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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 of constructing Artemisia selengensis cDNA library. Methods: Total RNA was extracted from young leaves of Artemisia selengensis using modified Trizol method and double-stranded cDNA was synthesized by reverse transcription polymerase chain reaction (LD-PCR). The PCR products were digested with proteinase K and treated with sfiⅣ The products were digested with CHROMA SPIN-400 and fractionated over 0.4 kb. The cDNAs were then ligated with λTripl E × 2 phages and packaged in vitro. The full-length cDNA library of Artemisia annua L. was constructed by SMART technique. The titer of the original library, the capacity of the library, the recombinant positive rate and the size of the cDNA insert were determined by electrophoresis. Results: The titer of the original library was 1.94 × 107pfu / mL, and the library capacity was 0.97 × 107pfu. The positive rate of cDNA inserts was 96% and the size was 0.5 ~ 2.0 kb with an average of 0.9 kb. Conclusion: The constructed library with high content and high quality can provide the foundation for the construction of Artemisia selengensis cDNA library.