嗜水气单胞菌诱导的池蝶蚌血细胞cDNA文库的构建和亲环蛋白基因序列的初步分析  

THE FULL-LENGTH cDNA LIBRARY OF HEMOCYTE INDUCED BY AEROMONAS HYDROPHILA AND MOLECULAR CHARACTERISTICS OF CYCLOPHILIN A FROM HYRIOPSIS SCHLEGELII

作　　者：谢凯[1] 徐灵[1] 盛军庆[1] 曾柳根[1] 王军花[1] 洪一江[1] 

机构地区：[1]南昌大学生命科学与食品工程学院,南昌330031

出　　处：《水生生物学报》2011年第5期783-789,共7页Acta Hydrobiologica Sinica

基　　金：国家公益性行业科研专项(200903028);江西省科技攻关项目(2009BNA07400);江西省自然科学基金(2008GZN0070);江西省教育厅科技项目(GJJ10306)资助

摘　　要：实验利用灭活的嗜水气单胞菌(Aeromonas hydrophila)诱导处于四龄池蝶蚌(Hyriopsis schlegelii)14h,将诱导后的池蝶蚌血细胞的总RNA进行逆转录,用LD-PCR法合成双链cDNA,从而首次成功构建池蝶蚌血细胞的全长cDNA文库。原始文库的滴度为4×106 cfu/cm3,重组率为90%,扩增后文库的滴度为3.55×109 pfu/mL。目前文库已随机测序672个样品,将所得双向序列进行拼接,去除载体,并多序列比对去除重复序列后,发现436条为已知功能序列,其余为未知功能序列。序列中最小长度270 bp,最大长度为2153 bp,平均大小608.6 bp,表明插入片段大小理想。从文库中筛选获得免疫相关基因池蝶蚌亲环蛋白A(HsCyp A)全长基因并进行序列分析。结果显示,HsCyp A全长1229 bp,序列包括52 bp的5′非编码区、495 bp的开放阅读框、682 bp的3′非编码区和29 bp的poly(A)尾,没有明显的加尾信号。对Cyp A氨基酸序列二级结构进行了较详细的分析并进行了三维建模,同时构建了其系统进化树,分析表明亲环蛋白家族是一个在进化上非常保守的蛋白家族。综合分析,Cyp A在水生动物中不仅仅只是一种组成型蛋白,而是可能在病原感染防御中发挥重要作用。Hyriopsis schlegelii,originated from the Lake BIWA of Japan,was introduced into China in 1997.In order to seek for genes related to the freshwater mussel on immune system,a full-length haemocyts cDNA library was constructed by using SMART（switching mechanism at 5′ end of the RNA transcript） technique.The total RNA was isolated from the four years old mussel blood hemocyte induced by Aeromonas hydrophila for 14 hours.The ＂anchor first-strand cDNA＂ containing a sites（A B） of symmetrical sfi I restriction enzyme was synthesized by reverse transcription,and the double-strand cDNA was synthesized and amplified by LD-PCR（long-distance PCR）.After digested by the proteinase K and sfi I restriction enzyme,size fractionation by CHROMA SPIN-400 columns,ligated with the sfi I digested pDNR-LIB vector and transformed into E.coli DH10B by thermal shock,the full-length cDNA library was constructed.It was the first time to construct a full-length cDNA library from blood hemocyte of the freshwater mussel.The titer of the primary constructed cDNA library was 4×10^6 cfu/cm^3 with a high recombination rate of 90% and the amplified one was 3.55×109 cfu/cm^3.672 clones were sequenced ran-domly selected from the library and 436 have the homologous gene information after analyzed by BLASTx in the website of NCBI.Among the sequences,the shortest one was 270 bp and the longest one was 2153 bp,and average length was 608 bp.Results indicated it was a high quality cDNA library.The full length of the Cyp A（HsCyp A）,which may provide immune stress function in some species,was isolated from the cDNA library by screening of H.schlegelii.It has 1229 base pair（bp）,containing a 52 bp 5′ untranslated region（UTR）,a 495 bp open reading frame,a 682 bp 3′-untranslated sequence,and a 29 nucleotide long poly（A） tail.The initiator codon（ATG） and the stop codon（TAA） can be found at the position of 53 and 545,but can not find the putative polyadenylation signal（AATAAA）.The predicted protein sequence consist

关 键 词：池蝶蚌 血细胞 CDNA文库 嗜水气单胞菌 亲环蛋白 

分 类 号：Q785[生物学—分子生物学]