大豆类黄酮糖基转移酶基因UGT73C19的功能研究

Functional Characterization of a UDP: Flavonoid Glycosyltransferase Gene UGT73C19 in Glycine max

【背景】类黄酮是大豆中积累的一类重要的植物次生代谢产物,参与大豆的生长、发育和抗逆等诸多生理活动。由UDP-糖基转移酶(UGT)催化的糖基化修饰是类黄酮生物合成的关键步骤。【目的】通过系统研究大豆UGT73C19编码重组酶的体外酶活特性和体内特性,完善大豆黄酮类化合物合成和积累的机制,为大豆品质的遗传改良提供基因资源和理论基础。【方法】通过高效液相色谱(HPLC)的方法检测大豆核心种质资源叶片中类黄酮的种类和含量,通过qRT-PCR的方法检测了UGT的表达水平。以大豆Williams 82叶片cDNA为模板,克隆得到UGT73C19的编码区序列。使用MEGA5和DNAMAN软件进行多重序列比对,并构建进化树。通过原核表达系统获得UGT73C19的重组蛋白,分析UGT73C19重组蛋白对各种类黄酮苷元的糖基转移活性,并通过高效液相色谱-质谱(HPLC-MS)对产物进行鉴定,确定重组蛋白的糖基化位点。利用qRT-PCR技术对UGT73C19在大豆不同组织的表达水平进行分析。构建植物过量表达载体,通过花序浸染法转化拟南芥,获得UGT73C19表达量高的纯合株系,检测转基因株系叶片和种子中类黄酮的种类和含量。【结果】通过HPLC分析大豆核心种质资源叶片类黄酮成分,发现不同品种中类黄酮的成分和含量存在明显差异。根据类黄酮成分的不同,将大豆核心种质分为12种不同的类型。大豆核心种质资源叶片中总黄酮的含量与UGT73C19的表达水平呈正相关关系。克隆得到UGT73C19的编码区序列,全长1482 bp,编码493个氨基酸,UGT73C19蛋白在C-端有一个保守的PSPG结构域。体外酶活分析表明,重组的UGT73C19蛋白对6种类黄酮苷元(山奈酚、槲皮素、杨梅素、芹黄素、大豆苷元和染料木素)都具有糖基转移活性,其中对槲皮素的催化效率最高;糖基化位点分别位于类黄酮的5位和7位羟基上,重组UGT73C19蛋白的糖基化底物和位点具有多样性。过量表达UGT73C19的拟南芥叶片和种子中的类黄酮总量明显升高,其中叶片中总黄酮含量提高49%—70%,种子中总黄酮的含量提高34%—37%;尤其是种子中槲皮素3-O鼠李糖的含量显著增加。【结论】UGT73C19蛋白是催化合成大豆中多个类黄酮糖苷的关键糖基转移酶,过量表达UGT73C19可以提高转基因植物中黄酮醇糖苷和类黄酮的含量。

【Background】Flavonoids are a group of important plant secondary metabolites accumulate in soybean,which are involved in many physiological activities,including soybean growth,development and stress resistance.Glycosylation catalyzed by UDP-glycosyltransferase is a key step in flavonoid biosynthesis.【Objective】The objective of the present study is to investigate the in vitro enzymatic activity and in vivo function of a soybean glycosyltransferase protein encoded by the UGT73C19 gene,the achievement of which will deep our understanding on the mechanism of the flavonoid biosynthesis in soybean.This study will provide gene resource and theoretical basis for the genetic modification in soybean.【Method】Flavonoids in the leaves of soybean core germplasm resources were detected by HPLC,and the expression level of UGT genes were detected by qRT-PCR.The coding region of the UGT73C19 gene was cloned from cDNA of soybean leaf(Williams 82).The amino acid sequences of UGT73C19 were searched in the NCBI database,and the software MEGA5 and DNAMAN were used for multiple sequence alignment and the construction of a phylogenetic tree.The recombinant UGT73C19 protein was expressed in E.coli and its enzymatic activity was determined towards various flavonoid aglycones.All the enzymatic products were identified by HPLC-MS.The expression profile of the UGT73C19 gene in soybean was analyzed by qRT-PCR.UGT73C19 was over-expressed in Arabidopsis thaliana by floral dipping method.Flavonoid content and composition were determined in seedlings and seeds in homozygous lines that showed the relatively high UGT73C19 expression level.【Result】Flavonoids in the leaves of soybean core germplasm showed significant differences in flavonoid composition and content in different varieties.Soybean core germplasm can be divided into 12 different types according to flavonoid composition.There was a positive correlation between the content of flavonoids and the expression level of UGT73C19 gene in the leaves of soybean core germplasm resources.The coding sequence of UGT73C19 gene was cloned,and the coding region was found to be 1482 bp,encoding a protein of 493 amino acids.The deduced UGT73C19 protein was found to have a conserved PSPG domain at the C-terminal.In vitro enzymatic activity analysis revealed that the recombinant UGT73C19 protein exhibited glycosyltransferase activity toward six flavonoid aglycones(kaempferol,quercetin,myricetin,apigenin,daidzein and genistein),and it showed the highest catalytic efficiency toward quercetin.The glycosylation sites were at the 5 and 7 hydroxy groups of flavonoid substrates,and the glycosylation substrates and sites of the recombinant UGT73C19 protein showed high diversity.It was found that the total flavonoid contents in the seedlings and seeds of the transgenic A.thaliana increased significantly,by 49% to 70% in leaves and 34% to 37% in seeds,in particular quercetin 3-O rhamnose in the seeds.【Conclusion】The recombinant UGT73C19 protein can catalyze the glycosylation of a group of flavonoid compounds and over-expression of UGT73C19 gene can increase the content of flavonols in plants like A.thaliana.