沙葱萤叶甲GdHsp70-2,GdHsp70-3的克隆鉴定与表达谱分析

Cloning,Identification and Expression Profiling Analysis of GdHsp70-2,GdHsp70-3 Genes in Galeruca daurica

为明确沙葱萤叶甲Galeruca daurica热激蛋白70(Heat shock protein 70,Hsp70)基因的序列结构及其系统进化关系,本研究通过PCR技术克隆沙葱萤叶甲Hsp70基因cDNA全长序列与基因组序列,并进行生物信息学与表达谱分析。结果显示,克隆获得了沙葱萤叶甲2条Hsp70基因GdHsp70-2(GenBank登录号:MZ853083)和GdHsp70-3(Genbank登录号:OK585088),基因全长分别为2410 bp和2242 bp,各自编码657和646个氨基酸,均含有3个保守的HSP70家族特征序列,预测蛋白三维结构均由N-端ATPase功能域和C-端底物结合功能域所组成;系统发育分析表明GdHSP70-2,GdHSP70-3分别与松墨天牛(Monochamus alternatus)MaltHSC70-1、玉米根萤叶甲(Diabrotica virgifera virgifera)DvirHSP70-2的亲缘关系最近;基因组DNA克隆获得GdHsp70-2的两段内含子序列,GdHsp70-3则不含有内含子;表达谱分析结果表明GdHsp70-2,GdHsp70-3均在蛹期的表达量最高,在成虫中分别在翅和足的表达量最高。研究有助于加深对沙葱萤叶甲Hsp70基因结构及其进化关系的了解,并为后续深入开展功能研究提供理论依据。

To identify the gene structure of the heat shock protein 70(Hsp70)of Galeruca daurica and further explore their phylogenetic relationship.Polymerase chain reaction(PCR)was used to clone the full-length cDNA and genomic sequence of Hsp70 gene from G.daurica.The sequence was subjected to bioinformatical and expression patterns analysis.The results showed that the two complete cDNA sequences were obtained and named GdHsp70-2(GenBank accession No.:MZ853083)and GdHsp70-3(GenBank accession No.:OK585088).Their full-length cDNA were 2410 and 2242 bp in length,encoding 657 and 646 amino acids,containing three highly conserved heat shock protein 70 family regions,and the 3D structure of the two HSP70 were composed of the N-terminal ATPase functional domain and C-terminal substrate binding domain,respectively.The phylogenetic tree showed that GdHSP70-2 and GdHSP70-3 have the highest amino acid sequence identity with MaltHSC70-1 of Monochamus alternatus,and DvirHSP70-2 of Diabrotica virgifera virgifera.The genomic sequence cloning results displayed that GdHsp70-2 has two introns,and the GdHsp70-3 contains no intron in the gene.Expression profile analysis showed that the expression levels of GdHsp70-2 and GdHsp70-3 in pupae were significantly higher than that in other developmental stages,and the relative expression level in wings and feet was the highest in adults.These findings improve the understanding of gene structure and its evolutionary relationships of G.daurica HSP70 family genes and provide a theoretical basis for gene function studies.