柑橘ATP柠檬酸裂解酶(ACL)在果实内在品质形成中的调控作用及其机制解析

It is very important to investigate the key point for the formation of fruit quality. ATP-citrate lyase (ACL)，catalyzing citrate into oxaloacetate and acetyl-CoA, is a cross-link among a diverse set of phytochemicals including carbohydrates, organic acids, isoprenoids, fatty acids, flavonoids, and so on. Although the previous researches in citrus confirmed that fruit has ACL activity and ACL participates in citrate utilization during fruit ripening, the effect of ACL on the accumulation of sugar, citrate, et al., and the factors affecting ACL are still not clear. In the present proposal, we will investigate the possible relation between the ACL activity and the formation of fruit quality by analyzing the ACL activity and the contents of sugar, citrate, isoprenoids, and et al. in different cultivars’ fruits. This role of ACL in fruit quality formation will be confirmed by hydroxycitrate injection or transgenic experiment with RNAi vector or overexpression vectors. Moreover, we will explore the underlying mechanism by RNA-seq technique and investigate the effect of different environmental factors on the ACL activity by using in vitro tissue culture with the explant of ‘Anliu’ orange callus and by using the field shading treatment. Taken together, the objective of this proposal will identify the role of ACL in the fruit internal quality formation, decipher the possible mechanism underlying the ACL-regulating fruit quality formation and the effect of different environmental factors on the ACL activity, which definitely provide a great theoretical and practical significance for the regulation of citrus fruit quality.

ATP柠檬酸裂解酶(ACL)催化柠檬酸生成草酰乙酸和乙酰辅酶A，是联系糖分、有机酸、脂肪酸、类黄酮和类异戊二烯类等物质的桥梁。前期研究证实柑橘果实中存在ACL，并参与柠檬酸降解，但是其活性变化对柑橘果实糖、酸、类异戊二烯等物质，即果实风味、着色和香气等品质的影响如何，自身又受哪些因素调控，目前均不清楚。本项目拟通过测定不同果实发育成熟过程中ACL活性和糖、酸等代谢物质的含量，明确ACL和果实内在品质相关代谢物质变化的关系；通过注射羟基柠檬酸（ACL抑制剂）和转基因试验，验证ACL在果实内在品质相关代谢物质积累中的作用；通过愈伤组织体外培养和田间遮光处理试验，明确不同环境条件对ACL活性的影响；通过转录组测序，解析ACL调控相关代谢物质积累的分子机制。本项目最终确定柑橘ACL在柑橘果实内在品质形成中的作用和对应的调控机制，其结果无疑对柑橘果实品质形成及调控措施研发具有重要的理论和实践意义。

ATP柠檬酸裂解酶（ACL）催化柠檬酸生成草酰乙酸和乙酰辅酶A，是决定碳流向不同代谢物（如糖、脂肪酸、类黄酮和类异戊二烯等）生物合成途径的关键酶。本课题按照预定研究计划系统比较了暗柳橙、纽荷尔脐橙、国庆1号温州蜜柑、华柑2号椪柑、早香柚和HB柚果实发育和成熟过程中，ACL、细胞质顺乌头酸酶活性以及柠檬酸、类黄酮和类胡萝卜素等代谢物质含量的变化，并通过转基因试验进行了验证；另外以HB柚为材料，全面比较了柑橘成熟叶片（ML）和果实汁胞（JS）之间的代谢产物和基因转录水平的差异，取得如下结果。（1）ACL活性变化与一些代谢物质积累之间的关系存在品种差异性，ACL和cyt-ACO的活性显著负相关（r = -0.443），ACL活性仅与细胞质乙酰辅酶A含量之间存在显著相关性（r = 0.433）；（2）在柑橘愈伤组织中超量表达或者RNA干涉ACL表明，增加或降低ACL活性可以分别降低和诱导cyt-ACO的活性，说明ACL和cyt-ACO一起协同控制果实细胞质柠檬酸的去路，但它们不是决定柠檬酸和柑橘果实中其他代谢物积累的关键因素；（3）ACL活性差异显著可能是柑橘成熟叶片（ML）和果实汁胞（JS）代谢物质积累差异的一个重要原因。成熟叶片高ACL活性，不仅增加了许多次生代谢物合成的中间前体物质，而且可能通过影响一些关键基因的表达来调控这些物质的合成或积累；（4）转基因研究证实ACL活性变化确实可以影响相关代谢物质的含量、以及一些基因的表达水平或酶活性；（5）ACL活性可以受到营养元素和环境因素的影响。.在本项目的资助下，先后发表论文6篇，其中SCI论文5篇；另外，先后有4名硕士或博士研究生的培养受到本课题经费的资助。总之，通过认真执行该项目，圆满完成了预定研究任务、实现了预定研究目标。所获得研究进展对柑橘果实品质形成及调控措施研发具有重要的理论和实践意义。