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

doi:10.3864/j.issn.0578-1752.2013.16.005

摘要/Abstract

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。

关键词: 稻曲病菌 , T-DNA插入突变 , 致病力 , 基因克隆

Abstract: 【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.

Key words: Ustilaginoidea virens , T-DNA insertion mutant , pathogenicity , gene clone

黄磊, 俞咪娜, 胡建坤, 于俊杰, 尹小乐, 聂亚锋, 陈志谊, 刘永锋. 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[J]. 中国农业科学, 2013, 46(16): 3344-3353.

HUANG Lei, YU Mi-na, HU Jian-kun, YU Jun-jie, YIN Xiao-le, NIE Ya-feng, CHEN Zhi-yi, LIU Yong-feng. Analysis of Biological Phenotypes and Molecular Cloning of T-DNA Integration Flanking Sequences of Ustilaginoidea virens Mutant Strain B-726[J]. Scientia Agricultura Sinica, 2013, 46(16): 3344-3353.

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