家蚕 BmChd64 基因的克隆与表达定位分析

Cloning,expression analysis and subcellular localization of BmChd64 in silkworm,Bombyx mori

Chd64是含有钙结合蛋白同源(Calponin homology,CH)结构域的蛋白,在果蝇蜕皮激素与保幼激素信号通路中发挥重要作用。本研究以家蚕Bombyx mori为研究对象,利用PCR克隆了与果蝇Drosophila melanogaster DmChd64同源的BmChd64基因,与表达载体pET-28a连接后,成功获得体外原核表达的BmChd64蛋白,并对其进行了亚细胞定位分析。家蚕BmChd64基因开放阅读框(ORF)的序列为567 bp,编码188个氨基酸,预测分子量大小为20.9 kDa,理论等电点为8.41,编码的蛋白在第27~130个氨基酸处存在CH结构域。同源性比对与进化分析显示,BmChd64与赤拟谷盗Tribolium castaneum和果蝇的Chd亲缘关系较近。qRT-PCR结果显示,BmChd64在家蚕5龄游走期的不同组织中均有表达,且在翅原基中的表达趋势与家蚕体内蜕皮激素(20-hydroxyecdysone,20E)滴度变化规律一致。亚细胞定位结果显示,BmChd64在细胞核与细胞质中均有分布,细胞核内荧光信号较强,且在细胞核外围较弱,推测细胞质中BmChd64也可入核,最终定位在细胞核中。研究结果表明BmChd64可能主要通过20E信号通路调控翅原基等的变态发育,这为进一步完善20E调控昆虫变态发育的分子机制提供了实验基础。

Chd64 is a protein containing calponin homology(CH)domain,which plays a key role in the signal pathway of JH and 20E in Drosophila melanogaster.In this study,the BmChd64 gene,which was homologous to DmChd64 of D.melanogaster,was cloned by PCR.After connecting with the expression vector pET-28a,the prokaryotic expression of BmChd64 protein in vitro was successfully obtained and its subcellular location analysis were carried out in Bombyx mori.The sequence of the open reading frame(ORF)of the BmChd64 gene was 567 bp,which encoded 188 amino acids,the predicted molecular weight was 20.9 kDa and the theoretical isoelectric point was 8.41.The encoded protein had a CH domain at the 27^(th) to 130^(th) amino acids.The homology comparison and evolutionary analysis showed that BmChd64 was closely related to Tribolium castaneum and D.melanogaster.The results of qRT-PCR showed that the gene was expressed in different tissues at wandering stage of the 5^(th) instar larvae of the silkworm,and the expression trend of BmChd64 in the wing discs were consistent with the change of 20E titer in silkworm.The subcellular localization results showed that BmChd64 was distributed both in the nucleus and the cytoplasm.The fluorescence signal in the nucleus was strong and it was weaker in the periphery of the nucleus.It was speculated that BmChd64 which was located in the cytoplasm could enter the nucleus and finally locate in the nucleus.These data indicated that BmChd64 may mainly regulate the metamorphosis of the wing discs through the 20E signaling pathway,providing an experimental basis for further improving the molecular mechanism of 20E regulating insect metamorphosis.