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目的构建金铁锁全长cDNA文库,为进一步开展金铁锁次生代谢合成途径相关基因的研究奠定基础。方法利用Rneasy Plant Mini Kit(Qiagen)试剂盒提取金铁锁根部总RNA,通过SMARTer(switching mechanism at 5’end of RNA transcript)技术构建全长cDNA文库,测定分析文库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分析。结果使用SMARTer技术构建的金铁锁cDNA文库的质量较高,在随机挑选的单菌落克隆中都有插入片段,重组率为100%;在15个单克隆中,扩增片段长度在750bp以上的有14个,占93.3%。结论利用SMARTer技术成功构建了金铁锁根部的全长cDNA文库,该文库可用于金铁锁今后功能基因组鉴定、新基因筛选及次生代谢产物生物合成的表达调控研究。
Objective To construct the full-length cDNA library of Jin Tie Su and lay the foundation for the further study of genes involved in the pathway of secondary metabolism of Jin Tie. Methods Total RNA was extracted from root of Rhizoma Ligustrum by using Rneasy Plant Mini Kit (Qiagen) kit. The full-length cDNA library was constructed by SMARTer (switching mechanism at 5’end of RNA transcript) technology. The recombination rate and insert size were determined and analyzed. Random sequencing and homology search and functional analysis in Genbank after Blastx analysis. Results The cDNA library of Atraz-Lock cDNA library constructed by SMARTer technique had high quality. The inserts were cloned from randomly selected single-colony clones with a recombination rate of 100%. Among 15 clones, the length of the cDNA fragments amplified above 750bp was 14 A, accounting for 93.3%. Conclusion The SMARTer technology was used to construct a full-length cDNA library of the roots of Jinkuijian, which can be used for the future functional genomics identification, new gene screening and expression regulation of secondary metabolites biosynthesis.