滞绿基因SlSGR1调控番茄果实类胡萝卜素积累的机理解析

Color character is an important aspect of quality of tomato fruit. Carotenoids are the pigments responsible for the external and internal coloration of fruit, and they are also important functional substance for human being. Previously research have confirmed that tomato stay-green protein SlSGR1 play a central role in regulation of chlorophyll degradation. Our lab research results show that SlSGR1 also plays an important role in regultion of the accumulation of carotenoids. Our research results show that knockout of SlSGR1 in tomato could lead the thylakoid stacks in chloroplast of fruits disappeared early than wild type during the ripening process and increase the globular noticeably at different ripen stages. Knockout of SlSGR1 in tomato could significantly improve the lycopene and β-carotene contents at different ripening stages. As the fruits turned red, the concentration of lycopene increased significantly to 380 μg g-1 FW in transgenic fruits which was 4.2-fold as high as in the wild type fruits. And approximately ten SlSGR 1 interacting proteins such as TIV1, SALK, PSY1, RanBP1, Fer1, and Sahh1 were screened by yeast two hybrid experiment. Based on these previous research results, this project will focus on the physiological and biochemical properties analysis of SlSGR1-RNAi transgenic tomato fruit. Combining with metabonomics and transcriptomics data, the key SlSGR1 interacting protein and the closely genes and metabolic pathways regulated by SlSGR1 should be determined. Then,the physiological mechanism and molecular regulatory networks of SlSGR1 in tomato fruit carotenoids accumulation would be well illustrated.

色泽是番茄果实的一个重要品质，类胡萝卜素是番茄主要的成色物质，也是一类重要的功能性物质。SlSGR1已被证明在叶绿素降解的调控方面发挥重要功能，本实验研究发现SlSGR1在番茄果实类胡萝卜素积累调控上也发挥重要作用，RNAi抑制SlSGR1基因的表达会加快番茄果实成熟过程中质体的转换过程、增加质体小球的积累量，从而导致SlSGR1-NAi转基因番茄果实的番茄红素和β-类胡萝卜素含量在各个成熟时期均显著高于对照，番茄红素的含量在红熟果实中甚至达到对照的4.2倍。酵母双杂实验表明SlSGR1与PSY1， RanBP1等蛋白存在互作。本项目研究拟在前期工作基础上，开展SlSGR1-RNAi果实发育过程中生理生化特性的分析研究，并结合代谢组学和转录组学研究，发掘与SlSGR1互作的关键蛋白基因和受SlSGR1调控的关键代谢途径，进而阐明SlSGR1调控番茄果实类胡萝卜素积累的生理机理和分子机制。

PSY1 是番茄果实中番茄红素合成的关键酶基因，本项目研究首次发现，番茄滞绿基因SlSGR1可以通过与PSY1互作，通过影响PSY1的活性，调控番茄果实中番茄红素和β-类胡萝卜素的积累。在CrtI 酶介导的番茄红素合成工程菌中，引入SlSGR1蛋白能抑制番茄红素生物合成工程菌中PSY1同源基因CrtI酶蛋白的生物活性，显著降低工程菌生产番茄红素的能力。而在番茄中通过RNAi抑制SlSGR1基因的表达，导致番茄果实中番茄红素和β-类胡萝卜素的异常积累，转基因果实的番茄红素和β-类胡萝卜素含量在各个熟期均有显著地提高，其中番茄红素和β-类胡萝卜素的含量在红熟期分别达到 355.03μg g -1 FW 和 76.32μg g -1 FW，为野生型对照的 4.2 倍和 37.4 倍，果实呈现为明显的铁锈色。同时，项目组还发现，抑制 SlSGR1 基因的表达可明显降低成熟番茄果实中过氧化氢的含量，在果实发育成熟的不同时期，果实中乙烯受体基因 ETR1、ETR3、ETR4、ETR5和CRT1 的表达水平均下调，受乙烯诱导的 PG、EXP1和PE 等基因的表达模式也发生了改变，番茄果实的耐贮藏性大大提高，贮藏时间由原来的 27天提高到45天以上。SlSGR1-RNAi转基因株系的转录组学和代谢组学研究结果表明，SlSGR1可能通过调控糖代谢来调控番茄果实叶绿素的降解和色泽形成。通过酵母单杂交，发掘出2个结合到SlSGR1基因启动子区域调控SlSGR1基因表达的重要转录因子。DNA多态性的研究结果则表明，SlSGR1基因功能缺失的突变与部分番茄果实成熟绿色相关。项目执行以来，共培养研究生2人，其中博士研究生1人，硕士研究生1人；有4名硕士研究生进入到博士研究生阶段学习。共发表标注本项目资助的SCI论文5篇。