关键词: 赤拟谷盗, 热激蛋白70, 竞争定量PCR, 内部竞争物, 同源性分析

Abstract:  To study the expression changes of heat shock protein 70 (a highly conserved protein) in Tribolium castaneum after exposure to heat stress, a fragment of 681 bp encoding for HSP70 was amplified and sequenced from T. castaneum. The fragment encoded 227 amino acid residues with the GenBank accession no. HM345948. The result of homology analysis showed that this fragment shared 97% identity with hsp70 from L. decemlineata (AF322911.1). Comparison of the deduced amino acid sequences of HSP70 in T. castaneum with those in Leptinotarsa decemlineata, Mamestra brassicae, Drosophila melanogaster and Liriomyza sativae indicated that they shared more than 94% identity. The internal competitor used in quantitative competitive PCR was obtained by RT-PCR. The detection system of hsp70 was constructed by PCR amplification using the same quantity of target cDNA and a series of diluted internal competitor as template. The linear equation of standard curve was Y=1.032X-1.618 (r²=0.975). This study provides a very convenient method for the quantitation of the hsp70 expression changes in T. castaneum and offers the basic data for the prevention and control of pests using thermal control technology.

Key words: Tribolium castaneum, heat shock protein 70, quantitative competitive PCR, internal competitor, homology analysis