摘要： UGT是一类糖基转移酶，它对黄酮类化合物进行糖基化修饰，合成异黄酮糖苷，在异黄酮的合成、转运、存储等方面发挥了重要的作用。为了探索UGT功能，通过RT-PCR方法从中豆27中克隆获得GmUGT73基因的CDS全长序列。利用ProtParam tool等在线软件对GmUGT73蛋白序列及理化性质进行分析，预测结果表明，GmUGT73基因的编码区编码464个氨基酸，分子质量为51.186 5 ku，理论等电点（pI）为6.27，总原子数为7 176，分子式为C₂₂₈₁H₃₅₈₀N₆₀₈O₆₈₅S₂₂。GmUGT73蛋白总正电和负电残基数分别为37和41，蛋白的不稳定系数为36.93，表明该蛋白较稳定，蛋白的脂溶指数为86.19，GRAVY值为-0.111，说明该蛋白为亲水性蛋白。将线性植物表达载体与目的片段进行连接，成功构建pCambia3300-GmUGT73植物表达载体，通过发根农杆菌介导法转化大豆毛状根并获得转基因阳性根系，利用高效液相色谱（HPLC）对根系进行异黄酮含量测定，结果表明，转基因阳性根中异黄酮含量与对照相比极显著提高（P<0.01），说明该基因可能具有促进合成大豆异黄酮的功能。

关键词: 大豆, 异黄酮类, 糖基转移酶, 候选基因, 农杆菌转化, 克隆, 基因功能鉴定

Abstract: UGT is a kind of glycosyltransferase,which performs glycosylation modification on flavonoids,synthesizes isoflavone glycosides,and plays an important role in the synthesis,transport and storage of isoflavones. In order to explore the function of related genes,the full-length CDS sequence of GmUGT73 gene was cloned from Zhongdou 27 by RT-PCR. The GmUGT73 protein sequence and physicochemical properties were analyzed by online software such as ProtParam tool. The results indicated that the coding region of GmUGT73 gene encoded 464 amino acids with a molecular mass of 51.186 5 ku,a theoretical isoelectric point(pI)of 6.27 and a total atomic number of 7 176. The molecular formula was C₂₂₈₁H₃₅₈₀N₆₀₈O₆₈₅S₂₂. The total positive electroreactive residue of GmUGT7 protein was 37,the total number of negatively charged residues was 41,and the instability coefficient of protein was 36.93,indicating that the protein was stable. The protein had a fat solubility index of 86.19 and a GRAVY value of -0.111,indicating that the protein should be a hydrophilic protein. The linear plant expression vector was ligated to the target fragment,and the pCambia3300-GmUGT73 plant expression vector was successfully constructed. Transformation of soybean hairy roots by Agrobacterium rhizogenes-mediated transformation and obtained transgenic positive roots.The isoflavone content of roots were determined by high performance liquid chromatography. The results showed that the content of isoflavones in the positive roots of transgenic plants was extremely significantly higher than that of the control(P<0.01),indicating that the gene may have the function of promoting the synthesis of soy isoflavones.

Key words: Soybean, Isoflavone, Glycosyltransferase, Candidate gene, Agroinfiltration, Cloning, Functional identification of gene

中图分类号: 

• Q78

• S565.03