稻曲病菌致病力丧失突变菌株B-1015的T-DNA标记基因的克隆

Molecular Cloning of T-DNA Targeted Genes of Ustilaginoidea virens Pathogenicity-Defective Mutant Strain B-1015

【目的】研究稻曲病菌(Ustilaginoidea virens)致病力丧失突变菌株B-1015,克隆和分析T-DNA插入位点的侧翼序列,为稻曲病菌的致病机制研究提供参考。【方法】分析稻曲病菌T-DNA插入突变体B-1015菌株的生长速率、产孢量、孢子萌发率以及致病力等生物学表型。用PCR检测T-DNA在B-1015基因组中插入的稳定性,用Southern杂交检测T-DNA插入的拷贝数,用hiTail-PCR扩增T-DNA侧翼序列,用RACE PCR克隆T-DNA标记基因,半定量RT-PCR分析所克隆基因的表达情况。【结果】与野生菌株P1相比,突变菌株B-1015的生长速率和产孢量都有较明显的降低,孢子萌发率无明显差异,致病力丧失。分子检测结果表明T-DNA在B-1015基因组中为稳定的单拷贝插入。hiTail-PCR得到的T-DNA两端侧翼序列在野生型菌株基因组上不相连,RACE PCR在两边的侧翼序列上分别克隆得到Uvt-1015R和Uvt-1015L。预测分析表明Uvt-1015R包含一个长度为948 bp的开放阅读框(open reading frame finder,ORF),可编码295个氨基酸,5′端非编码区(5′-untranslated regions,5′-UTR)长度为341 bp,3′端非编码区(3′-untranslated regions,3′-UTR)长度为271 bp。Uvt-1015L包含一个长度为351 bp的ORF,可编码116个氨基酸,5′-UTR为31 bp,3′-UTR长度为174 bp。在已知功能的蛋白中检索,没有发现与Uvt-1015R和Uvt-1015L基因同源的蛋白。序列分析发现T-DNA插入在两个基因序列中间,RT-PCR结果显示,在突变菌株B-1015中,Uvt-1015R表达量下降,Uvt-1015L不表达。【结论】稻曲病菌突变菌株B-1015致病力等重要生物学表型发生改变,可能是由于T-DNA的插入导致了B-1015基因组重排和Uvt-1015R、Uvt-1015L基因结构的破坏。

[Objective] The objective of this study is to analyze the biological phenotypes of the pathogenicity-defective mutant strain B-1015, to clone its T-DNA integration flanking sequences, and finally to reveal the functions of targeted genes in pathogenesis of Ustilaginoidea virens. The study will contribute to better understanding of the molecular pathogenesis of U. virens. [Method] Biological phenotypes of B-1015 were analyzed by testing growth rate, sporulation ability, spore germination rate and pathogenicity. The copy numbers of T-DNA inserted into B-1015 were identified by Southern blot. The flanking sequences of T-DNA were cloned by hiTail-PCR and the whole gene sequences of the T-DNA targeted genes were cloned by RACE PCR. The genes expression were detected by RT-PCR. [Result]By comparing with wild type strain P1, the sporulation ability and growth rate of the B-1015 were significantly declined, but the spore germination rate had no significant difference. The inserted mutant lost its pathogenicity in rice. Molecular detection analysis confirmed that the B-1015 was a stable single T-DNA insertional mutant. The T-DNA flanking sequences were obtained by hiTail-PCR. Two genes, Uvt-1015R and Uvt-1015L, were cloned by RACE PCR. Gene sequence analysis showed that Uvt-1015R contained a 948 bp open reading frame, which could encode a 295 amino acid protein, a 341 bp 5′-UTR and a 271 bp 3′-UTR. Another gene, Uvt-1015L, comprised a 351 bp open reading frame coding a 116 amino acid protein, a 31 bp 5′-UTR and a 174 bp 3′-UTR. By searching in the known protein, the Uvt-1015R and Uvt-1015L genes showed no homology of any protein. Sequence alignment showed that the flanking sequences of T-DNA were non-adjacent in the wild type genome and the T-DNA disrupted the two genes sequence. RT-PCR analysis confirmed that compared to P1 strain, the transcripts of Uvt-1015R was significantly decreased, and the transcripts of Uvt-1015L was not detected any more. [Conclusion] In B-1015, both the T-DNA insertion and chromosomal rearrangement caused important biological phenotype mutant, thus leading to the loss of pathogenicity.