草鱼肠道组织差异表达基因消减文库的构建.pdfVIP

第33卷第6期 湖南农业大学学报(自然科学版) Vbl-33 No．6 2007年 l2月 Journal of Hunan Agricultural University(Natural Sciences) Dec．2O07 文章编号：1007．1032(2007)06．0718．05 草鱼肠道组织差异表达基因消减文库的构建 苏建明h，陈 韬h，肖调义 ，陈立祥 ，章怀云 ’ (1．湖南农业大学a．动物医学院；b．动物科学技术学院，湖南 长沙 410128；2．中南林业科技大学 生物科学与 技术学院，湖南 长沙 410004) 摘 要：以致病性嗜水气单胞菌(Aeromonas hydrophila)人工感染的l龄草鱼(Ctenopharyngodon idellus)肠 道组织为材料，应用抑制性差减杂交技术，构建了肠道组织的消减eDNA文库．以草鱼管家基因O．actin作为 差减指标检测文库差减效率达 2 0倍，表明感染细菌后某些差异表达基因得到了相应倍数的富集．将获得的 eDNA片段连接到A／T载体，并转化大肠杆菌DH5a感受态细胞． 关 键 词：草鱼；嗜水气单胞茵；抑制性消减杂交；eDNA文库 中图分类号：Q785；$917 文献标识码：A ConstructiOn of a subtractive cDNA library from the intestinal tissue of grass carp experimentally infected byAeromonas hydrophUa SUJian-minglI。 CHEN Taoh。 XIAOaaao-yilb。CHENLi-xianglb． ZHANGHuai-yunIb·2． (1．a．CollegeofVeterinaryMedicine；b．CollegeofAnimalScience andTechnology，HNAU，Changsha410128，China； 2．College ofBiological Science and Technology。CSUFT，Changsha 410004。China) Abstract：Aeromonas hydrophila is one of the main causative agents resulting in serious infectious diseases of grass carp(Ctenopharyngodon idellus)and other animals．To understand anti-infectious response to bacteria in grass carp， a subtractive eDNA library Was constructed from the intestinal tract of grass carp experimentally infected with Aeromonas hydrophila using suppression subtractive hybridization(SSH)．Grass carp 13-actin gene was used as internal control to estimate the efficiency of subtractive eDNA．In this library，B-actin Was subtracted significantly at about 2 folds，suggesting that the subtractive eDNA library Was successfully constructed．The PCR products were inserted into pUCm-T vector and transformed to competent E．coli DH5a cells to set up a subtracted PCR fragment