苦瓜McMLO11基因克隆及其表达模式分析

Cloning and expression pattern of McMLO11 gene in Momordica charantia

【目的】克隆苦瓜McMLO11基因并研究其在白粉病和非生物胁迫下的表达规律,为阐明其在逆境调控中的作用提供参考。【方法】从苦瓜叶片中克隆McMLO11基因,利用生物信息学对其分子特征进行分析,并采用实时荧光定量PCR(qRT-PCR)方法,检测McMLO11基因在苦瓜不同组织(茎、卷须、老叶、嫩叶、根)及白粉菌侵染和盐、干旱胁迫下苦瓜真叶中的表达模式。【结果】苦瓜McMLO11基因含有1个1662 bp的开放阅读框(ORF),共编码553个氨基酸;McMLO11属于MLO基因家族,包含典型的Mlo超家族保守结构域。McMLO11蛋白的相对分子质量为63.83 ku,理论等电点为8.53,含7个跨膜域,无信号肽,属于不稳定蛋白;其二级结构主要由无规则卷曲和α-螺旋组成。系统进化树分析表明,McMLO11与黄瓜、南瓜、冬瓜等瓜类的MLO同源性较高。qRT-PCR检测发现,Mc-MLO11基因在苦瓜不同组织中的相对表达量依次表现为茎>卷须>老叶>根>嫩叶;与对照相比,白粉菌侵染后苦瓜叶片中的McMLO11表达量升高,至接种24 h达到峰值,推测其在白粉菌侵染苦瓜的早期产生应答反应;与对照相比,在盐胁迫6 h和干旱胁迫12 h时苦瓜叶片中的McMLO11相对表达量达到峰值,分别为对照的56.7和9.07倍,提示McMLO11基因可积极响应干旱和盐诱导。【结论】成功克隆了苦瓜McMLO11基因,其表达具有组织特异性,该基因可能参与白粉菌胁迫和多种非生物胁迫的调控过程。

【Objective】The McMLO11 gene of bitter gourd was cloned and its expression patterns in response to powdery mildew and abiotic stresses were analyzed to provide references for understanding its roles in regulating stress tolerance.【Method】McMLO11 gene was cloned from bitter gourd leaves,and its molecular characteristics were analyzed by bioinformatics.The expression patterns of McMLO11 in various tissues and under powdery mildew and abiotic stresses were detected by quantitative real-time PCR(qRT-PCR).【Result】McMLO11 gene contained an ORF of 1662 bp and encoded a protein with 553 amino acids.As a member of MLO family,McMLO11 contained typical Mlo superfamily conserved domains.Molecular weight and theoretical isoelectric point of McMLO11 protein were 63.83 ku and 8.53,respectively.McMLO11 was an unstable hydrophilic protein,which contained seven transmembrane domains without signal peptide and its secondary structure mainly consisted of random crimp andα-helix.Phylogenetic tree analysis revealed that McMLO11 had high homology with those of cucumber,pumpkin,winter melon.The qRT-PCR data showed that McMLO11 expressions in bitter gourd tissues were in decreasing order of stem>tendril>older leaf>root>younger leaf.Compared to the control,McMLO11 expression was up-regulated by powdery mildew pathogen,and the highest expression was observed 24 hours after inoculation,suggesting McMLO11 might play an critical roles in early responses to powdery mildew infection in bitter gourd.The expressions of McMLO11 gene reached the maximum at 6 h and 12 h under salt stress and drought stress,and they were 56.7 and 9.07 times higher than the control,respectively.It indicated that McMLO11 was also significantly induced under drought and salt stresses.【Conclusion】The full-length ORF of McMLO11 gene was successfully cloned in this study,and the tissue specificity of its expression was revealed.This study indicated that McMLO11 gene may be involved in regulating powdery mildew and abio-tic stresses.