新疆一枝蒿cDNA文库构建方法研究  

Study on the cDNA Library Construction Method for Xinjiang Artemisia rupestris

作　　者：刘冲 程波 何江 杨伟俊 地力努尔.吐尔逊江 满尔哈巴.海如拉 薛桂蓬 魏梅梅 王雪 

机构地区：[1]新疆维吾尔自治区药物研究所/新疆维吾尔药重点实验室,乌鲁木齐830004

出　　处：《中国药房》2017年第13期1793-1796,共4页China Pharmacy

基　　金：新疆维吾尔自治区公益性科研院所基本科研业务经费资助项目(No.KY2015121);新疆维吾尔自治区青年科技创新人才培养工程项目(No.2013721034)

摘　　要：目的:建立构建新疆一枝蒿cDNA文库的方法。方法:采用改良Trizol法提取一枝蒿幼嫩叶片总RNA,反转录成单链c DNA,长距离聚合酶链反应法(LD-PCR)合成双链cDNA;PCR产物经蛋白酶K消化,采用sfiⅣ酶切,酶切产物用CHROMA SPIN-400柱分级分离,回收0.4 kb以上的cDNA,以λTripl E×2噬菌体连接并进行体外蛋白包装,利用SMART技术构建一枝蒿全长cDNA文库;随机挑取文库中20个单克隆,电泳法测定原始文库滴度、文库容量、cDNA插入片段的重组阳性率与大小。结果:原始文库滴度为1.94×107pfu/mL,库容量为0.97×107pfu;cDNA插入片段重组阳性率为96%,大小为0.5~2.0 kb,平均为0.9 kb。结论:所构建的高库容、高质量的文库可为新疆一枝蒿cDNA文库的构建提供基础。OBJECTIVE：To establish a method for full-length cDNA library of Xinjiang Artemisia rupestris. METHODS：Mod-ified Trizol method was adopted to extract total RNA in young leaves of A. rupestris,it was transcribed into single-strand cDNA, and then synthesized into double-strand cDNA by long-distance polymerase chain reaction（LD-PCR）method. PCR product was di-gested by proteinase K and sfiⅠ,and then fractionated by CHROMA SPIN-400 columns. The cDNA longer than 0.4 kb were col-lected and ligated to phage λTriplE × 2,and then protein packaging was performed. Full-length cDNA library was established by SMART technology. 20 monoclonal were randomly selected from the library,and electrophoresis was used to determine the primary library titer,library capacity,recombinant positive rate and length of insert cDNA. RESULTS：The primary library titer was 1.94× 107 pfu/mL,library capacity was 0.97×107 pfu;recombinant positive rate of insert cDNA was 96% and length was 0.5-2.0 kb with an average of 0.9 kb. CONCLUSIONS：The established library is high in capacity and quality,which can provide basis for estab-lishing cDNA library of Xinjiang A. rupestris.

关 键 词：新疆 一枝蒿 CDNA文库 构建 功能基因 

分 类 号：R282.5[医药卫生—中药学]