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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1 413bp,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54 900,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 To study the pathways of polysaccharide metabolism of Codonopsis pilosula and clone the CpGGPase gene, a key enzyme in the metabolism of polysaccharide, from Codonopsis pilosula root, and analyze the sequence and prokaryotic expression. Methods Primers were designed according to the CpUGPase gene sequence of Codonopsis pilosula transcriptional group. The open reading frame (ORF) of CpUGPase gene was amplified by RT-PCR and cloned into pGEX-T vector. The prokaryotic expression vector PET-28a-CpUGPase After E. coli BL21 (DE3), expression was induced by isopropyl-β-D-thiogalactoside (IPTG). Results The sequence of CpUGPase ORF was 1 413bp in length and encoded 470 amino acids. Sequence analysis showed that the CpUGPase gene has a conserved UGPase_euk catalytic site and belongs to the type A glycosyltransferase family of proteins. Construct PET-28a-CpUGPase recombinant plasmid and obtain stable prokaryotic expression system. SDS-PAGE results showed that the size of the protein was about 54,900, which was consistent with the predicted protein relative molecular mass. Conclusion CpUGPase gene was cloned from Codonopsis pilosula and a stable prokaryotic expression system was established, which laid the foundation for the further purification of CpUGPase protein and its structure and function.