Cloning Of Heat Shock Protein Genes (hsp60、hsp70and Hsp90) In Leptinotarsa Decemlineata (Coleoptera: Chrysomelidae) And Its Expression Under Temperature Stress

As one of international major quarantine pest, Colorado potato beetle, Leptinotarsa decemlineata, has strong ability of stress resistance, especially for the extreme temperature stress resistance. The overall goal of this study is to better understand the molecular mechanism of temperature stress resistance in L. decemlineata. The full-length cDNA encoded the HSP60, HSP70and HSP90from L. decemlineata were cloned analysed by Bioinformations methods, RT-PCR and RACE approaches. The relative expression levels of Ld-hsp90and Ld-hsp60in adults L. decemlineata under a series of temperatures were detected by Real-Time PCR. The main results are as follows:This was the first time that full-length cDNA sequences encoded HSP90and HSP60from L. decemlineata were cloned, which were named to Ld-hsp90and Ld-hsp60respectively. The complete length cDNA were2489bp and2234bp respectively, the length of5’untranslated region (UTR) were113bp and101bp respectively, and the length of5’UTR were216bp and402bp respectively. The complete ORF (2160bp,1731bp) encodes a719and a576amino acid protein respectively. The amino acid sequences contain classical and highly conserved signature sequences of HSP90and HSP60family respectively.Meanwhile,3full-length cDNA sequences of encoded HSP70were cloned in L. decemlineata, which were named to Ld-hsp70a (2199bp), Ld-hsp70b (2397) and Ld-hsp70c (2482bp) respectively. The length of5’UTR were92bp,132bp and289bp respectively, and3’UTR were160bp,291bp and203bp. The complete ORF (1947bp,1974bp and1890bp) encodes a648, a657and a629amino acid protein respectively. The3amino acid sequences of HSP70share a71.50%identity, and they all contain the classical and highly conserved signature sequences of HSP70family. The Real-Time PCR results indicated that the relative expression levels of Ld-hsp90was significantly increased after1hour exposed to38℃and45℃when compared to the controls (25℃), while there was no significant different when it comes to the expression level of Ld-hsp60. The both expression levels of Ld-hsp90and Ld-hsp60had no significant different after1hour exposed to-10℃and0℃when compared to the controls (25℃). The results suggest that the up-regulation expression of Ld-hsp90may play an important role in high temperature tolerance, and the up-regulation expression of Ld-hsp60may couldn’t induced by thermal stress in L. decemlineata.