家蚕耐氟近等基因系SSH文库的构建及部分差异表达基因的分析  

Construction of SSH cDNA Library and Analysis of Differentially Expressed Genes of Near-isogenic Lines of Dominant Endurance to Fluoride(Def) Gene in the Silkworm,Bombyx mori

作　　者：王小强[1] 徐安英[2] 李刚[1] 张月华[2] 钱荷英[2] 孙平江[2] 郭锡杰[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》2011年第1期45-51,共7页ACTA SERICOLOGICA SINICA

基　　金：现代农业产业技术体系建设专项(蚕桑);国家重点基础研究发展计划"973"项目(No.2005CB121004);江苏省科技项目(No.BG2007322)

摘　　要：为了获取与家蚕耐氟性状相关的基因信息,利用家蚕耐氟品种T6和氟化物敏感品种733新建立耐氟近等基因系,以回交11代的家蚕耐氟近等基因系群体中的耐氟个体和敏感个体的中肠为材料,构建家蚕耐氟近等基因系抑制消减杂交(SSH)cDNA文库。通过对抑制消减杂交cDNA文库中随机挑选的153个阳性克隆测序和聚类拼接,得到19个重叠群(con-tig)、47个已知功能基因(un igene)和28个未知功能基因。获得的表达序列标签(EST)经BLASTx比对和同源性分析,初步发现这些基因参与家蚕体内物质转运、能量代谢、基因转录、蛋白质合成与降解、蛋白质加工、信号转导、机体免疫防御、细胞结构形成等诸多生物学过程。采用实时定量RT-PCR方法,对部分基因在家蚕耐氟近等基因系群体中的耐氟个体和敏感个体的不同组织的表达进行定量分析,发现磷酸根离子转运蛋白基因、三磷酸腺苷(ATP)合成酶基因、液胞型H+-ATP酶基因、脂肪酶基因以及主要过敏原蛋白基因在耐氟个体的中肠、马氏管、脂肪体组织中的表达量有较明显上调。To obtain information about dominant endurance to fluoride （Def） gene of the silkworm （ Bombyx mori）, silkworm near-isogenic lines （NILs） of Def gene were constructed using silkworm strain 733 Xin （sensitive to fluoride） and T6 （endurable to fluoride）. An SSH （suppression subtractive hybridization） cDNA library of silkworm NILs of Def gene was constructed using larval midguts of the individuals sensitive to fluoride and the individuals endurable to fluoride from the 11th back-cross generation. Sequencing and cluster assembly of 153 positive clones randomly selected from the constructed SSH cDNA library yielded 19 contigs, 47 genes with known functions （unigenes） and 28 genes with unknown functions. BLASTx searches and homology analysis with existing expressed sequence tags （ESTs） revealed that these genes were involved in various biological processes including substance transportation, energy metabolism, gene transcription, protein synthesis and degradation, protein processing, signal transduction, organismal immunological defense and formation of cellular structure. Real-time quantitative PCR analysis on expression of selected genes in various tissues from indi-viduals sensitive to fluoride and the individuals endurable to fluoride in silkworm NIL population of Def gene revealed that genes encoding phosphate transport protein, ATP synthase, vacuolar-type H^＋ATPase, lipase and major allergen protein were obviously up-regulated in midgut, Malpighian tubule and fat body of individuals which were endurable to fluoride.

关 键 词：家蚕 耐氟性状基因 抑制性消减杂交 同源性分析 实时定量RT-PCR 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]