中、意蜂Atg12基因克隆表达及生物信息学分析

试验旨在探究自噬相关基因Atg12(autophagy-related 12 gene)在中华蜜蜂(中蜂)、意大利蜜蜂(意蜂)中的表达差异,为探讨中蜂抗螨分子机理提供参考。参照GenBank中东方蜜蜂Atg12基因序列(登录号:XM_017048509.1)设计引物,扩增中、意蜂头部组织Atg12基因,并对其进行克隆、测序、原核表达及其氨基酸序列和蛋白结构分析,同时采用实时荧光定量PCR技术分析该基因在中、意蜂头部组织中的表达差异。结果显示,本试验成功扩增出大小为450 bp的目的片段,并表达出大小约42 ku重组蛋白;遗传进化树分析表明,在Atg12基因进化过程中,中蜂(Apis cerana cerana)、意蜂(Apis mellifera)、大蜜蜂(Apis dorsata)和小蜜蜂(Apis florea)亲缘关系较近;重组蛋白生物信息学分析显示,该蛋白的二级结构中共含有6个多肽结合位点、6个β-折叠和4个α-螺旋,分子质量约为16.13 ku,等电点为6.73;实时荧光定量PCR结果显示,Atg12基因在中蜂头部组织中表达极显著高于意蜂(P<0.01)。本试验结果为后续深入研究Atg12基因在中蜂抗螨上的作用机制提供了参考。

Cloning,Expression and Bioinformatics Analysis of Atg12 Gene in Apis cerana cerana and Apis mellifera

The purpose of this study was to investigate the expression differences of autophagy-related 12 (Atg12) gene in Apis cerana cerana and Apis mellifera,and provide references for studying the mechanism of Apis cerana cerana resitance to Varroa destructor.The primers were designed according to the Atg12 gene sequence of Apis cerana in GenBank (accession No.:XM_017048509.1).The Atg12 gene was amplified from the head of Apis cerana cerana and Apis mellifera,then was cloned,sequenced and expressed prokaryoticly,and the amino acid sequence and protein structure were analyzed.Meanwhile,Real-time PCR was used to analyze the different expression of Atg12 gene in head tissues of Apis cerana cerana and Apis mellifera.The results showed that the target fragment size was 450 bp and the recombinant protein size was about 42 ku.Genetic phylogenetic tree analysis showed that the relationship among Apis cerana cerana,Apis mellifera,Apis dorsata and Apis florea was closer in the evolution of Atg12 gene.Bioinformatics analysis of the recombinant protein showed that the secondary structure of the protein contained 6 polypeptide binding sites,6 β-sheets and 4 α-helices,the molecular weight was about 16.13 ku,and the isoelectric point was 6.73.The results of Real-time PCR showed that the expression of Atg12 gene in the head tissue of Apis cerana cerana was extremely significantly higher than that of Apis mellifera.The results of this study provided references for further study on the mechanism of action of Atg12 gene in Apis cerana cerana resitance to Varroa destructor.