一氧化氮调控桃果采后冷藏过程抗氰呼吸作用机理研究

Cyanide-resistant respiration has a prominent role in scavenging reactive oxygen species, inhibiting apoptosis and increasing resistibility of fruits and vegetables, and the terminal oxidase AOX has become the key point in the study of cyanide-resistant respiration. However, the sequence information, number, expression characteristics and regulation of AOX need to be further studied and solved. At present, it was proved that NO can restrain the respiration rate to extend ripening and senescence of fruits and vegetables, while little research has been done on the molecular mechanism of cyanide-resistant respiration in response to NO in fruits and vegetables. Therefore, the present study is to determine the capacity of respiration and AOX activity of peach after treatment with NO, analyze the contribution of cyanide-resistant respiration to total respiration, and elucidate the effect of NO on cyanide-resistant.respiration. We will clone the whole cDNA of AOX gene, analyze the sequence information, transcriptional activation activity and subcellular localization information to discuss the binding sites of NO and AOX, and the impact of NO on transcriptional activity and distribution in cell of AOX gene. We will also detect the amount of AOX mRNA using qPCR and western hybridization technologies, discuss the effect of transient over expression of AOX on cyanide-resistant respiration,and further clarify the molecule mechanism of NO on cyanide-resistant respiration of peach to provide theoretical basis peach for the effective regulation respiration of peach to enhance its storability.

抗氰呼吸具有清除活性氧、抑制细胞凋亡和增加抗病性等功能，与果蔬成熟衰老密切相关。NO作为生物活性分子被证实可以通过抑制呼吸速率来延缓果蔬成熟衰老，但NO对冷藏水蜜桃抗氰呼吸途径的调控机制、桃果AOX表达特性等科学问题尚不清楚。本项目以水蜜桃为研究对象，测定NO处理下果实冷藏过程呼吸容量以及AOX活性，分析不同呼吸途径对总呼吸的影响，明确NO对抗氰呼吸和AOX活性的影响，探讨NO处理对冷藏桃果果实抗氰呼吸的调控机制；克隆AOX基因全长序列，分析序列信息、转录活性和亚细胞定位信息，探讨NO与AOX基因的结合位点，揭示NO对AOX转录激活活性和在细胞中分布的影响；结合qPCR和Western杂交技术检测AOX的表达量，探讨AOX瞬时过表达对抗氰呼吸的影响，找出耐低温胁迫的调控相关的响应位点，阐明以AOX为中心抗氰呼吸在桃果冷藏过程中细胞内的调控路径，揭示NO调控桃果实抗氰呼吸的分子机理。

一氧化氮（NO）作为生物活性分子被证实可以通过抑制呼吸速率来延缓果蔬成熟衰老。高等植物抗氰呼吸是一条对氰化物不敏感的电子传递链途径呼吸，与果实总呼吸、成熟衰老密切相关。但NO对冷藏水蜜桃抗氰呼吸途径的调控机制、桃果实抗氰呼吸关键酶交替氧化酶（AOX）表达特性等科学问题尚不清楚，揭示NO处理对桃果采后抗氰呼吸途径运行的调控机制，靶向调控桃果贮藏保鲜具有重要科学意义。.本项目以水蜜桃为研究对象，开展了外源NO处理对冷藏桃果实呼吸作用影响的研究，析了不同呼吸途径的呼吸速率对总呼吸的影响，明确NO对抗氰呼吸和AOX活性的影响，探讨NO处理对冷藏桃果果实抗氰呼吸的调控机制，揭示NO调控桃果实抗氰呼吸的分子机理。.通过本项目研究，明确了NO对不同桃果实贮藏过程呼吸途径和抗氰呼吸的影响，建立了NO、抗氰呼吸和AOX的量效关系；NO处理延缓了桃果实冷害（CI）的发生，抑制了桃果实采后H2O2含量、O2-产生率的增加和硬度的降低。同时，通过RNA-seq分析，NO处理上调了PpG-6-PDH、Pp6-PGDH和PpAOX的基因表达，而下调了PpGPI和PpHK的表达，表明磷酸戊糖呼吸途径和抗氰呼吸途径得到促进，糖酵解途径受到抑制。此外，PpAOX的表达与PpPOD1/2表达和H2O2含量的趋势一致，表明AOX可能通过清除H2O2来增强其耐冷性。研究发现，茉莉酸甲酯（MEJA）和水杨酸甲酯（MESA）处理可诱导交替氧化酶（AOX）表达，外源NO通过增强桃果实三磷酸肌醇（IP3）、糖原合成酶激酶3（GSK-3）等活性，调控抗氰呼吸途径关键基因AOX活性和基因表达，诱导桃果实的抗氰呼吸途径，提高桃果实的抗氧化能力和耐冷性，阐明了外源NO处理对冷藏桃果实抗氰呼吸作用途径和分子机制。活性氧是导致果实成熟衰老的重要因素，贮藏过程中COX的表达量和酶活性降低，诱导交替氧化酶的活性增强，促进了抗氰呼吸速率，清除大量活性氧，减弱了乙烯的合成量，推迟了呼吸跃变的产生，最终延长了桃果实的贮藏期，达到较好的贮藏保鲜目的。