棉花凝集素类受体蛋白激酶基因GhLecRK1响应棉蚜危害的功能分析

Functional analysis of cotton lectin receptor-like kinase gene GhLecRK1 in response to damage caused by Aphis gossypii Glover

  • 摘要: 【目的】克隆棉花(Gossgpium hirsutum)凝集素类受体蛋白激酶(LecRK1)基因GhLecRK1,分析其在响应棉蚜危害中的功能,为培育棉花抗蚜新品种提供理论参考。【方法】在棉蚜诱导的棉花叶片全转录组测序数据分析基础上,克隆响应棉蚜危害的GhLecRK1基因,并对其序列进行生物信息学分析。采用实时荧光定量PCR检测GhLecRK1基因在棉蚜诱导下的表达模式,运用瞬时过表达技术研究其在棉花对棉蚜防御响应中的功能。【结果】GhLecRK1基因开放阅读框(ORF)长度1233 bp,编码410个氨基酸残基,理论等电点(pI)8.36,预测蛋白质分子量45948 Da,由具有1个α-甘露糖凝集素结合结构域、1个S-糖蛋白结构域(SLG)和1个纤溶酶原/苹果/线虫结构域(PAN)组成,属G型凝集素类受体蛋白激酶。同源序列比对分析结果表明,GhLecRK1与其他植物的G型LecRLKs序列具有高度相似性(94.39%)。系统发育分析结果表明,GhLecRK1与Gossypium raimondii的Gr012449778.1亲缘关系最近。在棉蚜取食诱导后24、48和72 h的棉花植株的子叶、根、茎中GhLecRK1基因的表达量均有不同程度上调,但是仅有子叶在棉蚜取食24 h时GhLecRK1基因相对表达量显著上调(P<0.05,下同)。选择性和非选择性试验结果显示,瞬时过表达GhLecRK1基因棉花上棉蚜的数量均少于WT植株和pMD植株,且在取食48 h时过表达GhLecRK1基因棉花上棉蚜蜜露的分泌量显著减少。【结论】GhLecRK1基因响应棉花对棉蚜的防御反应,GhLecRK1基因过量表达后可增强棉花对棉蚜的抗性。

     

    Abstract: 【Objective】To clone cotton(Gossypium hirsutum)lectin receptor-like kinase(LecRK1)gene GhLecRK1 and analyze its function in the response to Aphis gossypii Glover damage,so as to provide theoretical reference for breeding new cotton varieties resistant to aphids.【Method】Based on the analysis of whole-transcriptome sequencing data of cotton leaves induced by A. gossypiiGhLecRK1 gene which responded to A. gossypii damage was cloned and its sequence was analyzed through bio-information method. Real-time fluorescence quantitative PCR was used to measure GhLecRK1 gene expression pattern under the induction of cotton aphids. Transient overexpression technique was utilized to study its function in cotton defense response to cotton aphids.【Result】The open reading frame(ORF)of GhLecRK1 gene was made up of 1233 bp and encoded 410 amino acid residues. The theoretical isoelectric point(pI)was 8.36,and the predicted molecular weight of GhLecRK1 protein was 45948 Da. GhLecRK1 protein was consisted of one α-mannose lectin binding domain,one S-glycoprotein domain(SLG),and one plasminogen/apple/nematode domain(PAN),therefore,it was a Gtype lectin receptor-like kinase. Homologous sequence analysis showed that GhLecRK1 had high sequence similarity (94.39%)with G-type LecRLKs sequences from other plants. Phylogenetic analysis of indicated that GhLecRK1 was most closely related to Gr012449778.1 from Gossypium raimondii. The expressions of GhLecRK1 gene in the cotyledon, root and stem in cotton plants at 24 h,48 h and 72 h after A. gossypii feeding were up-regulated to varying degrees,but the relative expressio of GhLecRK1 gene only significantly increased in the cotyledon at 24 h after A. gossypii feeding(P<0.05,the same below). In addition,the results of selective and non-selective tests showed that the number of A. gossypii on transiently overexpressed GhLecRK1 gene cotton plants was less than those on WT plants and pMD plants respectively,and the amount of honeydew secreted by A. gossypii in overexpressed GhLecRK1 gene cotton plants was significantly reduced at 48 h after A. gossypii feeding.【Conclusion】GhLecRK1 gene responds to cotton defense response to A. gossypii. When GhLecRK1 gene is overexpressed in cotton plants,the resistance of cotton to A. gossypii is enhanced.

     

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