谷胱甘肽调控小白菜硫代葡萄糖苷代谢机理研究

Glucosinolates are the main secondary metabolism products of the Brassicaceae family, which is of value to the flavor and nutritional qualities of this category food crops. The degradation products of glucosinolates have been proved having strong anticarcinogenic properties. The glucosinolate system is also considered to be an efficient defense system for plant to be against many biotic and abiotic stresses. The tripeptide GSH (γ-Glu-Cys-Gly) is the most abundant low-molecular-weight thiol in the cell and is involved in numerous cellular processes. In addition to the proven biological roles of GSH, a role as sulfur donor in the biosynthesis of glucosinolates has been proven. However, the detail mechanism of GSH-regulated glucosinolate metabolism still remains to be proven. Since GSH and glucosinolate both are the important sulfur-containing metabolites of the order Brassicales, which includes the agriculturally important oilseed rape (Brassica napus), and the cruciferous vegetables. It is of great importance to study the GSH-regulated glucosinolate mechanism for understanding the sulfur metabolism in Brassicales plants, which can be a great use to guide agricultural practice and human health..In this program, pakchoi with different glutathione-S-transferase (GST) levels and γ-glutamyl peptidase (GGP) levels produced by transgenic method will be used as plant materials to study the effects of different GSH level (produced by foliar spray GSH or GSH inhibitor BSO) on glucosinolates metabolism. The relationship between glucosinolate metabolism and GSH metabolism will be analysized aiming to illustrate the glucosinolate mechanism modulated by GSH. We hope this study can help to understand the GSH-regulated glucosinolate metabolism in Brassicaceae, which can be further used as an effective strategy of modulating glucosinolates profile and content to enhance the plant resistance to environment stress and improve the quality of agricultural produces.

小白菜是我国最常栽培的蔬菜之一，栽培面积和消费量大。硫代葡萄糖苷（硫苷）是十字花科植物中重要的含硫次生物质，它的水解产物已被证实是十字花科蔬菜主要的风味物质、具有防癌抗癌等作用，同时还具有抗病虫功能、是植物防御体系的重要组成部分。硫苷代谢过程十分复杂，过去一直认为硫苷中的硫主要来源于半胱氨酸，最新文献报道证实谷胱甘肽（GSH）也作为硫源参与了硫苷合成，但GSH调控硫苷合成的具体机制仍有待研究。作为对十字花科蔬菜的生长发育、抗逆和营养品质都具有重要作用的主要含硫物质，研究GSH和硫苷调控机制对于了解此类作物的硫代谢以及防御机制具有重要意义。本项目以小白菜为试材，基因工程方法创建具有不同GST和GGP水平的实验材料，用外源GSH和GSH抑制剂BSO处理，研究GSH处理后小白菜中GSH和硫苷代谢情况，阐明GSH调控小白菜硫苷代谢的具体途径和机制，为后续GSH和硫苷调控的理论研究和应用打好基础。

小白菜是我国最常栽培的蔬菜之一，栽培面积和消费量大，硫代葡萄糖苷（硫苷）是其主要营养品质成分。硫苷是十字花科植物中重要的含硫次生物质，它的水解产物已被证实是十字花科蔬菜主要的风味物质、具有防癌抗癌等作用，同时还具有抗病虫功能、是植物防御体系的重要组成部分。谷胱甘肽（GSH）是植物体内广泛存在的生物活性物质，与植物的含硫代谢和抗性密切相关。本项目研究了谷胱甘肽（GSH）作为硫源参与硫苷合成调控的机制。.（1）分离鉴定了小白菜BcCYP79B2、BcCYP79F1、BcCYP83A1、BcCYP83B1、BcSUR1、BcUGT74B1、BcST5a、BcST5c、BcMYB28、BcMYB34等硫苷代谢相关基因，BcGSH1、BcGSTF10、BcGSTU20、BcGGP1等谷胱甘肽代谢相关基因，BcATPS1、BcAPR1、BcSIR、BcAPK1等硫初生代谢相关基因，构建了AntiCYP83A1、AntiCYP83B1和AntiBcGSTU20、AntiBcGGP1表达载体，经过遗传转化获得了硫苷低积累的AntiCYP83B1小白菜材料，为后续研究积累了丰富的材料。.（2）分析鉴定了小白菜中GSH代谢、硫苷代谢和初生硫代谢三条途径，建立了基于基因表达、酶活性分析和含量测定的代谢分析系统，为后续研究奠定基础。.（3）采用GSH、GSSG、BSO对小白菜叶片进行外源喷施的方法，从基因表达层面、酶活性和前体物质层面、物质含量层面研究了GSH及其抑制剂BSO处理对小白菜叶片GSH代谢系统、硫苷代谢系统和初生硫代谢系统的影响，找出了GSH影响硫苷合成和初生硫代谢的关键环节，综合分析了GSH影响小白菜硫苷代谢的主要途径及其调控机制。. 研究结果为小白菜等十字花科蔬菜的营养品质和抗性调控机制研究，为GSH代谢、硫苷代谢和硫代谢调控机制研究奠定了理论基础，对于了解此类作物的硫代谢以及防御机制具有重要意义；本项目所创制的植物材料为后续研究开展积累了材料；本项目的研究结果为后续通过外施肥料等调节剂改善十字花科蔬菜品质提供了应用基础。