可口革囊星虫cDNA文库的构建及蚯蚓血红蛋白基因序列的分析  

Construction and analysis of a cDNA library for Phascolosoma esculenta

作　　者：王孟前[1] 李晔[1] 苏秀榕[1] 李太武[1,2] 贺静静[1] 王中华[1] 周君[1] 

机构地区：[1]宁波大学生命科学与生物工程学院,浙江宁波315211 [2]宁波城市职业技术学院,浙江宁波315110

出　　处：《台湾海峡》2010年第1期20-26,共7页Journal of Oceanography In Taiwan Strait

基　　金：国家自然科学基金资助项目(40776075);浙江省重大科技攻关项目(2006C13089)

摘　　要：提取可口革囊星虫血总RNA,反转录成cDNA,将大于500bp的cDNA片段连接入载体pBluescriptII SK(+),电转化至DH10B宿主菌中,得到原始文库.对cDNA文库的滴度、重组率和插入片段的大小进行检测,并挑取500个单菌落进行测序.结果表明,cDNA文库的库容量为3.06×105cfu,重组率达97.2%.经测序共得到437条序列,分析得到211个单基因簇(unigene),其中115条序列有相关同源性.测序得到蚯蚓血红蛋白cDNA全长序列为823bp,ORF编码120个氨基酸,蛋白分子量为13.63kD,等电点为5.78.NCBI上同源性比对结果则表明,与Siphonosoma cumanense的蚯蚓血红蛋白同源性最高为67%.可口革囊星虫cDNA文库的构建为今后克隆和研究可口革囊星虫的相关功能基因奠定了坚实的基础.Total RNA was extracted from the blood of healthy Phascolosoma esculenta with Trizol reagent. The first- strand cDNA was synthesized from purified mRNA, and subsequently the second-strand cDNA was generated via E. coli DNA polymerase I. The fractionated cDNA fragments longer than 0.5 kb were ligated into pBluescript II SK （ ＋ ） express vector,and transformed into DH10B by electroporation. Then, the library titre, recombinant rate and length of inserted cDNAs were measured, respectively. The results indicated that this library reached 3.06×10^5 cfu in capacity and the percentage of recombination was as high as 97.2%. A total of 437 clones was sequenced, in which 211 cDNA were merged. 115 sequences were ass igned putative functions based on their similarity to known genes. The full-length cDNA of hemerythrin was 823 bp, including an open reading frame （ORF） encoding a polypeptide of 120 amino acids with predicted molecular weight of 13.63kD and theoretical isoelectric point of 5.78. The blasting in NCBI indicated that the homology between Siphonosoma cumanense and Phascolosoma esculenta was 67%. In conclusion, an excellent cDNA expression library had been constructed successfully in this report,which would lay solid foundation for further characterization of the genes of Phascolosoma esculenta.

关 键 词：海洋生物学 可口革囊星虫 CDNA文库 序列分析 蚯蚓血红蛋白 

分 类 号：Q178.53[生物学—水生生物学]