马铃薯甲虫热激蛋白基因Ld-HSP60的克

发布时间：2018-01-19 19:04

  本文关键词： 马铃薯甲虫 热激蛋白 HSP 基因克隆 温度胁迫 实时荧光定量PCR　出处：《昆虫学报》2017年05期 　论文类型：期刊论文

【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(Gen Bank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LdHSP60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。
[Abstract]:[objective] Potato beetle Leptinotarsa decemlineata is a worldwide quarantine pest with strong adaptability to temperature stress. In order to further clarify its adaptability to temperature stress molecular mechanism. The effect of heat shock protein (HSP60) on the response of potato beetles to temperature stress was studied. [methods] RT-PCR and RACE techniques were used to clone the c of HSP60 gene of potato beetle. DNA full-length sequence; The sequence characteristics of the gene and its encoded protein were analyzed by bioinformatics software. The expression pattern of this gene under temperature stress was analyzed by real-time fluorescence quantitative PCR. [results] the HSP60 gene of potato beetle heat shock protein was cloned. It was named Ld-HSP60(Gen Bank login number: KC556801.The length of cDNA was 2 234 BP, and the open reading frame (ORF) was 1 731bp long. It encodes 576 amino acids with a relative molecular weight of 61.27 KD and a theoretical isoelectric point of 5.51 'untranslated region (UTRR) of 101 BP. The amino acid sequence contains the typical characteristic sequence of HSP60 family. The results of real-time fluorescence quantitative PCR show that the amino acid sequence contains the characteristic sequence of HSP60 family. The induced expression of Ld-HSP60 in female and male adults of potato beetles was not detected under low temperature stress (-10 鈩，

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