春尺蠖热激蛋白AcinHsp70基因的克隆、分子特性与表达分析

Cloning,molecular characteristics and expression analysis of heat shock proteins AcinHsp70 in Apocheima cinerarius(Lepidoptera:Geometridae)

【目的】克隆春尺蠖热激蛋白Hsp70基因,分析其理化性质与表达情况,探究该基因在春尺蠖应对外界不利环境中的作用。【方法】基于前期所测转录组数据(春尺蠖幼虫不同低温胁迫),克隆获取Hsp70基因,命名为AcinHsp70(GenBank登录号:OP750249);利用生物信息分析软件对Hsp70基因的理化性质进行分析;采用qPCR技术检测AcinHsp70基因在不同温度(-10、-5、0、5和25℃)胁迫中回温与不回温及4℃不同时间处理下的表达谱。【结果】春尺蠖AcinHsp70基因完整CDS序列全长1899 bp,编码633个氨基酸,蛋白质预测分子量为69.91 kDa,预测等电点为5.51。在N端均不含信号肽且无跨膜区。磷酸位点检测分别发现丝氨酸、苏氨酸和酪氨酸磷酸位点24、26和8个;未发现糖基化位点。该蛋白的平均亲水性为-0.507,预测不稳定系数为33.19,故该蛋白属于亲水稳定蛋白,亚细胞定位结果显示,其主要位于细胞质。序列一致性比对与系统进化树分析结果显示,春尺蠖与庆网蛱蝶(Melitaea cinxia)亲缘关系最近,序列一致性达92.91%。基因表达结果显示,不同温度(-10℃除外)胁迫下,AcinHsp70基因表达量均无明显差异,回温后表达量显著上调。AcinHsp70基因表达量总体呈先升后降趋势,在4℃处理1 h后表达量达到最大值。【结论】AcinHsp70基因与春尺蠖应对低温胁迫的响应密切相关,结果有助于揭示该基因在低温胁迫下的分子功能。

【Objective】The purpose of the study was to clone heat shock protein Hsp70 gene from Apocheima cinerarius,analyze its physico⁃chemical properties and expression,and explore the role of this gene in coping with the adverse environment of A.cinerarius.【Method】In the study,Hsp70 was cloned and named as AcinHsp70(GenBank entry number:OP750249)based on the transcriptome data(under differ⁃ent low temperature stress of A.cinerarius larvae).The physical and chemical properties of Hsp70 gene were analyzed by using bioinformation analysis software.qPCR was used to detect the expression profiles of AcinHsp70 under different temperature stresses(-10,-5,0,5 and 25℃)and under different time treatments at 4℃.【Result】The total length of CDS of AcinHsp70 sequence was 1899 bp,encoding 633 a⁃mino acids,the molecular weight of protein was 69.91 kDa,and the isoelectric point was 5.51.The N⁃terminus of the protein lacked a sig⁃nal peptide and transmembrane region.Phosphorylation sites were identified at serine,threonine and tyrosine residues(24,26,and 8 sites respectively).No glycosylation sites were observed.The average hydrophilicity of the protein was-0.507,with a predicted instability coef⁃ficient of 33.19,indicating its stability as a hydrophilic protein.Subcellular localization analysis revealed predominant cytoplasmic distribu⁃tion.The results of sequence comparison and phylogenetic analysis showed that the A.cinerarius was closely related to Melitaea cinxia,and the sequence identity reached 92.91%.Gene expression results showed that AcinHsp70 expression was not significantly different under different temperature(except for-10℃)stress,and the expression was significantly up⁃regulated after recovery.The expression level of the AcinHsp70 exhibited an initial increase followed by a subsequent decrease,with maximum expression after 1 hour treatment at 4℃.【Conclu⁃sion】AcinHsp70 is closely related to the response of A.cinerarius to low temperature stress,and the results help to reveal the molecular func⁃tion of Acinhsp70 under low temperature stress.