仿刺参体壁创伤修复消减文库的构建与分析  

Construction and analysis of subtractive cDNA library of recovery body wall in sea cucumber Apostichopus japonicus

作　　者：秦艳杰[1] 李霞[1] 张慧敏[1] 王雪[1] 

机构地区：[1]大连海洋大学农业部北方海水增养殖重点实验室辽宁省海洋生物资源与生境恢复重点实验室,辽宁大连116023

出　　处：《大连海洋大学学报》2013年第3期224-229,共6页Journal of Dalian Ocean University

基　　金：辽宁省教育厅创新团队项目(2007T015);辽宁省教育厅实验室专项(2008S064)

摘　　要：应用抑制性消减杂交技术(SSH),构建了仿刺参Apostichopus japonicus(体质量为65～90 g)正常体壁及创伤修复(24、48、72、96、120 h后)体壁的消减cDNA文库,利用PCR和斑点杂交技术对文库进行筛选,随机挑取的768个克隆中发现292个阳性克隆,对其中信号强度较强的224个阳性克隆进行测序,得到208个有效EST序列。经BlastX工具对获得的EST与GenBank数据库进行比对分析,结果有171个EST序列与数据库中的基因同源(e≤0.001,相似性>40%),其中153个为未知基因,18个为已知功能或已命名基因,包括在创伤及修复的体壁中上调表达的β微管蛋白、微管蛋白α-1链、肌动蛋白、肌动蛋白ike 7B类似物、细胞色素c氧化酶亚基Ⅰ、tRNA假尿苷合成酶A、GTP酶、细胞分裂周期2类似蛋白、有丝分裂原活化蛋白激酶、homeobox蛋白、延伸因子1A、核糖体蛋白L30、核糖体蛋白L17、60S酸性核糖体蛋白P0、26S蛋白酶调节亚基、泛素特异性肽酶24、大肠癌血清抗原3、清道夫受体蛋白12等。本研究结果可为探讨刺参体壁再生过程和分子机理,以及筛选刺参体壁创伤修复过程中相关功能基因的研究提供基础依据。A subtracted eDNA library of sea cucumber Apostichopusjaponicus（body weight 65-90 g） was construeted by suppression subtractive hybridization technology （SSH） to screen EST associated with recovery body wall. The eDNA library of the test group has been constructed by the mRNA of the body walls 24, 48, 72, 96 and 120 h after the operation, and those with no operation as the control group. Differential EST from the subtracted eDNA library have been identified by both PCR technology and dot blot hybridization. Two hundred and ninety-two positive clones were observed from total 768 clones, and 224 positive ones were sequenced. Two hundred and eight EST were found and analyzed by BlastX tool in GenBank database, in which 171 EST were homologous with sequences in the database, and 18 were high homologous with known function genes including： beta-tubulin, tubulin alpha-1 chain, actin, tRNA pseudour/dine synthase A, GTPase, cell division cycle 2-like, Mitogen-activated protein kinase, homeobox protein, elongation factor 1 A, ribosomal protein L30 and L17, 60S acidic ribosomal protein P0, 26S protease regulatory subunit, ubiquitin specific peptidase 24, serologically defined colon cancer antigen 3 and scavenger receptor cysteine-rieh protein type 12 which upregulated in wounded and recovery body walls of the sea cucumber. In this study, the subtracted cDNA library was successfully constructed by SSH and a number of EST related to recovery body wall were identified, which laid a foundation for the molecular basis and the process of regeneration of body wall in the sea cucumber.

关 键 词：刺参 体壁 抑制性消减杂交 CDNA文库 创伤和修复 

分 类 号：Q785[生物学—分子生物学] S917[农业科学—水产科学]