中國農業科學 ?? 2019, Vol. 52 ?? Issue (20): 3507-3519.doi: 10.3864/j.issn.0578-1752.2019.20.002

? 作物遺傳育種·種質資源·分子遺傳學 ? 上一篇    下一篇

大豆類黃酮糖基轉移酶基因UGT73C19的功能研究

狄少康1,尹青崗2,夏亞迎1,2,龐永珍1()   

  1. 1 中國農業科學院北京畜牧獸醫研究所,北京100193
    2 中國科學院植物研究所,北京 100093
  • 收稿日期:2019-04-25 接受日期:2019-06-12 出版日期:2019-10-16 發布日期:2019-10-28
  • 通訊作者: 龐永珍 E-mail:[email protected]
  • 作者簡介:狄少康,E-mail:[email protected]
  • 基金資助:
    國家重點研發項目(2016YFD0101005)

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

ShaoKang DI1,QingGang YIN2,YaYing XIA1,2,YongZhen PANG1()   

  1. 1 Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193
    2 Institute of Botany, Chinese Academy of Sciences, Beijing 100093
  • Received:2019-04-25 Accepted:2019-06-12 Online:2019-10-16 Published:2019-10-28
  • Contact: YongZhen PANG E-mail:[email protected]

摘要:

【背景】 類黃酮是大豆中積累的一類重要的植物次生代謝產物,參與大豆的生長、發育和抗逆等諸多生理活動。由UDP-糖基轉移酶(UGT)催化的糖基化修飾是類黃酮生物合成的關鍵步驟。【目的】 通過系統研究大豆UGT73C19編碼重組酶的體外酶活特性和體內特性,完善大豆黃酮類化合物合成和積累的機制,為大豆品質的遺傳改良提供基因資源和理論基礎。【方法】 通過高效液相色譜(HPLC)的方法檢測大豆核心種質資源葉片中類黃酮的種類和含量,通過qRT-PCR的方法檢測了UGT的表達水平。以大豆Williams 82葉片cDNA為模板,克隆得到UGT73C19的編碼區序列。使用MEGA5和DNAMAN軟件進行多重序列比對,并構建進化樹。通過原核表達系統獲得UGT73C19的重組蛋白,分析UGT73C19重組蛋白對各種類黃酮苷元的糖基轉移活性,并通過高效液相色譜-質譜(HPLC-MS)對產物進行鑒定,確定重組蛋白的糖基化位點。利用qRT-PCR技術對UGT73C19在大豆不同組織的表達水平進行分析。構建植物過量表達載體,通過花序浸染法轉化擬南芥,獲得UGT73C19表達量高的純合株系,檢測轉基因株系葉片和種子中類黃酮的種類和含量。【結果】 通過HPLC分析大豆核心種質資源葉片類黃酮成分,發現不同品種中類黃酮的成分和含量存在明顯差異。根據類黃酮成分的不同,將大豆核心種質分為12種不同的類型。大豆核心種質資源葉片中總黃酮的含量與UGT73C19的表達水平呈正相關關系。克隆得到UGT73C19的編碼區序列,全長1 482 bp,編碼493個氨基酸,UGT73C19蛋白在C-端有一個保守的PSPG結構域。體外酶活分析表明,重組的UGT73C19蛋白對6種類黃酮苷元(山奈酚、槲皮素、楊梅素、芹黃素、大豆苷元和染料木素)都具有糖基轉移活性,其中對槲皮素的催化效率最高;糖基化位點分別位于類黃酮的5位和7位羥基上,重組UGT73C19蛋白的糖基化底物和位點具有多樣性。過量表達UGT73C19的擬南芥葉片和種子中的類黃酮總量明顯升高,其中葉片中總黃酮含量提高49%—70%,種子中總黃酮的含量提高34%—37%;尤其是種子中槲皮素3-O鼠李糖的含量顯著增加。【結論】 UGT73C19蛋白是催化合成大豆中多個類黃酮糖苷的關鍵糖基轉移酶,過量表達UGT73C19可以提高轉基因植物中黃酮醇糖苷和類黃酮的含量。

關鍵詞: 大豆, 糖基轉移酶基因, 類黃酮, 黃酮醇

Abstract:

【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.

Key words: soybean, UDP-glucosyltransferase, flavonoids, flavonols

福利彩票3d开奖结果