棉花黄萎病菌T-DNA插入突变体库的构建及其表型分析  

Construction of a T-DNA Insertional Mutant Library for Verticillium dahliae Kleb.and Analysis of a Mutant Phenotype

作　　者：贾培松[1] 丁丽丽[1] 周邦军[2] 郭惠珊[2] 高峰[1] 

机构地区：[1]石河子大学绿洲农作物病害防控重点实验室,石河子大学,新疆石河子832003 [2]中国科学院微生物研究所,植物基因组学国家重点实验室,北京100101

出　　处：《棉花学报》2012年第1期62-70,共9页Cotton Science

基　　金：国家自然科学基金(31160350);农业部转基因专项(2009ZX08010-001B)

摘　　要：以棉花强致病力黄萎病菌株V592为材料,利用农杆菌介导的T-DNA插入技术构建了一个含15000个转化子的突变体库,对突变体的生物学特性、致病力及T-DNA插入拷贝数进行研究,结果显示:(1)突变体的菌落形态分为菌核型、中间型、菌丝型和菌膜型,分别占92.12%,4.54%,3.19%和0.15%;(2)菌核型、中间型和菌丝型菌株,在菌落生长速度和孢子大小方面无明显差异;而在产孢量方面,菌核型普遍强于中间型和菌丝型;(3)致病性测定显示,菌核型的致病力普遍强于中间型和菌丝型;(4)Southern杂交显示,T-DNA单拷贝数插入的突变体比率约为70.99%,而菌核型的单插入率为80.00%,明显高于中间型(69.23%)和菌丝型(64.71%)。以上结果表明,农杆菌介导转化技术可用于棉花黄萎病菌突变体库的快速构建,突变体的菌落表型与其产孢能力、致病力及T-DNA插入拷贝数等之间存在一定的相关性。We constructed a mutant library by Agrobacterium tumefaciens-mediated transformation（ATMT）,containing 15000 mutants from a virulent defoliating V.dahliae Kleb.V592 isolated from cotton originating from Xinjiang Province,China.The biological characteristics,pathogenicity,and T-DNA insertion of some mutants were analyzed.（1） Mutant phenotypes were divided into four groups according to colonial phenotype,number of sclerotium,such as sclerotium type,intermediate type,hyphal type and velum type,which accounted for 92.12%,4.54%,3.19%,and 0.15%.（2） The sporulation abilities of sclerotial type mutants were generally stronger than that of intermediate and hyphal type mutants,while growth speed and spore size showed no distinct differences from the wild-type V592.（3） The pathogenicity of sclerotial type mutants was generally stronger than of intermediate and hyphal type mutants.（4） Southern blot analysis indicated that T-DNAs were inserted randomly into the V.dahliae genome,and about 70.99% of the 131 transformants were single copy.This indicates that Agrobacterium tumefaciens-mediated transformation could be used to create mutagenesis in Verticillium dahliae,and the mutant phenotype influences pathogenicity,sporulation,and T-DNA insertion.

关 键 词：棉花黄萎病菌 农杆菌介导转化 突变体库 菌落表型 致病力 

分 类 号：S435.62[农业科学—农业昆虫与害虫防治]