摘要
采用抑制性消减杂交技术构建了三角帆蚌肝脏瘟病感染期抑制性消减cDNA文库。经检验，差异表达基因均被富集了2^10倍左右，证明构建的cDNA消减文库具有很强的消减效率。PCR鉴定发现，在随机挑取的阳性克隆中，95％的克隆均含有0．2～1．0kb的插入片段，这些片段可能是三角帆蚌瘟病病毒感染后差异表达基因的cDNA片段。测序共获得214个有效cDNA序列，分别属于8大类，共98个基因。其中细胞分裂基因2个、细胞结构与运动基因9个、代谢基因10个、信号传导基因7个、细胞防疫基因10个、基因与蛋白表达基因20个、未知功能蛋白基因26个，GenBank中找不到任何同源序列的基因14个。结果说明，构建的差异表达cDNA文库，可较好地反映三角帆蚌瘟病病毒对三角帆蚌影响的基因信息。
This study used suppression subtractive hybridization （SSH） for construction of the cDNA subtractive library of the liver from the living body infected Hyriopsis cumingii plague virus. After testing, differentially expressed genes were enriched in the 2^l0 times that the proof of the cDNA library has strong subtractive efficiency. Three hundred positive clones were randomly picked and identified by PCR method; 95% clones contained 0.2 - 1.0 kb by inserts, which might be the cDNA flagmenu of differentially expressed genes in Hyriopsis cumingii plague virus infected group. Three hundred clones were sequenced and obtained 214 known function EST for 58 different genes were reported in H. cumingii for the first time in this study. According to the classification of functional genes by Adam, the identified EST in H. cumingii fell into eight categories relevant to 2 belong to cell division genes, 9 belong to cellular structure and movement genes, 10 belong to metabolism genes, 7 belong to signal transduction genes, 10 belong to cell immuneaction genes, 20 belong to gene and protein expression associates genes and 26 belong to other proteins with unknown functions. In addition, there are 14 no similar sequence in Genbank, conjecturing its new genes. Results show, the method to construct cDNA subtractive library could well reflect the gene information of abalone affected by H. cumingii plague virus. Results of the present study provide the basic data to research of relationship between resistance breeding and gene expression of liver.
出处
《水产学报》
CAS
CSCD
北大核心
2009年第5期856-864,共9页
Journal of Fisheries of China
基金
湖南省科技厅攻关项目(06NK3049)
湖南省教育厅重点项目(07A012)
关键词
三角帆蚌
肝脏
三角帆蚌瘟病病毒
抑制性消减杂交
消减cDNA文库
Hyriopsis cumingii
liver
Hyriopsis cumingii plague virus
suppression subtractive hybridization
subtracted cDNA library
