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目的:为了获得药用植物铁皮石斛功能基因的信息和筛选功能基因。方法:以SMART(switching mechanism at5’end of RNA transcript)方式合成全长cDNA,结合DSN(duplex-specific nuclease)均一化技术,构建铁皮石斛茎组织的均一化全长cDNA文库。结果:该文库重组率为93.9%,文库滴度1.3×106cfu.mL-1,插入片段平均大小1.5 kb左右。随机挑取163个阳性克隆进行单侧测序,通过生物信息学分析,获得150个单一序列,其中147个序列与相应的同源蛋白匹配,GO(geneontology)分类表明这些序列参与各种生化途径。8个序列包含了完整编码框,可判断为全长基因。结论:成功构建了铁皮石斛茎组织均一化全长cDNA文库,为满足铁皮石斛代谢途径等功能基因筛选和基因信息研究奠定了基础。
OBJECTIVE: To obtain information and screen functional genes of medicinal plants Dendrobium officinale. METHODS: Full-length cDNA was synthesized by SMART (switching mechanism at5’end of RNA transcript) and homogenized full-length cDNA library of Dendrobium officinale with tissue duplexization-specific nuclease (DSN). Results: The library recombination rate was 93.9%, the library titer was 1.3 × 106cfu.mL-1, and the average insert size was about 1.5 kb. One hundred and seventy-six positive clones were randomly selected for unilateral sequencing and bioinformatics analysis was performed to obtain 150 single sequences, of which 147 were matched with the corresponding homologous proteins. GO (gene ontology) classification indicated that these sequences were involved in various biochemical pathways. Eight sequences contain a complete coding frame, can be judged as full-length genes. CONCLUSION: A homozygous full-length cDNA library of Dendrobium officinale has been successfully constructed, which lays the foundation for functional gene screening and gene information research such as the metabolic pathway of Dendrobium candidum.