马铃薯甲虫热激蛋白基因Ld-HSP60的克隆、序列分析及温度胁迫下的表达

摘要

[目的]马铃薯甲虫Leptinotarsa decemlineata是一种世界性检疫害虫,对温度胁迫具有极强的适应性,为进一步明确其对温度胁迫适应性的分子机制,研究了热激蛋白HSP60在马铃薯甲虫温度胁迫应答过程中的作用.[方法]采用RT-PCR及RACE技术克隆马铃薯甲虫热激蛋白HSP60基因的cDNA全长序列;利用生物信息学软件分析该基因及其编码蛋白质的序列特性;运用实时荧光定量PCR技术分析该基因在温度胁迫下的表达模式.[结果]克隆得到马铃薯甲虫热激蛋白HSP60基因,命名为Ld-HSP60(GenBank登录号:KC556801),其cDNA全长2 234 bp,开放阅读框(ORF)长1 731 bp,编码576个氨基酸,相对分子量约为61.27 kD,理论等电点为5.51,5'端非翻译区(UTR)长101 bp,3'UTR长402 bp.氨基酸序列中含有HSP60家族典型的特征序列.实时荧光定量PCR结果表明,低温胁迫(-10和0℃)下未检测到马铃薯甲虫雌雄成虫中Ld-HSP60的诱导表达;高温胁迫(38和44℃)诱导马铃薯甲虫雄成虫Ld-HSP60上调表达,随着胁迫温度的升高Ld-HSP60表达量呈现先升高后降低的趋势,38℃高温胁迫下表达量最高,胁迫时间越长Ld-HSP60表达量也越高.[结论]相比其他热激蛋白,HSP60对温度敏感性较低,推测HSP60可能在马铃薯甲虫雄成虫抵御高温胁迫中发挥作用.%[Aim] As one of international major quarantine pests,the Colorado potato beetle,Leptinotars decemlineata,has strong resistance to temperature stress.The study aims to better understand the molecular mechanism of resistance to temperature stress in L.decemlineata by determining the role of heat shock protein HSP60 in response to temperature stress in this beetle.[Methods] The full-length cDNA encoding HSP60 from L.decemlineata was cloned by RT-PCR and RACE technologies.Bioinformatics programs were used to analyze the sequence characteristics of the gene and amino acid sequence.The mRNA expression levels of the gene in L.decemlineata under cold and heat stress conditions were detected by real-time PCR.[Results] The complete cDNA of the gene encoding heat shock protein HSP60 was obtained from L.decemlineata,and named Ld-HSP60 with the GenBank accession no.of KC556801.The complete cDNA (2 234 bp) is comprised of a 101 bp 5'untranslated region (UTR),a 402 bp 3'UTR,and a 1 731 bp open reading frame (ORF).The ORF of Ld-HSP60 encodes a protein of 576 amino acids,with the molecular weight of 61.27 kD and the theoretical pI of 5.51.The mature amino acid sequence contains the classical signature sequences of HSP60 family.The real-time PCR results showed that low temperature stress (-10 and 0℃) did not induce the expression of Ld-HSP60 in male and female adults of L.decemlineata;however,high temperature stress (38 and 44℃) induced the up-regulated expression of Ld-HSP60 in male adults,and the expression trend of Ld-HSP60 rose first and then fell with the increasing temperature.The expression level of Ld-HSP60 reached the maximum at 38℃,and the longer the stress time the higher the expression level.[Conclusion] HSP60 is less sensitive to temperature than other heat shock proteins,and it is inferred that HSP60 may play a role in resistance to high temperature stress in male adults of L.decemlineata.