烤烟品种南江3号均一化全长cDNA文库构建  

Construction Normalized Full-length cDNA library of Flue-cured Tobacco Variety of Nanjiang-3

作　　者：蔡刘体[1] 胡重怡[1] 张永春[1] 韩晓红[1] 

机构地区：[1]贵州省烟草科学研究所,贵州贵阳550018

出　　处：《生物技术》2011年第2期45-47,共3页Biotechnology

基　　金：贵州省烟草专卖局项目("烟草病圃微生物多样性分析及烟草抗病性基因研究";黔烟科2009-015);国家烟草专卖局项目("烟草cDNA文库构建及重要基因表达谱研究";110200701021)资助

摘　　要：目的:为了获得功能基因的信息,构建烤烟品种南江3号的均一化cDNA文库。方法:用RNeasy Plant Mini Kit提取烤烟南江3号叶片和花的RNA,用反转录酶逆转录合成第一链cDNA。以LD-PCR扩增获得的双链cDNA为模板,采用基于双链特异性核酸酶(Duplex-Specific Nuclease,DSN)的均一化cDNA文库技术,构建南江3号盛花期的均一化cDNA文库。结果:构建了南江3号盛花期的均一化cDNA文库,文库重组率大为97.47%,库容量约为1.26×106,插入片段平均长度大于1.2kb。从文库中随机48个克隆进行PCR检测,挑取20个克隆进行测序,序列通过BLAST比对结果显示文库可能包含大量基因和ESTs序列。结论:该文库为研究南江3号的基因功能和资源提供材料来源。Objective： In order to obtain information of functional genes,a normalized cDNA libray from Flue-cured tobacco variety of Nanjiang-3（Nicotiana tobacum cv.Nanjiang-3） was constructed.Method： RNA was extracted from leavies and flowers of Nanjiang-3 according to RNeasy Plant Mini Kit manipulation,the first cDNA was synthesized by reverse transcriptase.Double cDNA obtianed through long-distance PCR（LD-PCR） amplification method as template,the normalized cDNA library of Nanjiang-3 was constructed by the SMART technology which based on the double strand specific nuclease（Duplex-Specific Nuclease,DSN）.Result： The normalized cDNA library was constructed,which contained 1.26×106 independent clones,its recombination ratio was 97.47%,average insertion size was above 1.2kb.Random selected 48 clones were tested by PCR,then 20 positive clones were sequenced and BLAST,the results showed that many genes and expression sequence tags（ESTs） were contained in the cDNA pool.Conclusion： Basis material information of research for genetic resources and gene function of Nanjiang-3 were provided by the cDNA library.

关 键 词：烤烟 基因 均一化 CDNA文库 表达序列标签 

分 类 号：S572.032[农业科学—烟草工业]