草鱼肝胰脏组织部分EST的鉴定及分类

Identification of genes expressed in hepatopancrease from grass carp,Ctenopharyngodon idellus

本研究构建了草鱼(Ctenopharyngodon idellus)肝胰脏组织的cDNA文库,并对768个随机挑选的克隆进行部分测序,获得了567个ESTs。BLAST结果表明,457个ESTs与GenBank中已经鉴定的130个基因具有较高的同源性,其中有5个基因是已经报道的,125个是草鱼中首次鉴定的基因;110个和未鉴定的基因具有相对较低的同源性,称为假定蛋白。根据编码产物的功能可以将这些基因分为:代谢(41),基因表达及其蛋白质合成(56),细胞及机体防御(18),细胞信号传导与细胞通讯(11),细胞结构与运动(4)。

The hepatopancreas of cyprinoid fish plays an important role in carbohydrate, lipid, amino acid, protein, and prostaglandin metabolism as well. Due to its vital function and relative lack of cell type complexity, the hepatopancreas is an ideal model for studying cyprinoid fish expressed genes relating to some important physiological functions such as metabolism and immunity. However, far less is known of gene expression in hepatopancreas of the grass carp, Ctenopharyngodon idellus, a commercially important species in fresh water aquaculture in China. Most previous studies of grass carp have focused on the description of individual genes. To get a basic insight on gene expression, to obtain genetical and molecular biological information to the studies of some important physiological procession in grass carp, a rapid method of novel gene identification using partial sequencing of cloned cDNA to produce expressed sequence tag（EST） was explored in this context. Total RNA was isolated from grass carp hepatopancreas using TRIzol reagent（GIBCOBRL） and mRNA was purified by oligo（dT） magnet beads（Promega）. Then a cDNA library was constructed using a cDNA cloning kit（Stratagene）. After massive excise, the cDNA library was converted from λ-phage system into plasmid system and plated onto LB agar plate. Single colonies were randomly picked up and screened by PCR and only the clones with cDNA inserts more than 0.6 kb were used for sequencing. 768 cDNA clones were partially sequenced and 567 ESTs were obtained. BLAST result showed that 457 of the hepatopancrease ESTs could be ascribed to the transcriptional products of t 29 identified genes including 5 previously reported and 124 firstly identified in the grass carp, whereas 110 ESTs exhibited relatively low homology with unidentified sequences and were called hypothetical proteins. 129 known genes were classified into 5 function categories： 1 ） metabolism （41）;2） gene and protein expression （56）;3） cell signaling and communication（11）; 4） cell and organism defense（18） ;5 ） cell structure and motility（4）, which provide a genetical background for our molecular biology studies on metabolism and immunology in grass carp.