目的 克隆出金线莲Anoectochilus roxburghii尿苷二磷酸葡萄糖焦磷酸化酶（UDP-Glucose pyrophoshorglase，UGPase）基因全长，并对其表达量及酶活性与多糖积累量之间的相关性进行分析，以研究UGPase基因在金线莲多糖合成代谢途径中的调控作用。方法 利用实时荧光定量PCR（Quantitative Real-time PCR，qRT-PCR）技术和RACE（rapid-amplification of cDNA ends）技术克隆金线莲中UGPase基因序列；以金线莲尖叶、无纹等7个品种组培期及种植期茎段部位为材料，采用qRT-PCR、ELISA试剂盒法及苯酚-硫酸法分别测定UGPase基因表达量、酶活和多糖含量，并对其进行相关性分析。结果 克隆得到的序列全长1786 bp。开放阅读框为1425 bp，编码475个氨基酸。7个不同品种金线莲中UGPase基因表达量、酶活与多糖含量具有极显著正相关（相关系数分别为0.823、0.785）。结论 成功克隆出金线莲UGPase全长基因，初步证明UGPase基因是参与金线莲多糖合成代谢的关键基因之一。

Objective UDP-Glucose pyrophoshorglase (UGPase) gene of Anoectochilus roxburghii was cloned and the correlation between the expression content of gene and enzyme activity and polysaccharide accumulation was analyzed to explore the role of UGPase gene in the regulation of polysaccharide synthesis and metabolism. Methods In this study, the stem segments of A. roxburghii were used to clone the full-length sequence of UGPase gene via RT-PCR and RACE technology. RT-qPCR, ELISA technology and phenol-sulfuric acid process were used to determine the expression of UGPase andenzyme activity in seven different varieties of A. roxburghii and the content of polysaccharide. Finally, the correlation was analyzed. Result The cloned sequence is 1786bp in length, the ORF is 1425bp, encodes 475 amino acids. The results of correlation analysis showed that the expression level of UGPase gene and enzyme activity had significant positive correlation with the content of polysaccharide in seven different varieties of A. roxburghii (the correlation coefficients were 0.823 and 0.785, respectively). Conclusion The Full-length sequence of UGPase gene was successfully cloned in this study. Preliminary proof that UGPase gene is the one of the key genes involved in the anabolic polysaccharide metabolism.

R282.12

浙江省科技厅中药材育种专项（2016C02058）；浙江省重点研发项目（2017C02019）；浙江金线莲质量分析评价与特色功能产品开发项目（20202002a）和浙江万里学院省一流学科开放基金项目（KF2018001）共同资助