应用抑制性差减杂交技术构建铁皮石斛差减cDNA文库的探讨

【目的】探讨构建铁皮石斛抑制差减文库的方法，为克隆石斛碱生物合成相关功能基因奠定基础。【方法】采用抑制性差减杂交(SSH)技术，分别以4年生和1年生铁皮石斛叶片互补脱氧核糖核酸(cDNA)作为检测子(tester)与驱赶子(driver)，将所获得差减cDNA片段克隆入质粒表达载体(Point^TMXa-1 T-Vector)，转化大肠杆菌JM109，聚合酶链反应(PCR)扩增鉴定插入片段。【结果】成功地构建了与石斛碱生物合成相关的差减cDNA文库，获得的正、反向差减文库分别含560、220个重组子；插入片段的平均大小为550bp。【结论】所构建的差减cDNA文库适合进一步克隆分析石斛碱相关功能基因研究。

Construction of Subtractive cDNA Library from the Leaves of Dendrobium candidum Wall. ex Lindl. by Suppression Subtractive Hybridization Technology

Objective] Subtractive cDNA libraries from the leaves of D. candidum were constructed to supply evidence for the clone of dendrobine-biosynthesis-associated genes. Methods ] Suppression subtractive hybridization ( SSH) technology was used. With the cDNA of perennial leaf as the tester and that of annual leaf as the driver, forward and reverse hybridization was performed. The two obtained subtracted cDNA fragments were cloned into PointTMXa-1 plasmid vectors, and then the vectors were transformed into E. Coli JM109. The inserted fragments were amplified by PCR and then identified. Results] The subtractive cDNA libraries related with dendrobine-biosynthesis-associated genes were successfully constructed. The forward and reverse subtractive libraries included 560 and 220 clones respectively. Detected by PCR, the length of the inserted fragments was 550 bp on average. Conclusion] The constructed subtractive libraries are suitable for further study on the functional genes associated with dendrobine biosynthesis of D. candidum.