关键词: 朱砂叶螨, 热激蛋白90, 基因克隆, 序列同源性分析, 原核表达

Abstract: HSP90 gene of Tetranychus cinnabarinus was cloned and sequenced by the combination of reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) approaches. A cDNA fragment of 2 595 bp was obtained, including an open reading frame (ORF) of 2 169 bp which encodes a polypeptide of 722 amino acids with a predicted molecular weight of 83.45 kDa and a theoretical isoelectric point of 4.81, 3′-untranslated region (UTR) of 249 bp, and a 5′UTR of 177 bp. By using Antheprot analysis, five signature sequences of HSP90 family and C-terminal five amino acids of Hsp90 (MEEVD) were detected. Blast analysis indicated that the nucleotide sequence of HSP90 gene coding region of Tetranychus cinnabarinus shared high similarity with other known HSP90s, especially with those from insects of arthropods. The confirmed prokaryotic recombinant expression plasmids pET43a-TcHSP90 was transformed into Escherichia coli BL21 and expressed in prokaryotic cells. The fusion proteins of recombinant TcHSP90 in E. coli BL21(origami) were separated and identified by SDS-PAGE and Western blotting, and the result showed that the constructed prokaryotic expression plasmid could stably and correctly express in the host bacteria. Cloning and prokaryotic expression of TcHSP90 from T. cinnabarinus would provide useful experimental material for the further study of the nature and function of HSP90.

Key words: Tetranychus cinnabarinus, heat shock protein 90, gene cloning, sequence homology analysis, prokaryotic expression