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目的:构建滇重楼全长cDNA文库,为进行滇重楼生长发育相关基因及次生代谢产物合成途径相关基因的研究奠定基础。方法:改良Trizol法提取滇重楼根茎总RNA,通过SMART(switching mechanism at 5’end of RNA transcript)构建全长cDNA文库,测定分析文库滴度、重组率及插入片段大小,对文库进行随机测序并通过Blastx分析后在GenBank中进行同源性检索和功能预测。结果:文库的库容为2.5×107cfu/mL,文库重组率98.5%,插入片段大小平均为1.5 kb。随机挑取192个单克隆测序,其中有149条ESTs(Expressed Sequence Tags),其中9条序列与生长发育有关,5条序列与滇重楼次生代谢产物的合成、运输与代谢有关。结论:利用SMART技术成功构建了滇重楼全长cDNA文库,该文库库容量大、重组率高且插入的cDNA片段较长,可以满足滇重楼功能基因的鉴定、筛选及表达调控研究。
OBJECTIVE: To construct a full-length cDNA library of Polygonum pubescentis, which will lay the foundation for the study of the genes involved in the growth and development of secondary and secondary metabolites. Methods: Trizol method was used to extract the total RNA of rhizome of Dianzhong building. The full-length cDNA library was constructed by SMART (switching mechanism at 5’end of RNA transcript). The library titers, recombination rates and insert size were determined. The library was randomly sequenced After homology search and functional prediction in GenBank by Blastx analysis, Results: The library had a capacity of 2.5 × 107cfu / mL and a library recombination rate of 98.5%. The average insert size was 1.5 kb. Nineteen ESTs (Expressed Sequence Tags) were randomly selected, of which nine were related to the growth and development. The five sequences were related to the synthesis, transport and metabolism of secondary metabolites of D. officinalis. CONCLUSION: The full-length cDNA library of Lilianlou was successfully constructed by using SMART technology. The library has large capacity, high recombination rate and long inserted cDNA fragments, which can be used to identify, screen and express functional genes of Lilianlou.