淞江鲈肝脏组织cDNA文库的构建与部分EST测序  

Construction and Identification of Hepatic cDNA Library of Roughskin Sculpin

作　　者：许耀[1] 刘庆全[1] 罗武松[1] 秦志浩 王金秋[1] 

机构地区：[1]复旦大学遗传工程国家重点实验室,上海200438 [2]上海四鳃鲈水产科技发展有限公司,上海200438

出　　处：《复旦学报（自然科学版）》2015年第6期809-814,共6页Journal of Fudan University：Natural Science

基　　金：农业部公益性行业(农业)科研专项资金(201203065)

摘　　要：以成年雌性淞江鲈肝脏组织为材料,用TRIzol Reagent试剂提取总RNA.以总RNA为模板,先用逆转录酶合成cDNA第一链,再通过LD-PCR合成cDNA第二链,所得双链DNA分子用SfiⅠ酶切处理后,连接到同样经SfiⅠ酶切处理的改良pUC19载体上,获得重组质粒.将重组质粒导入大肠杆菌DH5α感受态细胞中,建成质粒cDNA文库.经鉴定,该文库库容为1.5×106,重组率为94.5%,平均插入片段长度为1.1kb,插入片段大小分布为0.5~2.0kb.从文库中随机挑选2 200个克隆进行5’端单向测序,剔除不合格序列后,得2 002条高质量表达序列标签（Expressed Sequence Tag,EST）序列,提交dbEST数据库.经筛选,从这些EST序列中共发现471个基因,分别对其进行了KOG功能分类预测（216个蛋白被注释上21种KOG分类）、KEGG注释（186个基因注释上175个KO,139个基因注释上195个pathway）和GO功能分类注释（312个蛋白被注释上GO分类）.Total RNA was isolated from the liver of an adult female roughskin sculpin （Trachidermus fasciatus Heckel） using TRIzol reagent. First-stranded cDNA was synthesized by reverse transcription and double-stranded eDNA was obtained using LD-PCR. Proteinase K and Sfi Ⅰ were employed to digest the ds-cDNAs, SPIN-400 column was also utilized to remove short cDNA fragments. Afterwards, the cDNAs were ligated to an improved pUC19 vector, the eDNA library was constructed. The storage of the library was 1.5 × 10^6 , the recombination rate was 94.5%, the average length of inserted fragments was 1.1 kb. 2 200 clones were randomly selected from the library and single pass sequenced from the 5＇ end, after removing low quality sequences, 2002 sequences left, they were submitted to the dbEST database of NCBI. 471 genes were screened out from these ESTs, 216 proteins were annotated into 21 KOG categories, 18 genes were annotated into 175 KO, 139 genes were annotated into 195 pathways, 312 proteins were annotated in C,O classification.

关 键 词：淞江鲈 肝脏 CDNA文库 EST序列 

分 类 号：Q785[生物学—分子生物学]