γ-氨基丁酸转氨酶基因家族在高粱盐胁迫响应中的功能研究

Sorghum, as an ideal C4 cereal model for food, feed and biomass energy crops, has strong tolerance to salt stress, but the molecular mechanisms underlying the Sorghum salt tolerance are poorly understood. Recent studies indicate that the γ-aminobutyric acid (GABA) metabolism pathway plays a crucial role in plant response to various biotic and abiotic stresses, but the molecular mechanism of GABA metabolism in response to stress remains unclear, and there is not any report to study the salt resistance mechanism from this pathway in Sorghum till now. Two homologous genes of GABA transaminase (SWGABA-Ts) have been previously identified from salt-tolerant Sweet Sorghum in our lab, and the results indicated that the expression levels of SWGABA-Ts were induced by salt stress, with expression patterns closely associated with the salt resistance of Sorghum varieties, but their functions in Sorghum salt-tolerant mechanism remain elusive. In this project,the Sorghum transgenic mutants will be created by RNAi gene silencing,gene overexpressing transgenic technology, respectively, and the comparative analysis between the wild type and mutants will be performed from morphological, physiological and biochemical assay, metabolites profiling analysis and genome-wide transcriptional analysis. The aim of these studies is to elucidate the biological function of SWGABA-Ts genes in response to salinity stress in Sorghum at the perspective of functional genomics. Our studies will not only be helpful to provide a theoretical basis for further studying the molecular mechanism of Sorghum responses to salinity stress, but also has very important practical significance in the excavation of functional genes, the improvement of crop yield and quality, and the effective utilization of saline-alkali soil.

高粱是一种重要的粮食、饲料、能源C4模式作物，耐盐性强，适应性广，但其耐盐分子机制的研究相对滞后。γ-氨基丁酸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未明确，且从该途径解析高粱耐盐机制还是个空白。本实验室前期从耐盐甜高粱中分离鉴定了两个γ-氨基丁酸转氨酶同源基因（SWGABA-Ts），结果表明SWGABA-Ts基因表达均受盐胁迫诱导，其表达模式与高粱品种的耐盐性密切相关，但其在高粱耐盐中的作用机制尚不清楚。本项目拟在此基础上通过RNAi基因沉默、过表达等技术创制高粱转基因突变体材料，对野生型和突变体进行形态鉴定、生理生化检测、代谢物图谱及基因组表达谱方面的比较分析，旨在从功能基因组学水平解析SWGABA-Ts基因在盐胁迫中的生物学功能，为深入研究GABA代谢途径在高粱耐盐分子机理中的作用提供理论依据，也为今后抗逆基因挖掘、作物品质改良、盐碱地开发利用奠定基础。

盐胁迫是影响植物生长，导致农作物减产的主要非生物胁迫因子之一。高粱是一种重要的粮食、饲料、能源作物，耐盐性强，适应性广，研究高粱的耐盐性对农业发展具有重要的现实意义，但其耐盐分子机制的研究相对滞后。γ-氨基丁酸代谢旁路在植物抵御逆境胁迫中发挥重要作用，但其抗逆分子机制尚未明确，且从该途径解析高粱耐盐机制目前还是个空白。本研究从高粱中克隆获得两个γ-氨基丁酸转氨酶同源基因（SbGABA-Ts），该基因主要负责GABA的代谢，是GABA代谢旁路中关键环节之一。通过原核表达、酶活鉴定结果表明：SbGABA-Ts蛋白均具有两种底物活性，其活性以α-酮戊二酸活性为主，而丙酮酸活性相对较低。亚细胞定位分析结果表明SbGABA-T1主要定位于细胞质、质膜上；SbGABA-T2则主要定位于细胞质和线粒体中。RT-PCR分析结果表明SbGABA-T1、SbGABA-T2基因在盐胁迫下均能够被诱导表达，但在不同品种、不同组织、不同盐胁迫条件下其基因表达模式有所不同。运用优化的农杆菌遗传转化方法，将SbGABA-Ts基因成功转入高粱基因组中，基因过表达对植株的生长发育并无影响，但在高盐（200 mM NaCl）胁迫下，能显著增强植株的抗逆性，而基因突变（利用CRISPR/Cas9编辑技术）则导致植株对盐胁迫更加敏感。进一步通过对野生型和突变体进行形态鉴定、生理生化检测、转录组及蛋白组等多方面地系统分析，结果表明SbGABA-Ts基因在高粱耐盐胁迫响应中发挥着重要的生物学功能。本研究为深入研究GABA代谢途径在高粱耐盐分子机理中的作用提供理论依据，也为今后抗逆基因挖掘、作物品质改良、盐碱地开发利用奠定基础。