彩叶草叶片cDNA文库的构建与分析

Construction and Analysis of cDNA Library from the Leaf of Coleus blumei Benth.

用植物(叶)总RNA抽提试剂盒提取彩叶草红色品种顶部叶片总RNA,用SMARTcDNALi-braryConstructionKit构建cDNA文库。经测定原始文库滴度为1.2×106pfu/ml,扩增总文库滴度为6.7×109pfu/ml,重组率达到98%,插入片段在0.5kb到2kb之间,1kb以上的占60%。通过PCR检测,从总文库中检测到了彩叶草CHS、DFR及ANS基因的特异片段。各项指标都表明,已获得较高质量的cDNA文库,为彩叶草叶色基因的分离克隆,彩叶草类黄酮类色素合成的分子调控的研究奠定了基础。

Total RNA was extracted from the top leaves of red forma of C.blumei using Plant Total RNA Extraction kit, and cDNA library was constructed with SMART cDNA Library Construction Kit. The titer of the primary library was 1.2×10^6 pfu/ml, in which 98% clones were recombinant and the inserted cDNA fragments ranged from 0.5 kb to 2 kb, 60% of them more than lkb. The amplified library has a titer of 6.7×10^9 pfu/ml. The positive signals of CHS, DFR and ANS gene were detected by PCR in the amplified library. The data indicate that the cDNA Library has high quality, and provides a useful tool for cloning leaf color genes and further study on the molecular mechanisms of the pigments biosynthesis of the flavonoids in C.blumei.