利用SMART技术构建歪头菜叶片全长cDNA文库  

Full-length cDNA library construction for leaves of Vicia unijugawith SMART technique

作　　者：韩瑜[1] 强维亚[1] 

机构地区：[1]草地农业生态系统国家重点实验室兰州大学生命科学学院,甘肃兰州730000

出　　处：《草业科学》2014年第1期83-88,共6页Pratacultural Science

基　　金：国家自然科学基金(31070353);甘肃省科技支撑项目(2007GS04976)

摘　　要：以歪头菜（Vicia unijuga）为研究材料, 用Trizol试剂提取歪头菜叶片组织总RNA, 用SMART TM cDNA Library Construction Kit构建cDNA文库。经测定, 初级文库滴度达到2.3×10^6 pfu·mL^-1, 扩增后文库总滴度为6.2×109 pfu·mL^-1, 重组率为92.7%。从扩增后的文库随机挑取16个噬菌斑进行PCR扩增鉴定, 结果显示, 插入片段大多分布在500～2 500 bp。用PCR法从该文库中扩增出了以往用RACE法克隆出的歪头菜木质素合成相关基因CAD 3′端全序列。通过各项指标验证成功构建了歪头菜叶片全长cDNA文库。Full-length cDNA Library for leaves of Vicia unijuga was constructed with SMARTTM cDNA library construction kit after total RNA of V.unijuga leaves was extracted by Trizol reagent. The primary library has titer of 2.3×10^6 pfu·mL^-1, while the amplified library has a high titer of 6.2×109 pfu·mL^-1 and 92.7% recombinant rate. Sixteen plaques were randomly selected from the amplified library and amplified by PCR. The results showed that the inserted fragment length ranged from 500 to 2 500 bp. The 3′end full-length cDNA of CAD, a Vicia unijuga gene related with lignin synthesis which has been cloned by RACE, was amplified in this cDNA library using PCR, which indicated that the cDNA library for leaves of V.unijuga had been successfully constructed using SMART technique.

关 键 词：歪头菜 CDNA文库 SMART技术 

分 类 号：Q943.2[生物学—植物学]