罗非鱼免疫前后白细胞cDNA差减文库的构建及鉴定  

Construction and Identification of Subtractive cDNA Library from Peripheral Blood Leucocytes of Tilapia

作　　者：李超[1,2] 陈明[1,3] 李莉萍[1] 黄维义[2] 余晓丽[1] 黄婷[1] 张彬[1] 雷爱莹[1] 陈福艳[1] 梁万文[1] 

机构地区：[1]广西水产研究所,广西南宁530021 [2]广西大学动物科学技术学院,广西南宁530005 [3]农业部水生动物遗传育种和养殖生物学重点开放实验室//中国水产科学研究院淡水渔业研究中心,江苏无锡214081

出　　处：《西南农业学报》2010年第4期1286-1292,共7页Southwest China Journal of Agricultural Sciences

基　　金：国家科技支撑计划项目(2008BADB9B04);农业部水生动物遗传育种和养殖生物学重点开放实验室开放基金项目(BZ2007-05);广西科技计划应用基础研究项目(桂科基0832029);广西区直属公益性科研院所基本科研业务专项资金项目(2060302GXIF-2009-04)

摘　　要：为筛选罗非鱼链球菌疫苗免疫前后白细胞表达变化基因及探讨鱼类白细胞免疫机理,利用抑制性差减杂交(SSH)技术,以链球菌疫苗免疫前后罗非鱼白细胞cDNA为材料,构建了罗非鱼免疫前后正、负2个cDNA差减文库,并采用地高辛(DIG)标记差减文库cDNA作为探针,进行差异表达片段的筛选鉴定。结果显示:2个文库重组率均在90%以上,插入片段在300～900 bp,接头连接效率超过50%,差减效率非常高效,阳性率均超过70%。杂交筛选后正、负文库各挑选30个阳性克隆进行测序组装聚类,正、负文库各获得16和18条非冗余EST(unigene),所得序列经GenBank同源性分析,正、负文库各有10和11条unigene获得功能注释,且各有6和7条unigene没有同源性匹配,为未知新基因。In order to screen the differentially expressed genes in the peripheral blood leucocytes and investigate the immune mechanism of leucocytes of Tilapia,which was i.p.vaccinated by S.iniae vaccine.This research used the suppression subtractive hybridization（SSH） technique constructed forward and reverse two libraries with the cDNA of Tilapia leukocytes.And the differentia1 expressed fragments were screened by reformed differential screening technique which the probes were labe1ed with DIG.The results showed that the recombination rate of the two libraries exceeded 90 %,the length of the inserted fragments was ranged from 300 to 900 bp.Efficiency of adaptor-ligation with digested cDNA exceed 50 %.Subtractive efficiency indicated that the libraries were in good quality,and the positive rates exceeded 70 %.After dot-blot,30 positive clones were sequenced,assembled and clustered from each library,and 16 and 18 unigenes were found in the forward and reverse library.After homology search in the GenBank,10 and 11 unigenes in the forward and reverse library were successfully annotated,respectively.They were related to immunization signal transduction protein,disease defence protein,tumor immunity protein and leucocytes protein.There were 6 and 5 unigenes without homology match,maybe novel gene.

关 键 词：罗非鱼 链球菌 白细胞cDNA 差减文库 抑制性差减杂交(SSH)技术 

分 类 号：S965.316[农业科学—水产养殖]