关键词: 沙葱萤叶甲, 丝氨酸蛋白酶, 基因克隆, 温度胁迫, 表达分析

Abstract: 【Aim】 Serine protease (SP) is an important proteolytic enzyme, with serine as its active center. This study aims to clone a serine protease gene from Galeruca daurica and to analyze its expression levels in response to temperature stress so as to lay a foundation for further investigation on the regulation mechanisms of temperature tolerance and other physiological functions in G. daurica. 【Methods】 Based on the transcriptome data of the 2nd instar larvae of G. daurica, the full-length cDNA of the serine protease gene was cloned from G. daurica by RACE, and its sequence was subjected to bioinformatics analysis. Its expression profiles in the 2nd instar larvae of G. daurica exposed to different temperatures (-10, -5, 0, 5, 25, and 35℃) for 1 h and recovered at 25℃ for 30 min were detected by qPCR. 【Results】 A serine protease gene was cloned from G. daurica, and named GdSP (GenBank accession no.: MG797556). GdSP is 1 110 bp in length with an open reading frame (ORF) of 969 bp, encoding a protein of 322 amino acids with the predicted molecular weight of 35.41 kD and pI of 5.61. The encoded protein shares the typical structural features of serine proteases, with a transmembrane domain but no signal peptide. Homologous sequence alignment and phylogenetic analysis showed that GdSP has the highest amino acid sequence identity (30.53%) with Anoplophora glabripennis SP. qPCR results showed that the expression levels of GdSP had no significant difference in the 2nd instar larvae of G. daurica exposed to different temperatures whereas increased significantly after recovery from the low (except -10℃) and high temperature stresses. 【Conclusion】 Rapid cold-hardening has no significant effects on the expression of GdSP while recovery from cold shock elicits its up-regulated expression.

Key words: Galeruca daurica, serine protease, gene cloning, temperature stress, expression profiling