桔小实蝇二硫键异构酶基因的克隆及其发育表达  

Cloning and developmental expression pattern analysis of PDI in Bactrocera dorsalis(Hendel)

作　　者：胡黎明[1] 申建梅[1] 胡美英[2] 宾淑英[1] 黄华枝[3] 廖泓之[1] 林进添[1] 

机构地区：[1]仲恺农业工程学院植物保护系/外来有害生物预警与控制研究所,广州510225 [2]华南农业大学资源环境学院/天然农药与化学生物学教育部重点实验室,广州510642 [3]广东省广州市园林科学研究所,广州510405

出　　处：《华中农业大学学报》2012年第4期457-462,共6页Journal of Huazhong Agricultural University

基　　金：国家自然科学基金项目(31171852);广东省大学生创新实验项目(1134711022);仲恺农业工程学院科研基金项目(G3100003)

摘　　要：利用RT-PCR和RACE方法克隆获得桔小实蝇Bactrocera dorsalis(Hendel)二硫键异构酶(pro-tein disulfide isomerase,PDI)基因的cDNA序列,命名为BdorPDI。测序结果表明,BdorPDI开放阅读框全长1 497bp,编码498个氨基酸。氨基酸序列结构分析表明,该序列有PDI蛋白家族的典型特征:N端含有信号肽序列;在序列的N端及C端具有二硫键/巯基氧化还原位点CGHC;在C末端含有内质网滞留信号肽KDEL。进化树分析表明:桔小实蝇的PDI蛋白序列与脊椎动物安乐蜥(Anolis carolinensis)的序列(XP_003217370)一致性最低,为49.3%;与双翅目昆虫刺舌蝇(Glossina morsitans)的序列(ADD20271)一致性最高,为76.3%。荧光定量PCR分析表明:BdorPDI mRNA在桔小实蝇1～3龄幼虫中的表达量相对较低,且呈逐渐增长的趋势;在1d蛹中的BdorPDI mRNA表达量达到最高峰,其表达量是基准含量的536.50倍,且随着蛹的发育,BdorPDI mRNA表达量呈逐渐降低的趋势。由此可见,桔小实蝇二硫键异构酶在化蛹过程中发挥了重要的生理功能。Protein disulfide isomerases （PDI） play in the protein folding, assembly, and posttrans- lational modification. In present study, a protein disulfide isomerase （PDI） cDNA sequence from Bactrocera dorsalis （BdorPDI） was cloned using the reverse transcription PCR （RT-PCR） compound with rapid amplification cDNA ends （RACE） method. The open reading frame （ORF） of BdorPDI is 1 497 bp in length and the deduced peptide contains 498 amino acids residues. Primary structure of the sequence reveals the typical characteristics of PDI family： signal peptide in the N-terminal, two CGHC active-site sequence motif in the N- and C- terminal, and retention signal KEDL at its C-end. Phylogenetic analysis show that Bactrocera dorsalis PDI shared the lowest sequence identity （49.3%） with PDI（XP_ 003217370） from Anolis carolinensis, and the highest sequence identity （76.3%） with PDI （ADD20271） from Glossina morsitans. Real-time PCR results indicated that BdorPDI expressed during whole developmental stages. The relative expression level in larvae was lower but gradually increased during the developing from 1-instar larva to 3-instar larva. The expression level in 1 d- old pupa reach a peak and the amount is about 536.50 folds of that in the baseline. The amount of the BdorPDI mRNA gradually decreases during the pupa development. The results implied that Bactrocera dorsalis PDI play an important physiological role in the pupation procession.

关 键 词：桔小实蝇 二硫键异构酶 基因克隆 实时定量PCR 

分 类 号：Q966[生物学—昆虫学] Q969.512.4