关键词: 沙葱萤叶甲, 热激蛋白70, 基因克隆, 表达分析, 温度胁迫

Abstract: 【Aim】 This study aims to clone the heat shock protein Hsp70 gene in Galeruca daurica, and to analyze the gene sequence and mRNA expression profiles so as to explore the function of Hsp70 gene in the development and its responses to thermal stress in G. daurica. 【Methods】 The full-length cDNA of a Hsp70 gene was cloned and identified from the 2nd instar larvae of G. daurica using RT-PCR and RACE technique, and the putative amino acid sequence was analyzed by bioinformatics methods. The relative expression levels of the Hsp70 gene in different adult tissues (head, thorax and abdomen), different developmental stages (egg, the 1st-3rd instar larva, pupa and female and male adult), the 2nd instar larvae exposed to different temperatures (-14, -10, -5, 0, 5, 10, 15, 20, 25 and 30℃ for 1 h), and eggs exposed to 0℃ for 0 min, 15 min, 30 min, 1 h, 1.5 h, 2 h, and 3 h, respectively, were measured using real-time quantitative PCR method. 【Results】 A heat shock protein 70 gene was cloned from G. daurica, and named GdHsp70 (GenBank accession no. KY460462), which is 2 340 bp in length and contains an open reading frame (ORF) of 1 899 bp, encoding 632 amino acid residues. The putative protein is 70.12 kD with an isoelectric point (pI) of 4.79, and has no transmembrane region and signal peptide. Subcellular localization prediction revealed that GdHsp70 is mainly located in the cytoplasm. Protein domain analysis showed that GdHsp70 has three highly conserved domains. Homologous alignment and phylogenetic analysis demonstrated that GdHsp70 has high similarity with the Hsp70 proteins from other insects which are highly close to G. daurica based on taxonomy. Tissue-and developmental stage-specific mRNA expression profiling showed that GdHsp70 had the highest expression levels in the adult thoraxes and eggs. Both heat and cold stress could induce the mRNA expression of GdHsp70 in the 2nd instar larvae with the highest expression level at -10℃ for 1 h. GdHsp70 was up-regulated in G. daurica eggs exposed to 0℃ for 15 min to 3 h, with the highest expression level at 1 h after treatment. 【Conclusion】 GdHsp70 might be associated with the development and play an important role in response to heat and cold stress in G. daurica.

Key words: Galeruca daurica; Hsp70, gene cloning, expression profiling, thermal stress