嗜水气单胞菌诱导中华绒螯蟹消减文库的构建及初步分析  

Construction and Primary Analysis of Suppression Subtractive Hybridization Library of Eriocheir sinensis by Aeromonas hydrophila

作　　者：赵大显[1,2] 陈敏文[1] 江坤[1] 吴小平[1,2] 

机构地区：[1]南昌大学生命科学与食品工程学院,江西南昌330031 [2]南昌大学食品科学与技术国家重点实验室,江西南昌330031

出　　处：《江西农业大学学报》2013年第2期398-403,共6页Acta Agriculturae Universitatis Jiangxiensis

基　　金：国家自然科学基金(31260642);中国博士后科学基金(2012M521292);江西省教育厅青年基金(GJJ12144);南昌大学食品科学与技术国家重点实验室开放基金(SKLF-KF-201001)

摘　　要：利用抑制消减杂交(SSH)技术分离嗜水气单胞菌(Aeromonas hydrophila)诱导中华绒螯蟹(Eriocheir sinen-sis)差异表达的基因,以血淋巴细胞为材料,提取感染前后总RNA,经mRNA分离、cDNA合成、酶切、加接头后分别以感染组为检测子(tester)、对照组为驱动子(driver)进行正向抑制性消减杂交,以对照组为检测子、感染组为驱动子进行反向消减杂交,成功获得了中华绒螯蟹正反双向差异消减cDNA文库;对构建的文库进行菌落PCR鉴定发现,克隆中插入的片段主要分布在100～1 000 bp之间;随机选取54个阳性克隆测序,共获得37条有效表达序列标签(EST)序列,将测序结果提交到GenBank进行Blastn比对发现,6条与其他物种的免疫基因序列具有较高的同源性。上述结果表明,构建的文库质量较好,且差异表达的与抗病相关的基因得到了富集,为进一步开展中华绒螯蟹抗病重要功能基因的筛选、克隆和功能分析奠定了基础,为揭示中华绒螯蟹抗病的分子机理提供了理论依据。Subtractive cDNA library of differentially expressed genes during the bacterial infection of Erio- cheir sinensis was constructed by using suppression subtractive hybridization （SSH） technique. The RNA and mRNA from hemocytes of normal crab and bacterial-challenging crab were firstly isolated. After mRNA was reversely transcribed into double strand cDNA, the two kinds of ds cDNA were denominated with Driver cDNA （ normal crab） and Tester cDNA （ bacterial challenging crab） separately. The results of analysis of Rsa I digestion, ligation and subtraction efficiency showed that the library was established successfully. Further PCR screening showed that the size of the inserts was mainly between 100 to 1000 bp. 54 positive clones were picked up and sequenced randomly,and the obtained 37 expressed sequence tags （EST） were compared with the sequences in NCBI with BLASTn algorithm,the results showed that 6 EST of them had homology with the immune-related genes of others species. Analysis of the libraries indicates that the constructed library per- formed wiht high quality, which can be used in further study on the immune mechanism in crab.

关 键 词：中华绒螯蟹 嗜水气单胞菌 消减cDNA文库 表达序列标签 

分 类 号：S968.25[农业科学—水产养殖]