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园艺学报 ›› 2015, Vol. 42 ›› Issue (3): 523-534.doi: 10.16420/j.issn.0513-353x.2014-0757

• 其它园艺植物 • 上一篇    下一篇

罗汉果葡萄糖基转移酶基因SgUGT4的克隆及表达研究

莫长明,马小军,唐 其,白隆华,潘丽梅,冯世鑫   

  1. 1广西大学农学院,南宁 530004;2广西药用植物园,南宁 530023;3中国医学科学院药用植物研究所,北京 100193;4湖南农业大学园艺园林学院,长沙 410128
  • 出版日期:2015-03-25 发布日期:2015-03-25
  • 基金资助:

    国家自然科学基金项目(30860379,31400275);国家科技支撑计划项目(2011BA101B03);广西自然科学基金项目(2013GXNFSBA019170);广西卫生厅中医药科技专项(GZPT1235)

Cloning and Expression Patterns of SgUGT4 Gene from Siraitia grosvenorii

MO Chang-ming1,2,MA Xiao-jun3,*,TANG Qi4,*,BAI Long-hua2,PAN Li-mei2,and FENG Shi-xin2   

  1. 1Agricultural College,Guangxi University,Nanning 530004,China;2Guangxi Botanical Garden of Medicinal Plant, Nanning 530023,China;3Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences,Beijing 100193,China;4College of Horticulture and Landscape Architecture,Hunan Agricultural University,Changsha 410128,China
  • Online:2015-03-25 Published:2015-03-25

摘要: 以授粉后70 d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1 726 bp,包含完整的开放阅读框1 344 bp,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65 kD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、AtUGT73C6、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,50 d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。

关键词: 罗汉果, 葡萄糖基转移酶, 基因克隆, 原核表达, 时空表达

Abstract: A glucosyltransferase gene,named as SgUGT4,was cloned from Siraitia grosvenorii fruits at 70 days after pollinating(DAP)by RT-PCR and RACE methods. The full-length cDNA of SgUGT4 is 1 726 bp and has an open reading frame(ORF)of 1 344 bp which encodes a predict protein of 447 amino acids. The recombinant plasmid pET32a-SgUGT4 was constructed in a prokaryotic expression system and then was transformed into E.coli strain Rossetta-gami(DE3),A 65 kD fusion protein was expressed after being induced by IPTG. Phylogenetic tree analysis showed that SgUGT4 was classified as the same subfamily as the glucosyltransferase of AtUGT73C3,AtUGT73C5,AtUGT73C6 from Arabidopsis thaliana and SrUGT73E1 from Stevia rebaudiana,which could catalyze mogrosides biosynthesis.qRT-PCR analysis demonstrated that the expression level of SgUGT4 in pulps where mogroside Ⅴ most abundantly accumulated was more higher than that in stems,leaves,flowers and peels. SgUGT4 did not nearly expressed in roots and seeds. From 40 DAP to 50 DAP,the content of mogrosideⅤgradually occurred and accumulated,the expression level of SgUGT4 rose gradually. The expression level of SgUGT4 up-regulated sharply after 50 DAP. The more higher the mogroside Ⅴ content in varieties was,the more higher its expression level of SgUGT4 in varieties was. Therefore,the SgUGT4 probably involved in mogroside Ⅴ biosynthesis.

Key words: Siraitia grosvenorii, glucosyltransferase, gene cloning, prokaryotic expression, temporal and spatial expression

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