γ-氨基丁酸代谢旁路在甜高粱盐胁迫响应中的功能研究

Sweet sorghum, as a model system for biomass energy crops, has strong tolerance to drought and saline-alkaline stresses. The previous studies were mostly concentrated on the biochemical and physiological mechanism of sweet sorghum response to salinity stress, while the molecular mechanism of salt tolerance was poorly understood. The recent studies indicate that the metabolic pathway of γ-aminobutyric acid (known as GABA shunt) plays a crucial role in plant response to various biotic and abiotic stresses, but its molecular mechanism in response to stress remains unclear. At present there is no any report from this pathway to study the resistance mechanism in sweet sorghum. In this study it will be the first report in sweet sorghum about the identification and characterization of the related genes involved in GABA shunt by full-length cDNA cloning and subcellular localization analysis. Then,the expression patterns of GABA shunt genes in different plant tissues, different developmental stages under different salinity stress condition will be evaluated via Real-time PCR analysis. Next, the biological function of GABA shunt in sweet sorghum will be verified by way of RNAi gene silence and overexpression transgenic strategy. Moreover, the structure and transcription activity of promoters will be investigated through the cloning and deficient mutant analysis of regulatory elements in the upstream regions of the GABA shunt genes. The aim of these studies is to elucidate the function and molecular regulatory mechanism of GABA shunt in response to salt stress in sweet sorghum. Our studies will not only be helpful to provide a theoretical basis for further study the molecular mechanism driving salinity stress response in sweet sorghum, but also has very important practical significance in the excavation of functional genes, in improvement of crop yield and quality, and in the development and effective utilization of saline-alkali soil.

甜高粱是一种重要的生物质能源模式作物，具有较强的抗旱、耐盐碱能力。目前的研究大都集中在甜高粱耐盐胁迫的生理生化机制方面，对其耐盐分子机制的研究相对滞后。已有的研究表明γ-氨基丁酸(GABA)代谢旁路在植物抵御各种逆境胁迫中发挥着重要的作用，但其抗逆分子机制尚不清楚。目前从该途径研究甜高粱耐盐分子机制还是个空白。本研究首次对甜高粱中GABA代谢旁路相关基因进行分离、鉴定及亚细胞定位；利用Real-time PCR技术分析这些基因在不同组织、不同发育时期及不同盐胁迫条件下的表达模式；通过RNAi、过表达等转基因技术明确GABA代谢旁路的生物学功能；并通过启动子克隆及其调控元件的缺失分析验证启动子的结构及转录活性。以上研究旨在从分子水平解析GABA代谢途径在盐胁迫下的表达调控特点及作用机制，为深入研究甜高粱耐盐分子机理提供理论依据，也为今后抗逆功能基因研究、作物品质改良、盐碱地开发利用奠定基础。

甜高粱作为禾本科中一种重要的C4模式作物，具有较强的抗旱、耐盐碱能力，但其耐盐分子机制的研究相对滞后。γ-氨基丁酸（GABA）代谢旁路在植物抵御各种逆境胁迫中发挥着重要的作用，但其抗逆分子机制尚不清楚。目前从该途径研究甜高粱耐盐分子机制还是个空白。本研究首次在甜高粱抗盐品种中分离鉴定出两个γ-氨基丁酸转氨酶GABA-T基因（分别命名为SWGABA-T1、SWGABA-T2），其核苷酸序列相似性为79%；对这两个同源基因分别进行了cDNA克隆、原核表达及Western Blot检测，结果表明SWGABA-T1、SWGABA-T2基因与His-tag融合蛋白均能够在大肠杆菌中正确表达，表达产物的酶活鉴定结果表明甜高粱γ-氨基丁酸转氨酶均具有两种底物活性，即以α-酮戊二酸活性为主，而丙酮酸活性相对较低；通过实时荧光定量PCR技术比较了SWGABA-Ts基因在不同品种、不同盐胁迫条件下的转录特点，结果表明SWGABA-T1、SWGABA-T2均受盐胁迫诱导表达，但具有组织特异性、时空表达特异性；通过SWGABA-T1、SWGABA-T2与GFP基因融合表达利用农杆菌注射烟草方法进行亚细胞定位，激光共聚焦检测结果表明SWGABA-T1、SWGABA-T2主要定位于细胞质、线粒体中；以高粱幼胚/幼穗愈伤组织为转化受体，通过优化转化条件，建立一个稳定、高效的甜高粱农杆菌遗传转化体系，在此基础上进行了SWGABA-T1、SWGABA-T2基因的遗传转化工作，现已经获得SWGABA-T1、SWGABA-T2基因过表达的甜高粱T0代转化材料，目前筛选鉴定实验正在顺利进行中。本项目基本按照研究计划进行，并完成了预期研究目标，取得了较好的研究进展，发表期刊论文2篇。该研究结果将为今后进一步验证GABA代谢途径在甜高粱抗逆机制中的生物学功能奠定了坚实的基础。