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以授粉后70d的罗汉果为材料,采用RT-PCR和RACE技术克隆到一个葡萄糖基转移酶基因,命名为SgUGT4。该基因cDNA全长1726bp,包含完整的开放阅读框1344bp,编码447个氨基酸。构建pET32a-SgUGT4原核表达载体,转化大肠杆菌Rossetta-gami(DE3),经IPTG诱导获得分子量约65k D的目的基因融合蛋白。系统进化分析表明,SgUGT4与具有罗汉果苷合成活性的拟南芥和甜叶菊葡萄糖基转移酶AtUGT73C3、AtUGT73C5、At UGT73C6、SrUGT73E1聚在一个亚家族。实时荧光定量PCR检测表明,SgUGT4在罗汉果甜苷Ⅴ主要积累部位果肉的表达量高于茎、叶、花和果皮,在根和种子中几乎不表达;果实发育40~50 d,甜苷Ⅴ开始积累,SgUGT4表达量则逐渐升高,50d时急剧上升;甜苷Ⅴ含量越高的品种,SgUGT4表达量也越高。SgUGT4可能在罗汉果甜苷Ⅴ生物合成过程中发挥作用。
After 70 days of pollination, we used a LuoGuoTuo as a material and cloned a glucosyltransferase gene by RT-PCR and RACE, named SgUGT4. The full length cDNA of this gene is 1726bp and contains the complete open reading frame of 1344bp and encodes 447 amino acids. The prokaryotic expression vector pET32a-SgUGT4 was constructed and transformed into Escherichia coli Rossetta-gami (DE3). The target gene fusion protein of about 65kD molecular weight was induced by IPTG. Phylogenetic analysis showed that SgUGT4 was localized in a subfamily with Arabidopsis thaliana and stevia glucosyltransferase AtUGT73C3, AtUGT73C5, At UGT73C6 and SrUGT73E1 with mogroside activity. The results of real-time fluorescent quantitative PCR showed that the expression of SgUGT4 in the main accumulation parts of mogroside Ⅴ was higher than that in stem, leaf, flower and pericarp, and almost no in roots and seeds; Accumulation, SgUGT4 expression gradually increased, rose sharply at 50d; the higher the content of glycosides Ⅴ, the higher the expression level of SgUGT4. SgUGT4 may play a role in mogroside Ⅴ biosynthesis.