关键词: 青杨脊虎天牛, 细胞色素P450酶系, CYP4G2基因, 克隆, 表达

Abstract: A pair of primers was designed based on the reported sequences of conservative amino acid regions of CYP4 protein family in insects. The cDNA encoding the cytochrome CYP4G2 protein was isolated from the intestines of Xylotechus rusticus L. by reverse transcription polymerase chain reaction (RT-PCR). The cDNA fragment was constructed to prokaryotic expression vector that named pET-CYP4G2 for overexpression in Escherichia coli JM109.Sequence analysis showed that the full-length of CYP4G2 (GenBank accession no. EF429250) open reading frame (ORF) was 387 bp, encoding 129 amino acid residues;  the predicted MW and pI were 16.9 kD and 5.75, respectively. The deduced amino acid sequence showed high identity to the reported sequences of CYP4 family amino acids from other insects（63%～86%） and shared the typical structural features of cytochrome from other insects. After induction with IPTG, its molecular weight was found to be about 22 kD by checking with SDS polyacrylamide gel electrophoresis. The molecular weight was the same as the prediction of fusion protein. Carbon monoxide (CO) difference spectrum analysis confirmed that the recombinant strain expressed pET-CYP4G2 with biological activity.

Key words: Xylotechus rusticus, cytochrome P450, CYP4G2, cloning, expression