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Cloning and sequence analysis of heat shock protein gene Ld-HSP60 in Leptinotarsa decemlineata(Coleoptera:Chrysomelidae) and its expression under temperature stress
Pages: 523-532
Year: Issue:  5
Journal: Acta Entomologica Sinica

Keyword:  Leptinotarsa decemlineataheat shock proteinHSP60gene cloningtemperature stressreal-time PCR;
Abstract: 【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.ThemRNA 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 Gen Bank accession no.of KC556801.The complete cDNA(2 234 bp) is comprised of a 101 bp 5’ untranslated region(UTR),a402 bp 3’ UTR,and a 1 731 bp open reading frame(ORF).The ORF of Ld-HSP60 encodes a protein of576 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 at38℃,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.
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