马铃薯甲虫热激蛋白基因Ld-hsp90的克隆及温度胁迫下的表达

cDNA cloning of heat shock protein 90 (Ld-hsp90) in Leptinotarsa decemlineata (Coleoptera:Chrysomelidae) and its expression under temperature stress

马铃薯甲虫Leptinotarsa decemlineata是一种抗逆性极强的世界性检疫害虫,对温度胁迫具有很强的适应性,为进一步明确其对温度胁迫适应性的分子生态机制,采用RT-PCR及RACE技术克隆得到了1个hsp90基因的cDNA全长序列,命名为Ld-hsp90,并利用实时荧光定量PCR技术检测其在温度胁迫后相对表达量的变化。Ld-hsp90全长为2489 bp,开放阅读框(ORF)长度为2160 bp,编码719个氨基酸,理论等电点为4.98,分子量约为82.09 kD,5'端与3'端非编码区长度分别为113 bp和216 bp。氨基酸序列中含有HSP90家族的5个签名序列及胞质特征序列MEEVD。实时荧光定量PCR检测结果显示:38℃和44℃高温热激1h,马铃薯甲虫成虫体内的Ld-hsp90的表达量均显著高于对照组,而0℃与-10℃低温处理1 h后的表达量与对照组均无显著差异。研究表明Ld-hsp90的上调表达在马铃薯甲虫抗高温中起着重要的作用。

As one of the international major quarantine pests, the Colorado potato beetle, Leptinotarsa decemlineata, has strong stress resistance, especially to the extreme temperature stress. The paper is aimed to better understand the molecular mechanism of temperature stress resistance in L. decemlineata. A full-length cDNA encoding the HSP90 from L. decemlineata was cloned using RT-PCR and RACE tech- nique. This cDNA was named as Ld-hsp90, and its relative expression levels in L. decemlineata adults under a series of temperatures were detected by real-time PCR. The complete cDNA （2489 bp） was com- prised of a 113 bp 5 ＇-untranslated region （UTR） , a 216 bp 3＇-UTR, and a 2 160 bp open reading frame （ORF）. The ORF of Ld-hsp90 encoded a protein of 719 amino acids, with a theoretical isoelectric point of 4.98 and molecular weight of 82.09 kD. The amino acid sequence contained five signature sequences of HSP90 family and a C-terminal cytoplasmic character sequence （MEEVD）. The real-time PCR results indicated that the expression level of Lcl-hsp90 was significantly increased 1 hour after exposed to 38 ~C and 45 ~C, compared to the control, while there was no significant difference between - 10 ~C and 0 ~C. The results suggested that the up-regulated expression of Ld-hsp90 might play an important role in high temperature tolerance in L. decemlineata.