摘要
本项研究以雌性草鱼肠道为实验材料,采用非均一化的Oligo-dT引物定向克隆技术构建了草鱼肠道组织的cDNA文库,对文库质量的分析结果表明:cDNA文库的库容量至少为2.3×105,重组率达95%,平均插入片断长度大于1000bp。挑取cDNA克隆进行5’端测序,总共进行了1571个成功反应,其中1411条ESTs长度大于100bp,初步拼接得到939个单基因簇(Unigene),其中包括188个重叠群(Contigs),751个单拷贝EST(Singletons)。使用BLAST软件将这些序列同GenBank等数据库进行比对、查询和注释,结果显示418条序列有相关同源性,其他的521(55.5%)条序列没有明显的同源性(E-valu 1.00E-10),但是也同时揭示出在这些ESTs中能够发现新功能基因的相当可能性。本研究结果对草鱼以及其他鲤科鱼类的消化系统功能基因的筛选和发现具有指导意义。此外,草鱼肠道cDNA文库的构建和EST测序工作将为草鱼基因组计划的实施奠定基础。
The technology of expressed sequence tag （EST） was broadly applied in analysis of gene function and expression pattern. Based on the construction technology of non-normalized cDNA library, the cDNA libraries of grass carp＇s intestinal cDNA Library was constructed. This library reaches 2.3 × l0^5 in capacity; the percenyage of recombination is as high as 95% . PCR results showed that the average size of inserts was larger than 1000 bp. And a total of 1571 successful sequencing reactions yielded 1411 which expressed sequence tags （ESTs） longer than 100 bp. In addition, 939 non-repetitive sequences were formed including 188 contigs and 751 singletons from these ESTs after initial assembly. Basic Local Alignment Search Tool （BLAST） analysis showed that nearly 44.5 % of the sequences were assigned putative functions because they showed similarity to known genes （ E-value 〈 1.00E-10）, mostly from other species, in one or more of the databases used, others suggesting a potential for further novel gene discovery. The study provides a significant number of ESTs for gene discovery and candidate genes for studying digestive system in grass carps and other carps. Furthermore, the large-scale ESTs sequencing provided a valuable resource for the functional genome research and the whole genome sequencing project of Ctenopharyngodon idellus.
出处
《水生生物学报》
CAS
CSCD
北大核心
2007年第2期251-258,共8页
Acta Hydrobiologica Sinica
基金
通威股份公司科技发展项目--草鱼基因组计划及产业化项目基金资助
关键词
草鱼
肠道
CDNA文库
EST
测序
Ctenopharyngodon idellus
Intestine
cDNA library
Expressed Sequence Tag
Sequencing
