摘要
从罗汉果(Siraitia grosvenorii)转录组中获得一条与罗汉果甜苷Ⅴ生物合成相关的葡萄糖基转移酶(UDPG)的unigene片段,以罗汉果授粉后70d的果实RNA为模板,利用RACE和RT-PCR技术克隆UDPG全长基因,将克隆得到的SgUDPG1基因连接到原核表达载体pEASY-E1上,构建融合表达载体,转化到大肠杆菌BL21(DE3),通过IPTG诱导表达,重组蛋白纯化,SDS-PAGE检测表达产物以及Western-blotting和质谱鉴定蛋白产物。结果表明,获得了1条SgUDPG1,全长为1959bp,开放阅读框ORF为1365bp,编码1条454aa的肽链,理论分子量为51.2kD,等电点为5.39,具有植物中次生代谢产物糖基转移酶特有的保守结构域PSPG-boxmotif。SgUDPG1在授粉后50d和70d的果实中表达逐渐升高,是对照授粉后3d的5.16倍和13.12倍,与果实中甜苷Ⅴ含量呈相同趋势。此基因的ORF可以在大肠杆菌中表达,并且可以纯化出比理论分子量大5.3kD的融合蛋白,通过Western-blotting和质谱鉴定,确定该蛋白属于罗汉果葡萄糖基转移酶。
Cloning, sequence analysis of the UDP-glycosyltransferase gene from Siraitia grosvenorii and its expression in E. coli were conducted to further explore the relationship between SgUDPG1 expression and mogroside V biosynthesis. The UDP-glycosyltransferase cDNA fragments amplified from Siraitia grosvenorii transcriptome by rapid amplification of cDNA ends （RACE） and RT-PCR, and then the cloned gene of SgUDPG1 was inserted into vector pEASY-E1. The recombinant plasmid pEASY-E1-SgUDPG1 was expressed in a prokaryotic expression system after it was transformed intoE. coli BL21 （DE3） .The fusion protein was analyzed by SDS-PAGE, Western-blotting and mass spectrometry. The results showed that a full-length SgUDPG1 cDNA of 1 959 bp including open reading frame （ORF） of 1 365 bp was isolated. The predicted SgUDPG1 protein has 454 amino acids with an estimated molecular mass of 51.2 kD and an isoelectric point of 5.39. The SgUDPG1 contains 6 conserved domains predicted by InterProScan, including PSPG-box motif which is a unique consensus sequence of glycosyltransferases involved in plant secondary metabolism. DGE analysis showed that the expression of SgUDPG1 in 50 d and 70 d increased 5.16 and 13.12 times respectively than the 3 d. The results of SDS-PAGE, Western-blotting and mass spectrometry demonstrated that the recombinant plasmid pEASY- E1-His-SgUDPG1 could express a fusion protein whose molecule mass is 5.3 kD larger than the predicted molecule mass. Therefore, the SgUDPG1 of Siraitia grosvenorii was cloned and successfully expressed in E. coli. This study will provide a foundation for studying the function of this UDP-glycosyltransferase.
出处
《园艺学报》
CAS
CSCD
北大核心
2013年第6期1195-1204,共10页
Acta Horticulturae Sinica
基金
国家自然科学基金项目(30960500)
国家‘十二五’科技支撑计划项目(2011BA101B03)
广西自然科学基金项目2013GXNSFBA019170)
广西卫生厅中医药科技专项项目(GZPT1235)
广西研究生科研创新项目(T32560)
关键词
罗汉果
葡萄糖基转移酶
RACE
原核表达
融合蛋白
Siraitia grosvenorii
UDP-glycosyltransferase
rapid amplification of cDNA ends：prokaryotic expression
fusion protein
