稻曲病菌 T-DNA 插入突变体 B2510的插入位点分析

摘要

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 B25 10 were analyzed.Compared with the wild-type U .virens strain P1 ,the pathogenicity of the mutant strain B25 10 in the field was significantly decreased.The growth rate of B25 10 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 B25 10 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 B25 10.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 B25 10 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.%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.