朱砂叶螨精氨酸激酶基因的克隆与表达特征

摘要

[Objective] This study aims to clone the arginine kinase gene in Tetranychus cinnabarinus,analyze the gene sequence and mRNA expression profiles,and then explore the function of TcAK gene during mite growth and its responses to diapause,thermal stress and UV stress.[Methods]The full-length cDNA encoding arginine kinase from T.cinnabarinus was cloned and identified via RT-PCR and RACE.The putative amino acid sequence was analyzed by bioinformatics methods.The relative expression levels of TcAK in T.cinnabarinus at different developmental stages and in diapausing adult females,as well as under thermal or UV stress conditions,were measured using real-time quantitative PCR.[Results] The results showed that TcAK gene contains an open reading frame of 1071 bp which encoding 356 amino acids and containing typical arginine kinase active site and substrate binding site.During the development of T.cinnabarinus,the expression of TcAK were analyzed.Compared with egg stage,the expression of TcAK increased at larva and nymph stage,and reached the highest level at the larva stage.And the expression of TcAK decreased at adult stage.In addition,the significant up-regulation of TcAK was observed in T.cinnabarinus in diapausing females,under high / low temperature stress or UV stress.[Conclusion]Arginine kinase may be involved in mite growth and development,and play an important role in T.cinnabarinus against the adverse environment.%[目的]拟克隆朱砂叶螨(Tetranychus cinnabarinus)精氨酸激酶基因,并分析其序列特征及表达模式,探讨该基因在朱砂叶螨生长发育、滞育及响应温度胁迫和紫外胁迫等方面的作用.[方法]通过RT-PCR技术和RACE方法,获得朱砂叶螨精氨酸激酶cDNA全长序列(TcAK),并进行生物信息学分析,采用实时荧光定量PCR技术检测TcAK基因在不同发育阶段、滞育状态和温度胁迫及紫外胁迫条件下的表达情况.[结果]结果表明,TcAK基因阅读框架全长1 071 bp,编码356个氨基酸残基,含有典型的精氨酸激酶活性中心位点和底物结合区.TcAK基因在幼螨期和若螨期的表达量较卵期显著增大,在幼螨期的表达量达到最大,远高于其他发育阶段,而TcAK基因在成螨期的表达量较卵期下降.此外,滞育状态、高温/低温胁迫、UV胁迫下朱砂叶螨的TcAK表达量均显著升高.[结论]朱砂叶螨精氨酸激酶与其生长发育相关,并可能参与抵御外界不良环境.