棉花黄萎病相关基因GhMYB6的克隆与功能分析

Cloning and functional analysis of GhMYB6 gene related to cotton Verticillium wilt resistance

  • 摘要: 【目的】克隆陆地棉MYB家族基因GhMYB6,并对其在棉花抗黄萎病反应中的功能进行初步探究,为挖掘棉花抗病相关基因及棉花抗病育种提供参考。【方法】基于棉花转录组测序数据,筛选并克隆了响应黄萎病菌侵染的基因GhMYB6,对其序列进行生物信息学分析,采用实时荧光定量PCR(qRT-PCR)检测其在黄萎病诱导下的表达模式,并利用病毒诱导的基因沉默(VIGS)技术初步验证棉花抗黄萎病中的生物学功能。【结果】克隆获得的GhMYB6基因开放阅读框(ORF)为258 bp,编码85个氨基酸残基,理论等电点(pI)为9.34,脂肪系数为73.41,平均疏水性为-0.793,相对分子质量为9.86 kD,不稳定指数为73.41,为亲水性、碱性的非跨膜蛋白,无信号肽,定位于细胞核,在第11~63氨基酸处含有1个SANT结构域。GhMYB6蛋白与雷蒙德氏棉GrMYB6聚在同一小分支上,说明二者的亲缘关系较近。GhMYB6基因在V991侵染后6和12 h时相对表达量较对照(未侵染处理,CK)极显著下调(P<0.01),72 h时显著上调(P<0.05,下同),24和48 h时与CK无显著差异(P>0.05)。与阴性对照植株TRV:00相比,GhMYB6基因沉默植株萎蔫程度和叶片黄化更严重,病情指数显著升高,茎秆的褐变程度更严重,且茎段在培养基上生长的真菌菌丝数量明显增多。【结论】GhMYB6基因响应黄萎病菌V991侵染,当抑制GhMYB6基因表达后,棉花对黄萎病菌的敏感性增强,抗性明显降低,推测GhMYB6是棉花抗黄萎病防御的一个正向调节因子。

     

    Abstract: 【Objective】To clone GhMYB6 gene of MYB family in upland cotton and analyze its function in response to cotton Verticillium wilt,so as to provide reference for exploring cotton genes related to disease-resistance and disease-resistant cotton breeding.【Method】Based on data of cotton transcriptome,the gene GhMYB6 in response to Verticillium wilt infection was screened and cloned,and its sequences was analyzed by bioinformatics. Its expression patterns induced by Verticillium wilt were detected by real-time fluorescence quantitative PCR(qRT-PCR). Biological function of cotton resistance to Verticillium wilt was preliminarily verified by virus-induced gene silencing(VIGS) technology.【Result】GhMYB6 open reading frame(ORF) was 258 bp,encoding 85 amino acid residues,with a theoretical point(pI) of 9.34,fat coefficient of 73.41,average hydrophobicity of-0.793,relative molecular mass of 9.86 kD,instability index of 73.41,indicating that it encoded non-transmembrane,alkaline,hydrophilic protein located in the nucleus,with a SANT domain in the 11th -63rd amino acids. Protein GhMYB6 and Gossypium raimondii GrMYB6 were clustered in the same small branch,indicating a close relationship between the two. After V991 infection,relative expression of GhMYB6 was extremely significantly down-regulated compared that of control(uninfected treatment,CK) (P<0.01) at 6 and 12 h,significantly up-regulated(P<0.05,the same below) at 72 h and no significant difference between it and CK was found at 24 and 48 h(P>0.05).After inhibiting GhMYB6 gene expression by VIGS technology,compared with the negative control plant TRV:00,wilting and leaf yellowing of GhMYB6 gene silenced plants was more serious,the disease index was significantly higher,stem brow-ning was more serious,and the number of fungal hyphae in stem segments was higher.【Conclusion】GhMYB6 gene responds to Verticillium dahliae V991 infection. After inhibiting GhMYB6 gene expression,cotton is more sensitive to Verticillium wilt and the resistance is significantly reduced,so it is speculated that GhMYB6 is a positive regulator of cotton Verticillium wilt resistance.

     

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