茶树谷氨酸合成酶参与茶氨酸代谢的调控机制研究

Tea plant, Camellia sinensis (L.) O. Kuntze, which belongs under the genus Camellia of the family Theaceae, is a perennial evergreen woody plant. Tea leaves are important raw materials for non-alcoholic beverage processing. Tea is rich in a variety of beneficial secondary metabolites, such as tea polyphenols, theanine and caffeine. L-theanine, or called as γ-glutamyl-L-ethylamide or γ-ethylamino-L-glutamic acid, is a unique non-protein amino acid in C. sinensis. All theanine that naturally exists in tea plant belongs to L-theanine, D-theanine can only be produced industrially. L-theanine is an important indicator in the quality evaluation of green tea because of the particular umami taste that largely influences the flavor of tea. As an important and highly abundant free amino acid in tea plants, L-theanine, almost can be detected in all tissues. However, L-theanine content in tea plants considerably varies among different cultivars and tissues.. At present, some enzymes have been confirmed directly participating the theanine synthesis and hydrolysis. Glutamine synthase/glutamate synthase (GS/GOGAT) cycle is the major pathway for ammonium assimilation, which produces the precursor glutamate for theanine production. Glutamate synthase play important role in theanine metabolism of C. sinensis. Till now, little information is available on the regulation mechanism of glutamate synthase involved in theanine metabolism in C. sinensis. Our previous results demonstrated that the glutamate synthase were strongly correlated with theanine metabolism and content in C. sinensis.. Based on the results of methods of morphological, physiological, anatomical, theanine and protein amino acids contents determination, gene clone, promoter clone, gene expression profiles, yeast hybrid system, Arabidopsis and Nicotiana tabacum transgenic plants with over-expression, genome, transcriptome and proteome analysis, an extensive picture of the regulation mechanism of glutamate synthetase gene involved in theanine metabolism in C. sinensis shall be attempted. Some areas were highlighted below: anatomic characteristics of C. sinensis; distribution and content of theanine; function of glutamate synthase genes in theanine biosynthesis. The correlation of glutamate synthetase gene and theanine metabolism; the function of glutamate synthetase gene studied by Arabidopsis and N. tabacum transgenic plants. The researches in the project have a good foundation. The results of the project shall not only help to ascertain the function of glutamate synthetase gene involved in theanine metabolism of C. sinensis., but also provide a scientific basis for improvement of quality of C. sinensis. The project shall also provides a scientific basis for the improvement of quality of tea.

茶树是多年生木本植物，有很高的经济价值。茶氨酸与茶叶品质密切相关，不同茶树品种和组织中茶氨酸含量相差很大。谷氨酰胺合成酶/谷氨酸合成酶循环是氨同化的主要途径，产生茶氨酸合成的前体谷氨酸，在茶树茶氨酸代谢中起重要作用。谷氨酸合成酶调控茶树茶氨酸代谢的机制还不清楚。申请者基于转录组和蛋白质组分析，证实茶树谷氨酸合成酶与茶氨酸代谢和含量之间存在密切的相关性。本项目在前期研究基础上，运用形态结构观察、茶氨酸和蛋白氨基酸的测定、基因克隆、启动子克隆、基因表达、酵母杂交、拟南芥和烟草遗传转化等技术，结合基因组、转录组和蛋白质组学分析等方法，开展谷氨酸合成酶对茶树茶氨酸代谢调控的机理研究。着重研究：茶树根与叶结构特征；茶氨酸积累动态变化；谷氨酸合成酶与茶氨酸代谢关系；转基因拟南芥和烟草验证茶树谷氨酸合成酶基因功能等。项目有基础，研究结果将进一步丰富茶树茶氨酸代谢调控的分子机制，也为茶树品质改良提供依据。

茶树是山茶科山茶属多年生常绿木本植物，含有丰富的次生代谢物质，如茶多酚、茶氨酸、芳香物质等。茶氨酸是茶叶质量评估的一项重要指标，它具有独特的鲜味，可以很大程度上影响茶的滋味。茶树中茶氨酸的含量与分布差异与关键酶之间的功能机制还不十分清楚，在不同茶树品种和组织之间，关键酶的结构和基因表达差异对茶氨酸代谢的影响机制复杂。谷氨酸合成酶（glutamate synthetase，GOGAT）在茶氨酸合成过程中扮演重要的角色。本项目围绕研究内容，主要开展了茶树谷氨酸合成酶参与茶氨酸代谢的调控机制研究，围绕茶树中茶氨酸代谢途径的分子机制开展了不同茶树品种、不同发育时期、不同器官以及不同处理茶氨酸及相关氨基酸含量测定与时空特点分析；观察与分析茶树叶和根形态、生理和结构特点及其差异；茶树茶氨酸代谢基因的表达与茶氨酸含量相关性分析；茶树谷氨酸合成酶结构和蛋白互作分析；开展茶氨酸代谢的相关组学研究，进一步阐释谷氨酸合成酶基因在茶氨酸代谢中的调控机制。随着茶树叶片成熟度增加茶氨酸含量逐渐降低，茎中含量最低，根中含量仅次于芽。茶氨酸含量分别在根和老叶中最高和最低，叶片中茶氨酸含量随着叶片成熟度的增加而逐渐降低。茶树氨基酸含量的动态变化总体来说，根和新稍的氨基酸总量远远多于老叶，随着时间的推移，氨基酸含量增加。茶树中茶氨酸的含量与不同N素水平和激素含量有关。CsFd-GOGAT在酵母中与CsNADH-GOGAT、CsTS1、CsNiR之间有直接互作。组学分析显示氮缺乏和氮充足条件下，茶树新稍与根之间茶氨酸代谢差异和响应缺氮胁迫密切相关。本项目开展茶树中茶氨酸合成规律、空间分布特征、谷氨酸合成酶在茶树茶氨酸代谢途径中的功能等的研究，为进一步明确关键酶在茶树次生代谢中的功能，为茶树品质的改良提供理论基础，同时也具有较大的潜在实际应用价值。