蓖麻蚕AGO1蛋白基因的克隆及序列与表达特征  

Cloning,Sequence Feature and Expression Pattern of Argonaute Gene 1 from Philosamia cynthia ricini

作　　者：何庆玲[1] 张月华[1,2] 钱荷英[1,2] 罗旭芳[1] 李刚[1,2] 孙平江[1,2] 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》2014年第5期824-829,共6页ACTA SERICOLOGICA SINICA

基　　金：现代农业产业技术体系建设专项(No.CARS-22)

摘　　要：小RNA介导的基因表达沉默机制在昆虫中非常保守。作为RNA诱导沉默复合体(RISC)的关键组分,Argonaute(AGO)蛋白家族成员在RNA干扰途径中起着关键性作用。以蓖麻蚕(Philosamia cynthia ricini)品种海蓝的幼虫头部cDNA为模板,利用RT-PCR和RACE技术克隆了一个编码AGO蛋白的基因,将该基因命名为pcr-AGO1(GenBank登录号:KJ676101)。该基因cDNA全长3 490 bp,包括182 bp的5'-UTR和572 bp的3'-UTR,ORF为2 736 bp,编码911个氨基酸,蛋白质分子质量为101.492 3 kD,等电点为9.31,蛋白质序列中含有AGO家族成员典型的PAZ结构域和PIWI结构域。实时荧光定量PCR检测pcr-AGO1基因在蓖麻蚕的幼虫期、蛹期、蛾期和卵期以及幼虫的头部、脂肪体、表皮、丝腺、中肠和血液等组织器官中均有表达。其中,在卵期的表达量最高,幼虫期最低,并且在除蚁蚕外的整个幼虫期的表达量没有太大变化;在幼虫各个组织中的表达量以表皮最高,中肠次之,在血液和头部的表达量都较低。研究结果提示,该基因可能在蓖麻蚕的整个发育期都发挥相应的功能,并可能参与幼虫表皮细胞的生长调控。Silencing of gene expression mediated by small RNA is a conserved mechanism in insects. As the core component of RNA-induced silencing complex（ RISC）,the Argonaute（ AGO） family proteins play a crucial role in RNA interference（ RNAi） pathway. An eri-silkworm（ Philosamia cynthia ricini） Argonaute family gene was cloned using cDNA from larval head of eri-silkworm variety Hailan as template by means of RT-PCR and RACE,and was named pcr-AGO1（ GenBank accession No. KJ676101）. Full length cDNA of this gene was 3 490 bp,including 182 bp 5＇-UTR and 572 bp 3＇-UTR. Its ORF was 2 736 bp and encoded a protein of 911 amino acids with molecular weight of 101. 492 3 kD and isoelectric point of 9. 31. The deduced protein contained two typical conserved domains of the AGO family,i. e. PAZ and PIWI domains. Fluorescent quantitative PCR revealed that pcr-AGO1 gene was expressed in various developmental stages of eri-silkworm including larval,pupal,adult and egg stages,and in various tissues of eri-silkworm larvae including head, fat body, epidermis, silk gland, midgut and hemolymph. Among various developmental stages,egg stage and larval stage had the highest and the lowest expression level respectively. The expression of pcr-AGO1 gene was not greatly varied throughout the larval stage except newly hatched larvae. Among various tissues,epidermis had the highest expression level followed by mid-gut. Both hemolymph and head had low expression level. These results suggest that pcr-AGO1 gene plays roles in all developmental stages of eri-silkworm and is involved in the regulation of epidermal cell growth.

关 键 词：蓖麻蚕 AGO家族蛋白 基因克隆 序列特征 表达谱 实时荧光定量PCR 

分 类 号：S885.2[农业科学—特种经济动物饲养] Q786[农业科学—畜牧兽医]