甘蔗抗病相关基因ShRPM1的克隆与表达分析

    Cloning and Expression Analysis of the Sugarcane Disease Resistance-related Gene ShRPM1

    • 摘要:
      目的 克隆甘蔗ShRPM1基因,并解析基因的结构、性质与表达特征,为甘蔗抗病分子育种提供基因源。
      方法 以甘蔗品种‘中蔗1号’为材料,通过RT-PCR克隆ShRPM1基因全长,结合生物信息学工具分析其编码蛋白的理化性质、结构域、亚细胞定位及启动子顺式作用元件;利用qRT-PCR检测甘蔗梢腐病菌胁迫下ShRPM1基因的表达变化。
      结果 成功克隆甘蔗ShRPM1基因,cDNA全长为3 747 bp,编码1 248个氨基酸;其蛋白理论分子量为141.8 kDa,等电点8.62,为亲水性非分泌蛋白,无跨膜结构,主要定位于细胞核。保守结构域分析显示,ShRPM1蛋白含有RX-CC-like、NB-ARC和LRR结构域,属于NLRs家族CNL类抗病蛋白。系统发育树表明其与高粱RPM1蛋白亲缘关系最近。启动子区鉴定到3类抗病相关顺式元件及多个激素应答元件,预示其受病原菌与激素信号调控。qRT-PCR结果显示,ShRPM1基因在甘蔗不同组织中的表达具有明显特异性,其中茎部表达量最高;ShRPM1基因的表达还具有明显的发育阶段特异性,在苗期表达量最高;ShRPM1基因在接种甘蔗梢腐病原菌24 h后,表达量显著上调,表明该基因受病原菌诱导表达。
      结论 首次从甘蔗品种‘中蔗1号’克隆获得ShRPM1基因,其编码的CNL类抗病蛋白参与甘蔗抗病响应,为甘蔗抗病分子育种提供新靶点。

       

      Abstract:
      Objective This study aimed to clone and characterize the ShRPM1 gene from sugarcane to elucidate its molecular features and provide a valuable genetic resource for the molecular breeding of disease-resistant sugarcane varieties.
      Method In this study, the full-length cDNA sequence of ShRPM1 was isolated via reverse transcription PCR (RT-PCR). Bioinformatics analysis was performed to characterize the physicochemical properties, conserved domains, and subcellular localization of the encoded protein, alongside the identification of cis-acting elements within the promoter region. Furthermore, the transcriptional response of ShRPM1 to stress induced by the sugarcane pokkah boeng pathogen was quantified using quantitative real-time PCR (qRT-PCR).
      Result The ShRPM1 cDNA is 3 747 bp in length, encoding a 1 248-amino-acid polypeptide with a predicted molecular weight of 141.8 kDa and a theoretical isoelectric point (pI) of 8.62. ShRPM1 is characterized as a hydrophilic, non-secretory protein lacking transmembrane domains and is primarily localized to the nucleus. Conserved domain analysis identified RX-CC-like, NB-ARC, and LRR domains, assigning ShRPM1 to the CC-NBS-LRR (CNL)-type disease resistance protein subfamily. Phylogenetic analysis demonstrated that ShRPM1 shares high homology with its ortholog in Sorghum bicolor. Moreover, ShRPM1 exhibited distinct spatio-temporal expression features, with the highest transcript levels observed in the stem and at the seedling stage, respectively. Notably, the expression of ShRPM1 was significantly up-regulated at 24 hours post-inoculation (hpi) with the pokkah boeng pathogen, indicating a strong inductive response to fungal infection.
      Conclusion In summary, the ShRPM1 gene was successfully cloned and characterized from sugarcane for the first time. Our findings demonstrate that ShRPM1 plays a pivotal role in the sugarcane defense response and represents a promising candidate gene for molecular breeding programs aimed at enhancing disease resistance.