葱蝇热激蛋白DaHSP23基因的克隆及在冬滞育和夏滞育蛹中的表达分析（英文）

Cloning and expression profiling of heat shock protein DaHSP23 gene in the winter and summer diapause pupae of the onion maggot,Delia antiqua( Diptera: Anthomyiidae)

【目的】低分子量（12~43 k Da）热激蛋白（s HSPs）具有抗逆应答的功能,滞育是昆虫抵抗不良环境的特殊发育形式,但s HSPs在昆虫滞育发育过程中的作用仍不清楚。本研究克隆和特征化葱蝇Delia antiqua s HSP基因,并研究它在夏滞育和冬滞育发育过程中的表达模式,为阐明s HSPs在滞育发育上的功能奠定基础。【方法】通过RACE-PCR方法克隆了葱蝇HSP23基因,通过相似性比较分析了其特征、结构域及与双翅目代表性同源基因的系统发育关系;采用实时荧光定量PCR研究了该基因在葱蝇冬滞育蛹和夏滞育蛹发育过程中的表达情况,通过表达的差异比较揭示了该基因与滞育发育的关系。【结果】克隆出了葱蝇HSP23基因,命名为Da HSP23（Gen Bank登录号：HQ392521.1）,其c DNA全长序列为904 bp,编码186个氨基酸,推测蛋白分子量为20.9 k Da,等电点为6.42。该基因的编码蛋白与其他双翅目昆虫的s HSPs有超过66%的氨基酸序列一致性,与已报道的其他双翅目昆虫的滞育相关HSP23基因同源。基因组测序显示该基因无内含子。Da HSP23基因在葱蝇非滞育蛹的发育过程中一直保持在较低的水平,各发育阶段间的表达量不存在显著差异。但在冬滞育和夏滞育蛹中,该基因从滞育起始期开始逐渐显著升高表达,到滞育维持期的中后期达到峰值,在滞育终止期逐渐降到较低的水平。【结论】Da HSP23基因在葱蝇冬滞育和夏滞育发育过程中明显上调表达,但存在差异,它在滞育期的调控可能是种专化的。Da HSP23可能在葱蝇两种类型的滞育上起重要作用。

[ 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.