T-DNA插入筛选棉花枯萎病菌致病力减弱突变体及其侧翼序列分析

Screening and flanking sequences of T-DNA insertion mutants with reduced pathogenicity in cotton Fusarium oxysporum

摘要:

【背景】 新疆是棉花生产的主要地区，枯萎病的发生严重影响着棉花的产量。目前，关于棉花枯萎病原菌的发病机制尚无详细报道。【目的】 在尖孢镰刀菌突变体库中筛选出致病力降低的突变体并分析其致病基因，以阐明棉花枯萎病菌的致病分子机制。【方法】 在前期已建立的枯萎病菌突变体库的基础上，对随机挑选的136份突变体致病力进行鉴定，并对其表型和侧翼序列进行分析。【结果】 获得5个致病能力明显降低的突变株，突变体B-18的形态与野生型有差异且菌落颜色变为紫色；5个突变体生长速率均降低，其中B-18下降最明显；5个突变体产孢数量均降低。扩增5个突变体，测序后经Blast比对分析，确定了T-DNA在枯萎病菌基因组的分布情况。【结论】 对筛选出的5个致病力缺陷的突变体进行分析并找到致病基因，为研究棉花致病基因的功能和克隆提供了坚实的理论基础。

关键词:棉花;尖孢镰刀菌;T-DNA插入突变体;高效热不对称交错PCR;致病力

Abstract:

[Background] Xinjiang is the main producing area of cotton, and Fusarium wilt seriously affects the cotton yield. However, the detailed pathogenesis of cotton Fusarium wilt remains to be reported. [Objective] To select the mutants with reduced pathogenicity from the Fusarium oxysporum mutant library and identity their genes conferring pathogenicity, so as to elucidate the molecular mechanism of the pathogen in causing Fusarium wilt in cotton. [Methods] On the basis of the previously established mutant library of F. oxysporum, By the identification of 136 randomly selected pathogenic mutants. Furthermore, we analyzed the phenotypes and flanking sequences. [Results] we screened out five mutants with significantly reduced pathogenicity, the morphology of mutant B-18 was different from that of the wild type, with purple colonies. The growth rate of the five mutants, especially B-18, decreased. In addition, the five mutants showed reduced spore production. After sequencing of the five mutants, Blast analysis was performed to determine the distribution of T-DNA in the pathogen genome. [Conclusion] From the five mutants with reduced pathogenicity, we identified the genes associated with the pathogenicity. The findings provide a solid theoretical basis for the functional research and cloning of the genes associated with the diseases in cotton.

Key words:cotton;Fusarium oxysporum;T-DNA insertion mutants;hi TAIL-PCR;pathogenicity