稻曲病菌T-DNA插入突变体5062的插入位点分析

【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。

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

【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.