关键词: 葱蝇, 滞育, 热激蛋白, 基因克隆, 表达模式, 实时荧光定量PCR

Abstract:  【Aim】 The sHSPs are a diverse of molecular chaperones with molecular weights ranging from 12 to 43 kDa in different species. These proteins have been shown to be crucial components of the diapause of some species, but need to be further elucidated. The study aims to clone and characterize the sHSP gene of Delia antiqua, of which orthologs were earlier reported to be diapause-related, and to investigate the expression patterns of the gene during both winter and summer diapauses of the species. 【Methods】 A sHSP gene was cloned from D. antiqua using RACE-PCR, and characterized through homology, domain and phylogenetic analyses. The existence of the intron was investigated with genome sequencing around the gene. The expression level of this gene during both winter diapause (WD) and summer diapause (SD) was detected real-time qPCR, and the expression difference was compared to reveal the association with diapause development. 【Results】 The cDNA of sHSP cloned from D. antiqua, which is named DaHSP23 (GenBank accession no. HQ392521.1), is 904 bp in length, encoding 186 amino acids with a molecular mass of 20.9 kDa and a theoretical isoelectric point of 6.42. DaHSP23 shares more than 66% amino acid identity with the known HSPs of other dipteran insects and is homologous with the known HSP23 genes of other dipteran insects. Genome sequencing around the gene revealed that it is intronless. The expression of DaHSP23 was up-regulated in both SD and WD pupae, with higher expression level detected in the late maintenance stage, and down-regulated when those diapauses were terminated. 【Conclusion】 The patterns of HSP23 regulation are substantially different between different developmental stages of both SD and WD, and its regulation in diapause might be species-specific. DaHSP23 might play an essential role in these two types of diapauses in D. antiqua.

Key words:  Delia antiqua, diapause, heat shock protein (HSP), gene cloning, expression pattern, real-time qPCR