荣昌猪肝组织全长cDNA文库的构建和EST测序及分析

Construction of full-length cDNA library and analysis of expressed sequence tags( ESTs) from the liver of Rongchang Pig

研究旨在为荣昌猪种质资源的保存、开发和挖掘提供重要的分子信息,同时也为荣昌猪作为生物医学模型,代谢疾病的研究提供基础数据.运用SMART法构建了荣昌猪肝组织的c DNA文库,并从文库中随机挑取部分克隆进行EST测序和功能注释.经检测,构建的原始文库滴度为4.19×10^6pfu/m L,重组率为96.31%,平均插入片段长度主要在400～2 000 bp之间.总共获得了226条有效EST序列,共计161个unigenes,包括19个重叠群（contigs）和142条单一序列（singlets）.101（62.73%）条为已知功能基因序列,60（37.27%）条为未知功能序列,其中40条为新基因序列.GO分类和KEGG途径分析揭示大量荣昌猪肝组织表达基因主要与细胞质部分、细胞外的区域、受体结合,化学反应及细胞分解代谢过程等相关;表达的基因主要涉及代谢、补足物和凝固级联和核糖体等功能信号途径.研究采用SMART法成功构建了荣昌猪肝组织全长c DNA文库,并进行EST测序及分析,综览了荣昌猪肝组织基因表达及功能分类特征.

To supply important molecular information for storing,exploring and digging genetic resources of Rongchang Pig and laying foundation for the research of biomedical model based on Rongchang Pig,the c DNA library of Rongchang Pig liver was constructed by using the SMART way and the sequencing and annotation of expressed sequence tags wascarried by selecting some clones at random.The results showed that the titer of primary library was 5.88×10^6pfu / m L and the proportion of recombinants from unamplified library was 96.31%; The average length of most exogenous inserts wais among 400-2 000 bp.Two hundred twenty-six effective expressed sequence tags were obtained,which included 161 unigenes with 19 contigs and 142 single sequence.Among those,101（ 62. 73%） sequences were known functional genes,and 60（ 37. 27%） sequences were unknown functional genes with 40 novel genes. Gene ontology analysis revealed that sequences expressed in Rongchang Pig livers mainly were relevant to cytoplasmic part,extracellular region,extracellular membrane-bounded organelle,receptor binding,chemical response and catabolic process. The expressive products of the genes were mainly involved in metabolic pathways,complement and coagulation cascades and ribosome.The research successfully constructed the full-length cDNA library and overviewed the expressed profiles and functional classified profile of Rongchang Pig by sequencing of expressed sequence tags.