AcSUSY1在调控猕猴桃果实蔗糖卸载能力与库强中的功能研究

CHO unloading in fruit highly controls its growth and development, and effects quality formation in fruit. In sink cell, sugar utilization efficiency sugar in the cytoplasmic feedbacks control sink strength. The studies showed that kiwifruit had higher SUSY activity and AcSUSY1 expression during the rapid unloading of sucrose, suggesting that SUSY may play an important role in the regulation of sucrose unloading. To further confirm the AcSUSY1 function in regulating sugar unloading and sink strength, this project will study the relationship of AcSUSY1 expression, enzyme activity and promoter activity with sugar and hormone signal, and detect its tissue expression characteristics, to explore relationship of the expression of AcSUSY1 with fruit sink strength and Suc unloading capacity; obtaining transgenic plants with over-expressing and silencing AcSUSY1 in kiwifruit or tomato to confirm the role of AcSUSY1 in regulation of fruit development and sugar content. Additionally, transcription factor ARF interacted with AcSUSY1 promoter will be screened and identified via in vitro and in vivo, which will support regulating mechanism of AcSUSY1 in sink strength and sugar metabolism. The purpose of this study was to investigate the role of SUSY mediated carbohydrate metabolism in regulating fruit sugar unloading and storage, and to lay a theoretical foundation for the improvement of fruit quality.

果实中CHO卸载能力对其生长发育和品质形成具有重要作用，而库细胞胞质中糖的利用效率反馈调控着库强。研究表明，猕猴桃SUSY活性和AcSUSY1表达与果实中蔗糖快速卸载密切相关，为进一步证实AcSUSY1在果实糖卸载和库强调控中的功能，本项目将研究AcSUSY1表达、酶活及其启动子活性与激素、糖信号之间关系，分析其组织器官表达特性，探讨AcSUSY1表达与果实Suc卸载能力和库强的关系；通过在猕猴桃中过量和干扰、在番茄中过量表达，证实AcSUSY1在果实发育和糖含量调控中的作用；同时验证与AcSUSY1启动子互做的转录因子ARF，并从体外、体内验证其与AcSUSY1启动子的互作及其启动活性，分析AcSUSY1调控库强和糖代谢的机理。其目的在于探明SUSY介导的糖代谢在果实糖卸载和库强调控中的作用，为果实品质遗传改良育种奠定理论依据。

库强调控对于果实发育和糖含量积累有主要意义，为了探明猕猴桃果实库强调控机制，本项目开展了猕猴桃SUSY与库强和糖含量的关系研究。果实猕猴桃果实发育过程中，对猕猴桃蔗糖韧皮部卸载后相关酶活性的研究发现， SUSY活性高，且AcSUSY1在果实中特异高度表达，SUSY在猕猴桃蔗糖的韧皮部后卸载中其关键作用。CPPU 处理后对猕猴桃库强显著增加，糖含量提升，增加了SUSY）和酸性转化酶（AINV）活性。进一步RNA-seq分析表明CPPU处理上调了蔗糖卸载途径中SUT，SPT，CWINV，SUSY，HXK等基因的表达水平，尤其与猕猴桃库强相关的SUSY1在CPPU处理后第7天有显著的上调，这可能与CPPU调控SUSY活性和库强有直接关系。这些结果证明SUSY1是猕猴桃果实库强和果实含量调控的关键基因。构建了蔗糖卸载后调控的关键基因NINV1 和SUSY1的过表达和干扰载体，获得了相应猕猴桃转基因株系和番茄SUSY1的过表达植株。发现过表达对过表达SUSY1生长发育有显著抑制，表现出矮化和座果率低。沉默AdSUSY1基因后降低了猕猴桃植株中SUSY的活性，影响着叶片糖代谢的改变，增加果糖和葡萄糖含量，但蔗糖和淀粉含量无显著变化，且沉默AdSUSY1转基因株系抗寒性降低。而过表达MdNINV1基因后提高了猕猴桃植株中NINV的活性，也增加了果糖和葡萄糖含量。通过ARF家族进行了全面表达分析，发现ARF5， ARF7和ARF12的表达与SUSY1的表达高度一致，可能是调控SUSY1的转录因子。利用酵母单杂技术、LUC-REN、GUS活性分析证明ARF7能与SUSY1启动子的互做，且ARF7定位与细胞核，瞬时过表达能够增加SUSY1，这些结果表明ARF7作为转录因子，参与了生长素信号对SUSY1表达的调控。项目的相关研究结果已在JAFC、Fruit Research等期刊发表研究性论文2篇，培养硕士生2人。上述研究结果深化了SUSY1在果实库强和糖含量调控的认知，挖掘了一批基因资源，为果实品质改良奠定了基础。