新疆一枝蒿cDNA文库构建方法研究-中国药房

新疆一枝蒿cDNA文库构建方法研究

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篇名：	新疆一枝蒿cDNA文库构建方法研究
TITLE：
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cDNA，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sfiⅠ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λTriplE×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1.94×107 pfu/mL，库容量为0.97×107 pfu；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。
ABSTRACT：	OBJECTIVE： To establish a method for full-length cDNA library of Xinjiang Artemisia rupestris. METHODS： Modified 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 digested by proteinase K and sfiⅠ， and then fractionated by CHROMA SPIN-400 columns. The cDNA longer than 0.4 kb were collected 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 establishing cDNA library of Xinjiang A. rupestris.
期刊：	2017年第28卷第13期
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
AUTHORS：	LIU Chong，CHENG Bo，HE Jiang，YANG Weijun，DILINUER·Tuerxunjiang，MERHABA·Heyrulla，XUE Guip- eng，WEI Meimei，WANG Xue
关键字：	新疆；一枝蒿；cDNA文库；构建；功能基因
KEYWORDS：	Xinjiang； Arternisia rupestris； cDNA library； Construction； Functional gene
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