稻曲病菌突变体B-726生物学性状分析及其T-DNA插入位点侧翼序列的克隆  

Analysis of Biological Phenotypes and Molecular Cloning of T-DNA Integration Flanking Sequences of Ustilaginoidea virens Mutant Strain B-726

作　　者：黄磊[1,2] 俞咪娜[1] 胡建坤[1,2] 于俊杰[1] 尹小乐[1] 聂亚锋[1] 陈志谊[1] 刘永锋[1] 

机构地区：[1]江苏省农业科学院植物保护研究所,南京210014 [2]南京农业大学生命科学学院,南京210095

出　　处：《中国农业科学》2013年第16期3344-3353,共10页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31171802);江苏省农业自主创新基金(CX(12)1003-10)

摘　　要：【目的】分析稻曲病菌T-DNA插入突变体库中致病力减弱突变菌株B-726的生物学性状,分析其T-DNA插入位点的侧翼序列,克隆因外源基因插入被破坏正常表达的基因,为明确该基因在稻曲病菌致病过程中的作用奠定基础,并为稻曲病防治新途径的制定提供理论依据。【方法】通过测定突变菌株B-726生长速率、产孢能力及致病力检测,分析其生物学性状;Southern杂交分析B-726外源基因插入的拷贝数;利用HiTail-PCR获得T-DNA插入位点的侧翼序列;运用RACE-PCR技术,克隆与插入位点毗邻的基因全长;用半定量RT-PCR分析该基因表达情况。【结果】突变菌株B-726与野生菌株P1相比致病力极显著下降,产孢能力、生长速率显著下降。Southern杂交结果显示T-DNA在菌株B-726中以单拷贝形式插入。经过序列分析发现,T-DNA插入在1个命名为Uvt-726上游344 bp处,半定量PCR结果表明,Uvt-726在B-726表达量较P1显著下降。【结论】克隆了1个可能与稻曲病菌致病力相关的基因,推断该基因在稻曲病菌致病过程中起着重要的作用。【Objective】 The objective of this study is to analyze the phenotypes and T-DNA integration flanking sequence of a mutant strain B-726 and to clone a novel gene which normal expression is disrupted by T-DNA insertional event,understand the role of the gene in U.virens molecular pathogenic process,and to develop a new strategy for controlling rice false smut.【Method】 Biological phenotypes of B-726 were analyzed by testing growth rate,sporulation ability and pathogenicity.The copy number of T-DNA inserted in B-726 was identified by Southern blot.The flanking sequence of T-DNA was cloned using TAIL-PCR and the completed gene sequence in the flanking was cloned by RACE-PCR.The gene expression was detected by RT-PCR.【Result】 Phenotypic analysis of B-726 showed that the pathogenicity of B-726 was significant reduced,the sporulation ability and growth rate were declined.Genomic Southern bolt analysis confirmed that B-726 was a single T-DNA insertional event.The T-DNA insertion site was in the 344 bp upstream of a gene named Uvt-726.RT-PCR analysis confirmed that Uvt-726 transcriptions were expressed in B-726 significantly decreased compared to P1.【Conclusion】 A novel gene maybe associated with pathogenicity of U.virens was cloned,and these results imply that the Uvt-726 might play an important role during the pathogenic process of U.virens.

关 键 词：稻曲病菌 T-DNA插入突变 致病力 基因克隆 

分 类 号：S435.111.4[农业科学—农业昆虫与害虫防治]