水通道蛋白基因PIPs在柑橘采后贮藏期的生物学功能及其调控机制

Citrus is the dominant fresh fruit in South China, however, serious economic loss was usually caused by the uncontrollable postharvest quality degradation. In the past decade, we found that stress caused by water loss was the original cause for citrus fruit senescence and postharvest quality degradation, whereas, rare information underlying the biological bases to water loss is available yet. Aquaporins play key roles in plant water transfer, and solid evidences have demonstrated that the plasma membrane intrinsic proteins (PIPs), a main subgroup in aquaporins family, showed intimate relation to citrus postharvest quality degradation. In present project, fruit of Newhall navel orange (Citrus sinensis) will be adopted as experimental materials, storage tests imitated to citrus industry will be conducted. Candidates of CsPIP genes that participated in citrus postharvest water loss will be screened by integrating of physiological, biochemical, and bioinformatic analyses. Further function identification will be conducted in yeast, Arabidopsis, and tomato. Meanwhile, the gene and promoter structure of CsPIPs will be detailedly analyzed, and the potential interaction proteins and key regulation factors will be screened. The regulation mechanisms underlying CsPIPs biofunction will be explored from the levels of transcription, translation, and posttranslational modifications (especially, phosphorylation and glycosylation), as well as the protein crystal structure analyzing. And ultimately, we expected to get the key regulation factors, such as transcription factors or miRNAs that directly participate in CsPIPs regulations. Understanding the molecular mechanisms to citrus postharvest water loss will supplying scientific evidences to the innovations for postharvest biology and technology.

柑橘是我国南方最重要的鲜食水果，采后品质劣变给产业带来巨大的经济损失。前期的研究结果表明失水胁迫是引起柑橘采后衰老和品质劣变的重要原因，但对影响柑橘采后失水速率的生物学基础认识还十分有限。水通道蛋白在植物水分转运中发挥着重要作用，亦有充分证据表明PIPs类水通道蛋白与柑橘采后品质劣变的关系十分密切。本项目拟以纽荷尔脐橙果实为材料，模拟产区生产流程进行贮藏试验，结合生理生化指标测定和生物信息学分析，筛选直接参与果实采后失水的CsPIPs候选基因，并在酵母、拟南芥和番茄中对其进行功能验证。同时分析CsPIPs基因和启动子的结构，筛选上游调控蛋白，从转录、翻译、翻译后修饰（磷酸化和糖基化）以及蛋白质晶体结构等不同水平解析CsPIPs对水分转运的效率及可能的调控机制，获得参与柑橘果实采后失水的CsPIPs关键调控元或互作分子，阐明柑橘采后失水的生物学基础，为柑橘贮藏保鲜理论和技术创新提供科学依据。

柑橘采后失水是引起果实品质下降及采后衰老的关键非生物因素。植物的水分转运过程主要由水通道蛋白（Aquaporins，AQPs）调节。AQPs在采后贮藏过程中的差异表达暗示了这类蛋白影响柑橘果实贮藏性能，然而关于AQPs的具体作用机制尚不清楚。本研究以贮藏期纽荷尔脐橙果实为主要实验材料，结合多种实验手段对柑橘AQPs开展了研究，系统解析AQPs在采后失水过程中的生物学功能及相关调控机制。取得以下进展：1）维持贮藏环境湿度及使用酸性溶液处理有利于延缓柑橘果实采后失水：低湿度贮藏环境加速果实采后失水和质地下降；高湿度贮藏环境能够有效延缓果实采后失水，维持果实品质。碱性溶液（pH=9）处理加速果实采后失水和质地下降，而酸性溶液（pH=6）处理能够有效延缓果实采后失水、维持果实品质。2) 柑橘AQPs在采后贮藏过程中的表达模式具有时空和品种特异性：甜橙基因组中共检索到39个柑橘AQPs。多数AQPs（PIPs和TIPs）在紧皮柑橘（纽荷尔脐橙和HB柚）及温州蜜柑果皮中下调表达，而在椪柑和Fairchild橘果皮中普遍上调表达，表达模式在贮藏过程中呈现品种特异性。3) CsNIP5;1通过转录水平参与调控柑橘水分平衡，并且受到CsWRKY4/28调控：CsNIP5;1在爪蟾卵母细胞中定位于细胞膜，并且具有水分转运活性。转录因子CsWRKY4和CsWRKY28共同参与调控CsNIP5;1表达：CsWRKY28在贮藏初期抑制CsNIP5;1表达，而CsWRKY4在贮藏后期激活CsNIP5;1表达。本研究为深入了解柑橘采后失水进程和调控机制提供了新见解，为后续使用基因工程等手段维持柑橘贮藏品质和遗传育种改良提供了理论依据。在该项目资助下，本课题组已在Journal of Experimental Botany等国内外学术期刊发表论文5篇；并获全国农牧渔业丰收奖一等奖、神农中华农业科技一等奖等2项。