茶叶调控谷氨酸脱羧酶合成γ-氨基丁酸的机制

γ-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in mammalian. GABA-rich products can be used to treat epilepsy and other diseases. GABA production is usually from bioaccumulation. GABA in plants is related to response to the stress. In the study of Arabidopsis and other model plants, it was found that under anaerobic stress, there had been no difference for the gene expression levels or the enzyme activities of glutamic acid decarboxylase (GAD), which was responsible for the synthesis of GABA. It was confirmed that GAD was stimulated by pH and activated by calmodulin (CaM). GABA levels were increased by dozens of times in postharvest tea leaves under anaerobic stress, but its regulation mechanism is still unclear. In our previous studies, it was found that the expression level of one GAD gene member had been increased by 70 times, which indicated that there might be a different regulation mechanism in tea from that in other plants. Therefore, in this project, we intend to express three GAD members of tea in vitro, detect their tissue specificity, and confirm their catalytic function in vivo; Meanwhile, a CaM-binding domain-deletion mutants of GADs will be constructed and examine their regulation in tobacco. This project will help uncover the molecular mechanism of GABA accumulation in postharvest tea, and reveal whether the regulation mechanisms of different GAD members in tea are the same, and whether they are consistent with other plants.

γ-氨基丁酸（GABA）是哺乳动物中重要的抑制性神经传递物质，富含GABA的产品可用于治疗癫痫等病症。GABA生产通常利用生物富集的途径。植物中的GABA与逆境下的应激反应有关。在拟南芥等植物的研究中发现，合成GABA的谷氨酸脱羧酶（GAD）在厌氧胁迫下的基因表达量和酶活力都没有变化，其活性是受pH和钙调蛋白（CaM）的激活。茶叶采后在厌氧处理下的GABA含量可以提高几十倍，但对其调控机制尚不清楚。本项目组在前期研究中发现厌氧处理后茶叶GAD三个成员中GAD1基因的表达量上升了70倍，预示着可能存在着不同于其他植物的调控机制。因此，本项目拟将茶叶GAD进行体外表达，检测其组织特异性，并在烟草中验证其催化功能；同时构建CaM结合位点缺失的GAD突变体，在烟草体内探明GAD受调控的机制。本项目有助于探明茶叶采后GABA富集的分子机制，明确GAD不同成员的调控机制是否一致，与其它植物有何异同。

γ-氨基丁酸（GABA）是哺乳动物中重要的抑制性神经传递物质，富含GABA的产品可用于治疗癫痫等病症。茶叶采后在低氧处理下，其GABA含量可以提高70多倍，但目前对其调控机制尚不清楚。本项目通过原核表达和烟草瞬时表达证明茶叶的GAD中只有GAD1和GAD2有酶活，而酶活反应和钙调蛋白（CaM）柱洗脱实验表明只有GAD1受钙离子/钙调蛋白系统的激活。拟南芥原生质体中的亚细胞定位结果表明茶树的GAD1、GAD2和CaM都定位于细胞质，存在蛋白互作的场所条件。荧光定量PCR检测表明只有GAD2基因的表达水平在低氧条件下显著上升。对C端的钙调蛋白结合域（CBD）的结构分析表明，GAD2和GAD3都存在着保守位点的变异。通过构建C端缺失的GAD突变体，发现三个GAD的C端都有抑制酶活的能力。在对不同茶树品种、季节、叶龄的检测中，发现调控水平各有不同，但都体现出了低氧处理下GAD2表达水平的上调。通过与茶树整株低氧处理的比较发现，茶叶采摘后才表现出更为强烈的应激反应，因此可能受到机械伤和低氧的双重诱导。同时我们以拟南芥为对照，也发现了双重胁迫效应的存在。本研究结果不仅丰富了植物中GAD的调控机制，也为今后GABA茶生产过程中的原料选择和工艺处理提供了参考条件。由于植物中不受CaM调控的GAD很少，因此，茶叶中的GAD2和GAD3对今后深入研究CaM的调控机制提供了很好的基因资源。我们构建的高活性的茶树GAD突变体也为生物生产GABA开辟了新的途径。