GABA支路调控采后柑橘果实有机酸代谢的机理

Gamma-aminobutyric acid (GABA) plays peculiar roles in maintaining postharvest citrus fruit quality. To date, the regulation mechanisms underling the metabolism of GABA shunt in postharvest citrus fruit are still unclear. In previous studies, we obtained two groups of hybrid strains with high and low acid contents in the fruits from the F1 population of HB pummelo × Fairchild tangerine. In present project, the high and low acid contents fruits with very similar genetic background will be selected as materials. To explore the rate-limiting step and key genes related to GABA shunt, the bio-processes of GABA biosynthesis, GABA transfer from the cytoplasm to mitochondria and the subsequent response of the mitochondria to exogenous GABA will be studied systemically. Further work will focus in the bio-function of CgGABP, a mitochondrial specific GABA transfer protein. In order to unravel the regulatory mechanisms of GABA shunt activity differences between high and low acid strains, the sequences structures, gene expressions, promoter elements, and activities of transcription will be parallelly analyzed. Foremore, the space-time characters of GABA distribution after it been transferred into the mitochondria will be tracked by using immune colloidal gold technique. In addition, intact mitochondria will be isolated (or enriched), and mitochondria response to exogenous GABA will be investigated via mitochondrial ultra structure observation, as well as proteomics and metabomics approaches. Elucidating organic acid metabolism mechanism regulated by GABA shunt was expected to have important theoretic and practical impacts on both citrus postharvest biotechnology and novel cultivar breeding in citrus industry.

GABA支路在柑橘果实采后品质保持中行使了某些特殊功能，但调控GABA代谢的机制还不清楚。前期研究中我们获得了HB柚×Fairchild橘F1分离群体的高酸和低酸果实，本项目拟其为材料，亲本为参照，从GABA生物合成、向线粒体的转运及线粒体对GABA的响应三个环节系统研究柑橘采后GABA代谢的限速步骤和关键基因，在此基础上着重研究线粒体GABA转运蛋白质CgGABP的生物学功能。对比分析GABP在高酸和低酸柑橘果实中的序列结构、表达水平、启动子元件及转录活性；从DNA、转录和翻译等不同水平揭示柑橘GABP的转运活性调控机制。另外，我们还将利用免疫胶体金技术跟踪GABA进入线粒体后的时空分布特征，同时分离和富积线粒体，从线粒体超微结构、蛋白质组和代谢组水平解析线粒体对外源GABA的响应。阐明GABA对柑橘果实有机酸代谢的调控机制对柑橘贮藏保鲜和耐贮藏新品种培育均具有重要的理论意义和应用价值。

有机酸是鲜食柑橘风味品质的重要组成部分，但有机酸降解对其含量的影响研究较少。γ-氨基丁酸（GABA）支路是柠檬酸降解的主要途径之一，本研究以不同来源的高酸和低酸柑橘果实为材料，研究GABA代谢支路对贮藏期柠檬酸含量的影响。并以柑橘GABA转运蛋白GABP为切入点，对其调控柠檬酸代谢的机制展开深入研究，取得以下主要进展：1）外源GABA处理可以显著提高采后贮藏期夏橙（Citrus sinensis Osbeck ‘Valencia’）和纽荷尔（Citrus sinensis Osbeck ‘Newhall’）脐橙果实中柠檬酸含量，改善贮藏性能，提高贮藏品质。外源GABA处理可抑制GABA上游谷氨酸脱羧酶（GAD）的表达，同时在贮藏初期还降低了GABP的表达，从而抑制柠檬酸的降解。此外，与柠檬酸代谢相关的氨基酸在处理的果实中也出现不同程度的积累。同时显著降低贮藏期果实的腐烂率，提高了果实的贮藏性能。2）GABA支路基因的表达活性参与决定HB× Fairchild 杂交群体高酸和低酸果实采后贮藏期酸水平。柠檬酸代谢相关基因的表达分析表明大部分GABA代谢支路基因以及顺乌头酸酶（Aco）的表达在高酸果实中显著低于低酸果实，是高酸果实维持高酸含量的主要原因。3）柑橘GABA转运蛋白基因（CgGABP）参与调控柠檬酸代谢，且转录因子CgbHLH13和CgBBX32能够通过负调控GABP的表达参与调控柠檬酸水平。超量表达CgGABP的番茄果实中，内源SlAco3a和SlAco3b的表达被抑制，而柠檬酸合酶（SlCS）和磷酸烯醇式丙酮酸羧化酶（SlPEPC）上调表达，因而促进柠檬酸的积累。通过酵母点对点验证，双荧光素酶实验证明CgBHLH13和CgBBX32均能结合到CgGABP启动子上并显著抑制其转录活性，进而参与调控柑橘果实柠檬酸水平。本研究对揭示采后柑橘果实酸的降解及GABA支路调控柠檬酸代谢的机制等方面均获得了新认识，具有重要的理论意义和潜在的实践价值。在本项目资助下，本课题组已在New Phytologist，Journal of Agricultural and Food Chemistry，Horticulture Research等国内外学术期刊发表论文5篇。