目的 为研究党参多糖代谢途径，从党参Codonopsis pilosula根中克隆多糖代谢关键酶尿苷二磷酸葡萄糖焦磷酸化酶CpUGPase基因，并进行序列分析和原核表达。方法 根据党参转录组中CpUGPase基因序列设计引物，通过RT-PCR扩增得CpUGPase开放阅读框（ORF）序列，并进行TA克隆、测序和序列分析；构建原核表达载体PET-28a-CpUGPase，转入大肠杆菌BL21（DE3）后，在异丙基-β-D-硫代半乳糖苷（IPTG）诱导下进行表达。结果 CpUGPase ORF序列全长1413 bp，编码470个氨基酸。序列分析表明，CpUGPase基因具有保守的UGPase_euk催化位点，属于A型糖基转移酶蛋白家族。构建PET-28a-CpUGPase重组质粒，获得稳定的原核表达体系。SDS-PAGE结果显示该蛋白的大小约54900，与预测的蛋白相对分子质量一致。结论 从党参根中克隆得到CpUGPase基因，并建立了稳定的原核表达体系，为进一步纯化CpUGPase蛋白，研究其结构和功能奠定了基础。

Objective In order to study the metabolic pathway of Codonopsis Radix polysaccharide, the key enzyme gene UDP-glucose pyrophosphorylase involving in the polysaccharide metabolism, a CpUGPase gene was cloned from the roots of Codonopsis Radix, and its sequence analysis and prokaryotic expression were performed.Methods According to the CpUGPase gene sequence of transcriptome of Codonopsis Radix, a pair of primers were designed, and the open reading frame (ORF) of cDNA sequences was obtained by RT-PCR. Then TA cloning, sequencing, and sequence analysis were performed. Prokaryotic expression vector PET-28a-CpUGPase was constructed and transformed into Escherichia coli BL21 (DE3) for the expression under the induction of isopropyl β- D-1-thiogalactopyranoside (IPTG).Results The ORF of CpUGPase had a length of 1413 bp coding for 470 amino acids. Sequence analysis showed that CpUGPase had a conserved UGPase_euk catalytic site and belonged to A type of glycosyl transferase protein family. PET-28a-CpUGPase recombinant plasmid was constructed to obtain a stable prokaryotic expression system. The SDS-PAGE results showed that the expressed protein were about 54 900 in size and consistent with the anticipated size.Conclusion The CpUGPase gene is successfully cloned, and the stable prokaryotic expression system is established. This study will provide a foundation for the further purification, structural and functional researches of CpUGPase protein.

国家自然科学基金项目（81072987）；国家科技支撑计划项目（2011BAI07B07）；山西省高等学校中青年拔尖创新人才支持计划（晋教材 [2012] 146号）