酸性转化酶DlSAI调控龙眼果肉退糖的机理研究

After missing the best harvest period, the “sugar receding” characterizated by sharply decreasing sugar content happened in longan pulp, seriously affected its edible quality and the on-tree preservation. The breaking through the limitation of understanding on sugar receding and exploring the molecular regulation mechanism of sugar receding are very important for the technology innovation of maturity controlling and fresh-keeping. The rate of sugar receding in longan fruit was positively correlated with the degradation of sucrose in pulp. Invertases including neutral invertases, cell wall invertases and soluble acid invertases, which catalyze the sucrose into fructose and glucose irreversiblly, are the key enzymes for controlling sink strength and the content of hexose in plants. The relationship between the rate of sugar receding and the activity of sucrose-metabolic enzymes in different varieties indicated that the sugar receding was mainly affected by the activity of invertase SAI. At present, the roles of SAI and its regulation are unclear. This study aims to explore the effect of DlSAI on the sugar metabolism by analyzing the sequence of DlSAI genes and promoters in longan with different desugar rate, prokaryotic expression of the recombinant DlSAI protein and functional verification from two sides by the agrobacterium-mediated over-expression manipulation of DlSAI in tomato and virus-induced gene silencing in longan fruits. In addition, interaction screening and expression analysis of transcription factors will be used to explore the regulation mechanism of DlSAI expression. The study will help clarify the roles of DlSAI in sugar metabolism of fruits and provide theoretical basis for delaying the sugar receding in longan.

错过最佳采收时期后，龙眼果肉出现含糖量急剧下降的“退糖”现象，严重影响其食用品质和挂树保鲜。突破对退糖认识的局限，探究退糖的分子调控机制，对龙眼果实成熟期调控和保鲜技术的创新至关重要。龙眼退糖速度与果肉蔗糖降解速度呈正相关。转化酶包括中性转化酶、细胞壁转化酶和可溶性酸性转化酶，催化蔗糖不可逆地分解为果糖和葡萄糖，是控制果实的库强度和己糖含量的关键酶。申请人前期研究不同品种龙眼退糖速度和蔗糖代谢酶活力的关系发现，退糖快慢主要受酸性转化酶SAI活力影响。目前SAI在退糖中的作用和调控机制尚不明确。本研究拟分析DlSAI基因及启动子在不同退糖类型龙眼果实中的序列差异，结合原核表达、农杆菌介导转化番茄和病毒介导基因沉默转化龙眼，从正反两面探究DlSAI的生物学功能；通过互作筛选转录因子及表达模式分析，探究调控DlSAI表达的机制。旨在解析DlSAI在果实糖代谢中的作用，为延缓龙眼退糖提供理论依据。

错过最佳采收时期后，龙眼果肉出现含糖量急剧下降的“退糖”现象，严重影响其食用品质和挂树保鲜。突破对退糖认识的局限，探究退糖的分子调控机制，筛选出延缓龙眼果实退糖的有效配方，对龙眼果实成熟期调控和保鲜技术的创新至关重要。可溶性酸性转化酶（SAIs）催化液泡中的蔗糖不可逆地分解为果糖和葡萄糖，对果实的品质发挥重要调控作用。然而，目前SAIs对龙眼果肉糖比例和退糖过程的调控作用仍缺乏系统研究。我们的结果表明，不同品种龙眼果肉的蔗糖/己糖比例和退糖速率差异显著。分析各个品种果肉中蔗糖合成和分解相关酶活力、基因表达和蔗糖/己糖比，发现酶活和基因表达两个水平上，DlSAI均与蔗糖/己糖比呈现最高负相关性。发育到成熟阶段高的DlSAI酶活力和基因表达可能是造成‘罗伞木’果肉极低蔗糖/己糖比的原因，而上调的DlSAI酶活力和基因表达可能是造成‘石硖’果肉留树期间退糖的原因。因此，DlSAI酶是通过转录水平和酶活调控蔗糖/己糖比和退糖的关键因子。利用TMT-标记的蛋白质组学分析‘石硖’龙眼果肉留树期间的差异表达蛋白发现，糖酵解、三羧酸循环、发酵和能量代谢途径关键蛋白质的显著上调可能促进了己糖的消耗，与DlSAI酶协同促进退糖。重复2年的GA3喷施处理试验表明，50 mg L-1 GA3可有效延缓留树期间‘石硖’龙眼果肉的退糖，维持品质和抗氧化物质含量。激素谱分析表明，GA3处理显著提高了龙眼果肉中GA3、N6-异戊烯腺嘌呤和反式玉米素核苷的含量。分析蔗糖糖代谢和激素信号转导相关基因的表达发现，GA3处理显著提高了留树期间龙眼果肉中蔗糖合成相关基因（DlSPS和DlSPS1）与GA和CTK合成相关基因的表达，抑制了DlSAI-2的表达。综上所述，GA3处理有效延缓了‘石硖’龙眼退糖，延长其留树保鲜，有望解决集中上市和积压等产业问题，为进一步研究激素对DlSAI的转录调控提供了研究基础。