意大利蜜蜂甘油醛-3-磷酸脱氢酶基因amGAPDH2的cDNA克隆与序列分析

Cloning and Sequence Analysis of am GAPDH2 Gene from Apis mellifera

应用RT-PCR技术克隆了意大利蜜蜂(Apis mellifera)甘油醛-3-磷酸脱氢酶基因amGAPDH2,利用MEGA5.1、DNAMAN、PredictProtein和I-TASSER等软件对该基因进化关系以及其所对应的蛋白的同源性、理化性质和结构进行了预测和分析。从意大利蜜蜂cDNA文库中克隆得到了长1188 bp的amGAPDH2序列,GenBank登录号为MH152402。该序列具有完整的开放阅读框(ORF,114~1115 bp),其编码333个氨基酸。该基因编码的氨基酸序列与其它昆虫的GAPDH有较高的序列相似性(80%以上),系统进化分析表明amGAPDH2与中华蜜蜂、小蜜蜂的序列相似性最高。利用ProtParam等软件对amGAPDH2编码的蛋白质分析结果显示,amGAPDH2属于不稳定、亲水性蛋白;二级结构属于混合型,Helix占25.53%、Strand占24.62%、Loop占49.85%;am GAPDH2具有两个保守结构域:一个是N端的NAD(P)结合结构域,另一个是C端的催化结构域Gp_dh_C。该研究为后期amGAPDH2基因的生理功能研究提供了理论依据。

The full-length cDNA of the am GAPDH2 of Apis mellifera was cloned by RT-PCR technology,then a lot of softwares such as MEGA5.1,DNAMAN,PredictProtein and I-TASSER was used to predict and analyze the evolutionary relationship of this gene and the homology,physical and chemical properties and structure of the corresponding protein.The 1188 bp long amGAPDH2 sequence was cloned from Apis mellifera cDNA library,and the GenBank accession numbers is MH152402.The sequence has a complete open reading frame(ORF,114~1115 bp),which encodes 333 amino acids.The amino acid sequence encoded by this gene is highly similar to the GAPDH sequence of other insects.Phylogenetic analysis showed that the amGAPDH2 has the highest homology with the Apis cerana and Apis florea.Using ProtParam and other software to analyze the protein encoded by amGAPDH2,the result showed that it was an unstable hydrophilic protein.The secondary structure belongs to the mixed type,and Helix is 25.53%,Strand is 24.64%,Loop is 49.85%.The GAPDH2 has two conserved domains:One is a NAD(P)binding domain at the N-terminus,the other is catalytic domain Gp_dh_C at the C-terminal.This research laid the foundation for the study of the physiological function of the amGAPDH2 gene.