[目的]本研究旨在克隆沙葱萤叶甲Galeruca daurica热激蛋白Hsp70基因,并对其进行序列和表达模式分析,探讨该基因在沙葱萤叶甲生长发育及响应温度胁迫方面的作用.[方法]采用RT-PCR和RACE技术从沙葱萤叶甲2龄幼虫中克隆Hsp70基因,并进行生物信息学分析;用WoLF PSORT在线软件进行亚细胞定位预测;采用实时荧光定量PCR检测该基因在沙葱萤叶甲成虫不同组织(头、胸和腹)中、不同发育阶段(卵、1-3龄幼虫、蛹、雌雄成虫)、不同温度(-14,-10,-5,0,5,10,15,20,25和30℃)处理1h的2龄幼虫及0℃下分别处理0 min,15 min,30min,1h,1.5h,2h和3h时卵中的相对表达量.[结果]克隆获得一个沙葱萤叶甲Hsp70基因并命名为GdHsp70(GenBank登录号:KY460462),该基因全长2 340 bp,开放阅读框(ORF)1 899 bp,编码632个氨基酸,预测蛋白质分子量为70.12 kD,等电点(pI)为4.79,无跨膜区,无信号肽.蛋白质亚细胞定位预测该蛋白主要位于细胞质内.蛋白质结构域分析表明,GdHsp70有3个功能保守区.同源比对与系统进化分析表明,GdHsp70与分类学关系上较为接近的昆虫的同源蛋白间有较高的相似度.组织特异性和不同发育阶段表达分析表明,GdHsp70在沙葱萤叶甲成虫胸部和卵期表达量最高;高温和低温胁迫均能诱导沙葱萤叶甲2龄幼虫体内GdHsp70的表达,其中-10℃处理1h表达量最高;0℃低温处理卵15 min至3h后均能诱导GdHsp70不同程度的表达上调,其中处理1h上调幅度最大.[结论]沙葱萤叶甲GdHsp70与该虫的生长发育相关,并对高低温胁迫的响应有重要作用.%[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 1 st-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 upregulated 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.
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