南移养殖的刺参(Apostichopus japonicus)cDNA文库的构建及原肌球蛋白基因的研究  

CONSTRUCTION OF cDNA LIBRARY WITH APOSTICHOPUS JAPONICUS CULTURED IN THE SOUTH AND SEQUENCE ANALYSIS OF ITS TROPOMYOSIN GENE

作　　者：崔静[1] 李太武[2] 苏秀榕[1] 李晔[1] 徐静[3] 周君[1] 应琪[1] 王中华[1] 李松伟[1] 

机构地区：[1]宁波大学生命科学与生物工程学院,宁波315211 [2]宁波城市职业技术学院,宁波315100 [3]温州职业技术学院,温州325035

出　　处：《海洋与湖沼》2010年第6期850-856,共7页Oceanologia Et Limnologia Sinica

基　　金：国家农业科技成果转化资金;2007GB2C220359号;浙江省重大科技专项(优先主题)重大农业项目;2008C02009-Ⅱ-2号;宁波市农业科技成果转化资金;2007C30001号

摘　　要：以南移养殖的刺参为实验材料,采用非均一化的Oliga-dT引物定向克隆技术构建了南移刺参混合组织的cDNA文库。对文库质量的分析结果表明,cDNA文库的库容量为2.2×107cfu,重组率达95.8%,平均插入片段长度750bp左右。挑取cDNA克隆进行5’端测序,成功测序185个反应,分析得到136个单基因簇(Unigene),其中包括40个重叠群(Contigs)、96个单拷贝EST(Singletons)。结果表明,克隆南移养殖的刺参原肌球蛋白(tropomyosin,Trp)cDNA全长序列为1112bp,ORF编码284个氨基酸,其预测蛋白的分子量为33.27kDa,等电点为4.56。该氨基酸序列与紫石海胆、南极大磷虾、锯缘青蟹、文昌鱼、河蟹、家蚕同源性分别为62%、51%、46%、49%、47%、46%。Based on the construction technology of non-normalized cDNA library, the cDNA libraries of Apostichopus japonicus cultured in the south was constructed. This library reached 2.2×107 in capacity; the percentage of recombination was 95.8%. PCR results showed that the average size of inserts was larger than 750bp. A total of 185 successful sequencing reactions yielded. In addition, 136 non-repetitive sequences were formed including 40 contigs and 96 singletons from these ESTs after initial assembly. The tropomyosin gene of A. japonicus, which was 1112bp long, its ORF encoding 284 amino acids with a predicted molecular weight of 33.27kDa and a theoretical isoelectric point of 4.56. The amino acid sequence was compared with Strongylocentrotus purpuratus, Euphausia superba, Portunus sanguinolentus, Branchiostoma belcheri, Eriocheir sinensis, and Bombyx mori; and high similarities of 62%—46% were revealed.

关 键 词：刺参 CDNA文库 基因克隆 原肌球蛋白基因 

分 类 号：Q346[生物学—遗传学]