GhMAPKKK2基因在棉花抗黄萎病中的功能分析

Functional analysis of Gh MAPKKK2 gene in cotton resistance to Verticillium wilt

【目的】探索Gh MAPKKK2基因在棉花抗黄萎病中的功能。【方法】利用生物信息学方法分析GhMAPKKK2基因的结构特征和进化关系。通过实时荧光定量聚合酶链式反应(Quantitative real-time polymerase chain reaction, qRT-PCR)分析GhMAPKKK2基因在黄萎病菌侵染以及外源茉莉酸(Jasmonic acid,JA)和水杨酸(Salicylic acid, SA)处理下的表达模式。利用病毒诱导的基因沉默技术结合阳性植株表型鉴定以及qRT-PCR技术,对GhMAPKKK2基因在棉花抗黄萎病中的功能及可能参与的抗病机制进行初步分析。【结果】以陆地棉cDNA为模板克隆得到Gh MAPKKK2基因,其编码区全长为1 341 bp,编码446个氨基酸,与雷蒙德氏棉Gr MAPKKK2同源性最高。GhMAPKKK2基因的转录水平受黄萎病菌及JA和SA诱导。沉默GhMAPKKK2基因后增强了棉花对黄萎病菌的敏感性,与阴性对照植株相比,沉默植株的病情指数显著增加、维管束褐变程度明显加重、茎段菌丝数量明显增多。qRT-PCR分析表明,沉默植株中JA和SA信号传导途径相关基因的表达量均显著低于阴性对照植株。【结论】GhMAPKKK2基因可能通过参与JA和SA信号通路在棉花抗黄萎病反应中发挥正调控的作用。

[Objective] The purpose of this study is to explore the function of GhMAPKKK2 gene in resistance to Verticillium wilt in cotton. [Method] The structural characteristics and phylogeny of GhMAPKKK2 gene were analyzed by bioinformatics methods. The expression patterns of GhMAPKKK2 gene under Verticillium wilt infection and exogenous jasmonic acid(JA)and salicylic acid(SA) treatment were analyzed by quantitative real-time polymerase chain reaction(qRT-PCR) technology.Using virus-induced gene silencing(VIGS) technology combined with phenotypic identification of gene-silenced plants and qRT-PCR, the function of GhMAPKKK2 gene in cotton resistance to Verticillium wilt and the possible disease resistance mechanism were preliminarily analyzed. [Result] The GhMAPKKK2 gene was cloned and its coding sequence(CDS) was1 341 bp, encoding 446 amino acid residues, which has the highest homology with Gr MAPKKK2. The transcription level of GhMAPKKK2 gene was induced by Verticillium dahliae and exogenous JA and SA treatment. The silence of Gh MAPKKK2gene enhanced the sensitivity of cotton to V. dahliae. Compared with the negative control plants, the disease index of the silenced plants was significantly higher;the browning degree of vascular bundles in silenced plants was more severe;and the number of hyphae in stem segments of silenced plants was increased obviously. qRT-PCR analysis showed that the expression levels of genes-involved in JA and SA signaling pathway in silenced plants were significantly lower than that in control plants.[Conclusion] Gh MAPKKK2 gene may play a positive regulatory role of Verticillium wilt resistance in cotton by participating in the JA and SA signaling pathways.