金花茶类黄酮糖基转移酶基因的克隆和功能初步研究

Cloning and Preliminary Functional Study of UDP-flavonoid Glycosyltransferase Genes of Camellia nitidissima Chi

类黄酮糖基转移酶(UDP-flavonoid glycosyltransferase,UFGT)催化黄酮醇、花青素等形成稳定的糖苷,是黄酮醇苷、花青素苷合成的最后一步反应的关键。该研究以金花茶的花瓣为材料,采用PCR扩增的方法,获得了2个金花茶转录组中筛选到的类黄酮糖基转移酶基因。结果显示:(1)CnUFGT14基因(登录号为MT370521)全长1562 bp,开放阅读框1380 bp,编码459个氨基酸;CnUFGT15基因(登录号为MT370520)全长1546 bp,开放阅读框1368 bp,编码455个氨基酸;两个蛋白序列均具有UFGT蛋白特有的PSPG保守区域。(2)系统进化树分析发现,CnUFGT14和CnUFGT15分别与茶树UFGT78A14和UFGT78A15亲缘关系最近。(3)荧光定量PCR分析发现,CnUFGT14基因的表达量与多种多酚组分的含量呈正相关,CnUFGT15基因的表达量与花瓣黄酮醇、多酚等的含量相关性不显著。(4)亚细胞定位研究发现,CnUFGT14、CnUFGT15蛋白在细胞核膜、细胞质、细胞膜部位均呈现明显的定位。(5)叶盘法转化烟草发现,CnUFGT14基因表达量较高的转基因株系中总多酚含量及多种多酚组分含量升高,而CnUFGT15基因的转基因株系中黄酮、多酚组分变化不显著。研究表明,CnUFGT14基因具有促进多酚合成的作用,而CnUFGT15基因对类黄酮通路不具有明显作用。

UDP-flavonoid glycosyltransferase(UFGT)catalyzes the formation of stable glycosides such as flavonols and anthocyanins,which is the key to the last step of the synthesis of flavonol and anthocyanin glycosides.In this study,we used the petals of Camellia nitidissima Chi as materials,cloned two UDP-flavonoid glycosyltransferase genes selected from the transcription by PCR amplification.The results show that:(1)CnUFGT14 gene,with GenBank numbers MT370521,was 1562 bp in length,1380 bp in open reading frame,and encoded 459 amino acids.CnUFGT15 gene,with GenBank numbers MT370520,was 1546 bp in length,1368 bp in open reading frame,and encoded 455 amino acids.Both protein sequences had a conserved region of PSPG characteristic of UFGT proteins.(2)Phylogenetic tree analysis showed that CnUFGT14 and CnUFGT15 were closely related to UFGT78A14 and UFGT78A15 in C.sinensis,respectively.(3)Quantitative PCR analysis showed that the expression levels of CnUFGT14 were positively correlated with the contents of multiple polyphenol components,while CnUFGT15 expression levels were not significantly correlated with the contents of flavonols and polyphenols.(4)The subcellular localizations showed that CnUFGT14 and CnUFGT15 proteins were in the nuclear membrane,cytoplasm and cell membrane.(5)The transform of tobacco with leaf disc method found that the total polyphenol content and the contents of multiple polyphenol components in the plants with high expression level of CnUFGT14 increased,while the changes of flavonoids and polyphenol components in the transgenic lines of CnUFGT15 were not significant.All the results indicated that CnUFGT14 gene can promote the synthesis of polyphenols and CnUFGT15 had no obvious effect on the flavonoid pathway.