摘要
以湖北钉螺肝胰腺为材料构建cDNA文库。文库初始滴度为5.6×104 cfu/mL,库容量为6.72×105 cfu/mL,重组率为93%。插入片段长度为400—2700 bp。对其中1728个克隆进行测序,得到1520个序列,其中长度大于100 bp的ESTs为1393条,初步拼接得到698个单基因簇(Unigene),其中包括234个重叠群(Contig),464个单拷贝(Singleton)EST。使用BLAST软件进行同源性分析,结果显示有111个单基因簇具有不同程度的同源性,约占15.90%;587个单基因簇与所有已发现的蛋白质的基因没有或者有着相当低的同源性,约占84.10%。研究为湖北钉螺的基因学研究提供了重要的基础资料,同时亦为筛选和鉴定湖北钉螺生长发育、免疫等相关的功能基因以及血吸虫与螺之间的相互关系奠定了基础。
The snail Oncomelania hupensis is the unique intermediate host of Schistosoma japonicum which causes schistosomiasis endemic in the Fast East,especially in China's Mainland.After miracidium penetrated into the snail host,the schistosome larvae develop into sporocyst,move to snail hepatopancreas and develop into cercariae at last,which is the infection stage for the final host including human.Hepatopancreas is not only the necessary organ for development of schistosome larvae in snail host,but also be rich of hemocyte,which was thought to mediate the defense system of O.hupensis in response to schistosome infection.The present study was thus designed to construct a cDNA library of O.hupensis hepatopancreas,and give some information to deeply understand the interaction between the snail host and schistosome parasite through obtained genomic data.Total RNA were extracted from O.hupensis hepatopancreas and mRNA were purified by using PolyATtract mRNA Isolation Systems Ⅳ.The cDNA library was then constructed from purified mRNA by using CloneMinerTM cDNA Library Construction Kit under the manual direction.Totally,library had 5.6×104 cfu/mL in initial titer,and 6.72×105 cfu/mL in capacity.The percentage of recombination was about 93%,with the length of inserts being 400—2700 bp.1728 positive clones were sequenced,and a total of 1520 were successfully sequenced with the yield of 1393 expressed sequence tags（ESTs） longer than 100 bp.Through initial gene splicing,698 unigenes were formed from 1393 ESTs,including 234 contigs and 464 singletons.After BLAST（Basic Local Alignment Search Tool） analysis of these sequences,111（15.90%） unigenes were assigned to have homology with some genes in the GenBank,which could be divided into four types,including Cellular Processes and Signaling（21.62%）,Information Storage and Processing（46.85%）,Metabolism（27.93%） and Poorly Characterized（3.60%） based on the protein function through COG analysis.Other 587（84.10%） unigenes had no homology with any genes in present GenBank data,which might be potential for further novel gene discovery.O.hupensis hepatopancreas cDNA library have been constructed,with plenty of new genes have been discovered,which supply genomic platform for further study.In addition,some immunology related genes such as glutathione S-transferase,chitinase,ferritin-like protein,glutathione peroxidase and heat shock protein family have been found,which may indicate that the hepatopancreas participates in the immunology activity of snail against parasite.In summary,this study provides plenty of genomic data of O.hupensis,which can supply some information for further investigations such as discovery of new immunology of snail host,and interaction between O.hupensis and S.japonicum.
出处
《水生生物学报》
CAS
CSCD
北大核心
2011年第4期672-680,共9页
Acta Hydrobiologica Sinica
基金
中国科学院知识创新工作重要方向性项目(KSCX2-YW-N-055,KSCX2-W-2-0808)
国家自然科学基金(30770353)资助
关键词
湖北钉螺
肝胰腺
CDNA文库
EST
Oncomelania hupensis
Hepatopancreas
cDNA library
Expressed sequence tag
