稻曲病菌T-DNA插入突变体5062的插入位点分析  

Molecular Characterization of T-DNA Integration of the Ustilaginoidea virens Mutant 5062

作　　者：俞咪娜[1] 胡建坤[1] 黄磊[1] 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

出　　处：《中国农业科学》2013年第9期1790-1798,共9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】研究稻曲病菌致病力增强的ATMT突变菌株5062,为稻曲病菌致病机制解析、致病相关基因克隆提供方法基础。【方法】测定和观察5062的菌落直径、菌落形态、产孢能力等生物学特性,进一步利用TAIL-PCR和RACE技术克隆5062基因组的T-DNA侧翼基因,并比对分析基因的信息,最后利用RT-PCR检测突变基因的表达量。【结果】与野生型菌株相比,突变菌株5062田间接种表现为致病力增强,在MM和水琼脂培养基上生长速率减慢,产生大量分生孢子,而在PSA和TB3培养基上,其菌落形态、色素等方面与野生型无明显差异。TAIL-PCR扩增得到的紧邻T-DNA两侧的侧翼序列在野生型中不相邻。TAIL-PCR结合RACE技术克隆了5062基因组的T-DNA侧翼基因,RT-PCR表明T-DNA插入位点右侧1个基因在突变体中上调表达。【结论】突变菌株5062的致病力增强,在营养贫乏条件下产孢能力增强,其表型的变化可能是染色体重排和T-DNA插入共同影响的结果。[Objective] The objective of the experiment is to establish a method for cloning genes of the Ustilaginoidea virens mutant, and to shed light on the pathogenic mechanism of the U. virens, depending on a new mutant 5062, which has increased virulence. [Method] With the wild type strain 70-22 as a control, phenotypic analysis, such as growth rate, colonial morphologies and sporulation, were analyzed. TAIL-PCR combined with RACE were used to identify the T-DNA integration site and the genes of flanking right site of the T-DNA. The expression levels of the genes were analyzed by RT-PCR as well. [Result] Colonial morphologies and pigment on PSA and TB3 medium were no different between 70-22 and 5062. But on MM and water-agar medium, 5062 grew slower and produced more conidiophores. The flanking sequences of T-DNA were non-adjacent in the wild type. The expression of the gene flanking right site of the T-DNA was upregulated. [Conclusion] In 5062, both the T-DNA insertion and inversion of chromosomal fragment caused a mutation of the increased virulence.

关 键 词：稻曲病菌 致病力 T-DNA 突变 染色体重排 

分 类 号：S435.111.4[农业科学—农业昆虫与害虫防治]