蓖麻蚕乙酰胆碱酯酶基因Pcrace2的克隆及序列与表达分析  

Molecular Cloning,Sequence Feature and Expression Pattern of Acetylcholinesterase Gene from Philosamia cynthia ricini

作　　者：刘娟[1,2] 张月华[1,2] 张华光[1] 高丽[1] 钱荷英[2] 孙平江[2] 徐安英[1,2] 

机构地区：[1]江苏科技大学,江苏镇江212003 [2]中国农业科学院蚕业研究所,江苏镇江212018

出　　处：《蚕业科学》2013年第3期460-466,共7页ACTA SERICOLOGICA SINICA

基　　金：现代农业产业技术体系专项(No.CARS-22)

摘　　要：昆虫乙酰胆碱酯酶(acetylcholinesterase,AChE,EC 3.1.1.7)是有机磷和氨基甲酸酯类杀虫剂的作用靶标。利用RT-PCR和RACE技术从蓖麻蚕(Philosamia cynthia ricini)幼虫头部组织克隆了乙酰胆碱酯酶基因Pcrace2。该基因cDNA全长为2 051 bp,包含1 497 bp的ORF,编码498个氨基酸,蛋白质的分子质量为56.25 kD,等电点(pI)为5.11,含有由23个氨基酸组成的前体信号肽,推测为分泌型蛋白质。基于PcrAChE2同源氨基酸序列的系统进化分析表明:蓖麻蚕与小菜蛾(Plutella xy-lostella)、棉铃虫(Helicoverpa armigera)、烟青虫(Helicoverpa assulta)的亲缘关系最近,与家蚕(Bombyx mori)和野桑蚕(Bombyxmandarina)的亲缘关系也比较近。利用Real-time PCR方法分析Pcrace2基因在蓖麻蚕5龄第3天幼虫的头部、脂肪体、丝腺、中肠和血液中均有表达,以头部的表达量最高;在蓖麻蚕不同发育时期,Pcrace2在蛾期的表达量明显高于其它发育时期,在5龄幼虫期的表达量最低。研究结果有助于进一步分析蓖麻蚕乙酰胆碱酯酶基因的功能。Insect acetylcholinesterase（AChE,EC 3.1.1.7） is the target of organophosphorus and carbamate insecticides.An eri-silkworm（Philosamia cynthia ricini） acetylcholinesterase gene Pcrace2 was cloned from head tissue of eri-silkworm larva by means of RT-PCR and RACE.Full-length cDNA of this gene is 2 051 bp long with an ORF of 1 497 bp that encodes 498 amino acids.The encoded protein is predicted to have a molecular weight of 56.25 kD and pI of 5.11.The protein has a signal peptide precursor of 23 amino acids and is predicted to be a secretory protein.Phylogenetic analysis based on homologous amino acid sequences showed that Philosamia cynthia ricini shared the closest genetic relationship with Plutella xylostella,Helicoverpa armigera,and Helicoverpa assulta.It also had close genetic relationship with Bombyx mori and Bombyx mandarina.Real-time PCR analysis showed that Pcrace2 gene was expressed in head,fat body,silk gland,midgut and hemolymph of day 3 eri-silkworm larvae of the 5th instar with the highest expression level in head.Among various developmental stages of eri-silkworm,transcriptional level of Pcrace2 was obviously higher at moth stage than all other stages with the lowest level at the 5th instar larval stage.The obtained results would facilitate further studies on function of Philosamia cynthia ricini acetylcholinesterase gene.

关 键 词：蓖麻蚕 乙酰胆碱酯酶 基因克隆 序列特征 表达谱 实时定量PCR 

分 类 号：S885.2[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]