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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 PCRcDNA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。
Objective To construct subtracted cDNA library of root of Dendrobium officinale induced by symbiotic mycorrhizal fungi and select differentially expressed genes from them. Methods SMARTer PCR cDNA synthesis was used to directly synthesize and enrich rare cDNAs using total RNA as a template. Subtractive subtractive cDNA library of D. officinale induced by mycorrhizal fungi was constructed by suppression subtractive hybridization (SSH). Results The subtracted cDNA library of D. officinale induced by mycorrhizal fungi was successfully constructed and a total of 1 975 positive clones were obtained. After testing, more than 90% of the clones can amplify the effective product, and the size of the inserted fragment is between 150 and 1 000 bp. A total of 20 clones were randomly selected and sequenced and analyzed. Most of them were plant defense-responsive genes for environmental stress. Conclusion The subtractive subtractive cDNA library constructed by this technology system is of good quality and easy to operate, especially suitable for rare and endangered plant materials.