稻曲病菌T-DNA插入突变体B2510的插入位点分析

以稻曲病菌T-DNA插入突变体库中致病力减弱突变菌株B2510为材料,通过分析T-DNA插入位点的侧翼序列和突变基因,分离出在稻曲病菌致病过程中起作用的基因。通过测定突变菌株B2510的生长速率、产孢能力及致病力发现,与野生型菌株P1相比,B2510田间接种表现为致病性减弱;在MM培养基上生长速率下降,而在PSA和TB3培养基中生长速率与野生型没有显著差异,但丧失产孢能力。Southern杂交显示T-DNA在突变菌株B2510中以双拷贝形式插入,利用TAIL-PCR技术扩增紧邻T-DNA两侧的侧翼序列,经过比对分析发现,T-DNA分别插在基因UV8b_1412的启动子区域和UV8b_1386的下游3′端,且稻曲菌基因组序列均未丢失,T-DNA上只有几个碱基发生变化。半定量RT-PCR分析基因的表达情况,显示两个基因在突变体B2510的表达量较P1均显著下降,推测T-DNA插入位点处的基因与稻曲病菌致病性相关,可能在某一阶段参与调控稻曲病菌在水稻上的致病过程。

Molecular Characterization of Ustilaginoidea virens T-DNA Insertion Mutant B2510

To isolate the gene concerned with molecular pathogenic process of Ustilaginoidea virens (U. virens) , T DNA integration flanking sequence and the mutant genes of a mutant strain B2510 were analyzed. Compared with the wild type U. virens strain P1, the pathogenicity of the mutant strain B2510 in the field was significantly decreased. The growth rate of B2510 on MM medium was slower than that of P1, but was not significantly different on PSA and TB3 medium which were nutritionally endowed. The mutant strain B2510 did not produce conidiophores in PS broth medium. Genomic Southern bolt analysis confirmed that there were double T DNA events inserted in genome of mutant strain B2510. The flanking U. virens sequences of T DNA obtained by TAIL PCR were adjacent in the wild type and with no sequences lost, and only a few bases of T DNA were changed. The T DNA insertion site was in the promoter region of UV8b_1412 and downstream 3′region of UV8b_1386, respectively. RT PCR analysis confirmed that the expression of both of two genes in B2510 were significantly decreased. The genes which were affected by T DNA insertion may be associated with pathogenicity and participate in the regulation of pathogenic process of U. virens in rice.