菌根真菌诱导的铁皮石斛根差减cDNA文库构建

 目的 构建菌根真菌共生诱导的铁皮石斛根的差减cDNA文库,从中筛选出差异表达基因。方法 采用SMARTer PCR cDNA合成方法直接以总RNA为模板合成并富集稀少cDNA,再利用抑制消减杂交（SSH）方法构建受菌根真菌共生诱导铁皮石斛根的差减cDNA文库。结果 成功构建了受菌根真菌诱导铁皮石斛根的差减cDNA文库,共获得1 975个阳性克隆。经检测,90%以上克隆能扩增出有效产物,其插入片段大小在150~1 000 bp之间。随机挑取了20个克隆菌液测序并进行了比对分析,大部分为植物应对环境胁迫防御性基因。结论 该技术体系所构建的差减cDNA文库质量较好,操作方便,尤其适用于珍稀濒危的植物材料。

Construction of Differential cDNA Library on the Roots of Dendrobium candidum Induced by a Symbiotic Mycorrhizal Fungus

OBJECTIVE To screen differentially expressed genes on the roots of Dendrobium candidum induced by a symbiotic mycorrhizal fungus, the differential cDNA library was constructed. METHODS The ds cDNAs were synthesized and enriched by SMARTer PCR cDNA synthesis kit using total RNA of D. candidum roots symbiotic with the fungus as templates. The differential cDNA library was constructed by using suppressive subtraction hybridization (SSH). RESULTS The differential cDNA library, containing 1 975 clones in the storage capacity, was constructed successfully. It was detected that above 90% clones could be amplified effective products. The lengths of the differential cDNA fragments cloned were 150 bp to 1 kb by electrophoresis detection to 20 randomly selected clones. Then, the 20 ESTs similarity analysis based on BLASTx software was carried on and most of the cloned genes were the defensive genes of plant responding to environmental stresses. CONCLUSION Using this technology system could construct a fine differential cDNA library and be operated easily, especially suitable to the rare species.