GABA对盐胁迫促进大麦芽苗酚酸富集的生物信号传导机理

Phenolic acids are main phenolic compounds in cereal grains. Their antioxidant effect on human body has been becoming a research hotspot. Salt (NaCl) stress can induce accumulation of phenolic acids in barley seedlings. Γ-aminobutyric acid (GABA) is involved in this process as a bio-signal molecule, but its transduction mechanism is still unclear. This study will detect compositions and contents of phenolic acids, their activities and gene expressions of the key enzymes to clarify active mechanisms of GABA in bioaccumulation of phenolic acids as well as its downstream biomessengers of nitric oxide (NO) synthesis and Ca2+ transmembrane under the stress. It will also adopt methodologies of both external additive and endogenously inhibitor substances to investigate functions and efficiencies of NO and Ca2+ on GABA signal transduction and the order of their downstream biomessengers. Thus, it will enable us to elucidate their interactive bio-signaling pathway and regulatory effects on relevant gene and protein expressions for bioaccumulation of phenolic acids. This study will reveal transduction mechanisms of GABA/NO/Ca2+ signals in bioaccumulation of phenolic acids under salt stress at four levels of biological enzymology, gene regulation, protein expression and bio-signal transduction, respectively. As a result, it will provide a scientific basis and theoretical support to the development of the germinated- grain food with healthy functions.

酚酸是谷粒中主要的酚类物质，因其对人体的抗氧化作用正在成为研究热点。盐（NaCl）胁迫可诱导大麦芽苗富集酚酸，γ-氨基丁酸（GABA）作为生物信号分子参与其中，但作用机理不明。本研究将检测NaCl胁迫下GABA合成对大麦芽苗中酚酸组成和含量、及其合成关键酶活性与基因表达的影响，明确GABA在酚酸富集中的作用；考察一氧化氮（NO）和Ca2+作为次级生物信号因子对GABA信号的传导和对酚酸合成的调控作用；运用外加法和内源抑制法确定NO和Ca2+对GABA信号传导的顺序定位；阐明GABA、NO和Ca2+组成的生物信号通路及其对酚酸合成关键酶活性和基因与蛋白表达的调控作用。本项目将在酶学、基因调控、蛋白表达和生物信号四个水平上分别揭示GABA/NO/Ca2+对NaCl胁迫下的大麦芽苗富集酚酸的生物信号传导机理，为研发谷物芽类功能食品提供科学依据和理论支撑。

本项目首先研究了NaCl胁迫下GABA对大麦芽苗酚酸富集的介导作用。大麦芽苗经60 mM NaCl处理后造成氧化损伤，产生应激反应。GABA处理可缓解NaCl胁迫对大麦芽苗造成的氧化损伤，诱导了苯丙氨酸解氨酶（PAL）、肉桂酸-4-羟化酶（C4H）、4-香豆酸辅酶A连接酶（4CL）和阿魏酸-5-羟化酶（F5H）蛋白的表达，促进大麦芽苗酚酸的累积。可见，盐胁迫下GABA对大麦芽苗中酚酸的富集具有正向调节作用。在此基础上，研究了盐胁迫下NO对GABA生物信号的传导作用。硝普钠（SNP）显著诱导了酚酸合成关键酶活力及其基因、蛋白表达水平，促进了酚酸的累积。NO清除剂（cPTIO）显著抑制了这些酶的活力和基因表达，降低了酚酸含量。回添GABA后，大麦芽苗中NO含量显著提高，有效缓解这种抑制作用。可见，NO和GABA在大麦芽苗酚酸合成中有互作关系。GABA诱导了大麦芽苗根尖细胞Ca2+的流入和总钙、水溶性钙的增加，激活酚酸合成代谢通路，同时酚酸得到累积。CaCl2处理亦逆转内源GABA合成酶抑制剂3-MP对PAL、C4H和F5H蛋白表达的抑制作用。表明与NO类似，Ca2+作为信号分子，参与介导了盐胁迫下GABA对大麦芽苗中酚酸代谢通路的调控。最后，研究了NO-Ca2+在NaCl胁迫下大麦芽苗酚酸合成中的作用关系。发现NaCl胁迫下大麦芽苗酚酸合成中Ca2+在NO的上游发挥作用。.综上，本项目揭示了盐胁迫下GABA/NO/Ca2+作为胁迫信号通路促进大麦芽苗酚酸累积的传递途径，为今后大规模、商品化、高效生产富含酚酸的功能性全谷物大麦芽苗食品提供领先的技术和理论指导。.