大黄鱼肌肉组织cDNA全长文库的构建及EST分析  

Construction of a full length cDNA library from the skeletal muscle of Larimichthys crocea and its EST analysis

作　　者：黄伟[1] 薛良义[1,2] 李婷[1] 杨斌[1] 

机构地区：[1]宁波大学生命科学与生物工程学院,浙江宁波315211 [2]宁波大学应用海洋生物技术教育部重点实验室,浙江宁波315211

出　　处：《台湾海峡》2010年第2期189-195,共7页Journal of Oceanography In Taiwan Strait

基　　金：国家863计划资助项目(2006AA10A405);国家自然科学基金资助项目(30871916);长江学者和创新团队发展计划项目(IRT0734)

摘　　要：以大黄鱼肌肉组织为材料,利用Creator Smart cDNA Library Construction Kit构建了大黄鱼肌肉组织的全长cDNA文库.文库质量分析表明:cDNA文库容量不低于1.68×106cfu/mm3,重组率达96%,插入片断的平均长度大于1 000bp.随机挑取512个cDNA克隆进行5’端测序,其中430条ESTs的长度大于100bp;并初步拼接得到了230个单基因簇(unigene),其中包括58个重叠群(contigs),172个单拷贝ESTs(singletons),冗余度为46.51%.经检索,35个ESTs在BLASTx上无明显的同源性(E值≤1.00×10-10),为新基因.195个ESTs与已报道的基因有较高的同源性;其中与能量相关基因的ESTs丰度最高,占总数的20.00%.蛋白质合成、细胞骨架和信号传导次之,比例分别为13.48%、12.17%、10.00%,其中包括高迁移率族蛋白B1、瘦素受体、β1-热激蛋白等.这些EST数据为进一步筛选和克隆大黄鱼肌肉特异性表达基因提供了平台.A full-length cDNA library was constructed for the skeletal muscle of Larimichthys crocea using the Creator Smart cDNA Library Construction Kit.Library quality analysis showed that the library reached 1.68×106 cfu/mm3 in capacity with a recombinant efficiency as high as 96%.PCR results showed that the average size of inserts was larger than 1 000 bp.512 clones were randomly selected and sequenced.Of these 430 were expressed sequence tags（ESTs） longer than 100 bp.From these ESTs,230 non-repetitive sequences were acquired,including 58 contigs and 172 singletons after initial assembly and with a redundancy rate of 46.51%.From this retrieval 35 ESTs seemed to be novel genes（E-value≤1.00×10-10） as no clearly homologous known sequence was found in GenBank databases;195 ESTs were found to have considerable homology to reported genes,of which energy-related genes had the highest abundance,accounting for 20.00%,while protein synthesis,cytoskeleton and signal transduction genes made up 13.48%,12.17% and 10.00%,respectively.These included the high-mobility group protein B1,leptin receptor and β1-heat shock protein.The results provide a foundation for further screening and cloning of specific expression genes in the skeletal muscle of Larimichthys crocea.

关 键 词：海洋生物学 大黄鱼 肌肉组织 cDNA全长文库 表达序列标签(ESTs) 

分 类 号：Q178.53[生物学—水生生物学]