contained 6 tandem Kelch motif and a galactose oxidase domains,and Kelch repeats formed a 6-bladed propeller construction.Thus,the gene was designated as VdKeR(GenBank No.MZ855770)due to the encoding protein containing Kelch repeats.On potato dextrose agar(PDA)media,VdKeR knockout mutant produced more aerial hyphae on the edge of the colony than the wild-type strain V592 and complementary strain,but the colony growth rate of VdKeR knockout mutant exhibited obviously lower than those of V592 strain and complementary strain,indicating that VdKeR gene knockout resulted in increased formation of aerial hyphae and slower growth rate in V.dahliae.VdKeR knockout mutant produced significantly less conidia than those of V592 strain and the complementary strain,suggesting that VdKeR gene knockout affected the conidiation in V.dahliae.In cotton seedling,Vd

关 键 词：大丽轮枝菌 Kelch重复(KeR) VdKeR基因 产孢 致病力 

分 类 号：S432.1[农业科学—植物病理学]