硫化氢调控LOX延缓果实成熟衰老进程的生理机制研究

The mechanism of maturation and senescence of fruits during postharvest storage has always attracted the concerning of scientists. The new gaseous signal hydrogen sulfide (H2S) was found to participate in the regulation of fruit maturation and senescence, while the underlying mechanism is still obscure. In this project, the climacteric fruit tomato and the non-climacteric fruit strawberry are used to study the physiological mechanism of H2S in modulation of lipoxygenase (LOX) mediated maturation and senescence in fruits. The methods of q-PCR, Western blotting, Native PAGE, Fluorescence in situ Hybridization (FISH) and immune colloidal gold techniques are used in this research, and the gene expression, protein contents, enzyme activity changes and subcellular localization of H2S/ethylene-regulated LOX and the key enzymes in the ethylene bio-synthesis pathway (ACC synthase and ACC oxygenase) will be investigated in the levels of transcription, translation and activity regulation. Meanwhile, the real time changes of H2S and ethylene releasing are monitored. The spatiotemporal patterns of H2S/ethylene changes, ethylene synthesis and the regulation of LOX gene expression/protein level and activity will be built, thereby revealing the role of LOX in response to H2S and ethylene in modulation of fruit maturation and senescence. This study will explore the new role of H2S, enrich the knowledge of LOX?ethylene pathway, and provide new insight to the mechanism of postharvest fruit preservation.

采后果实成熟衰老机理的阐述一直被人们所关注，新型气体信号分子硫化氢（H2S）最近被发现参与了果实成熟衰老进程，但相关的作用机制尚不清楚。本项目以呼吸跃变型果实番茄和非呼吸跃变型果实草莓为材料，拟探查H2S调节脂氧合酶（LOX）介导果实成熟衰老进程的生理机制。采用q-PCR、蛋白印迹、活性电泳、免疫荧光原位杂交及胶体金或荧光标记技术，从转录、翻译和活性调节等水平，考察受H2S/乙烯信号调节的LOX和乙烯合成关键酶（ACC合成酶、ACC氧化酶）的基因表达、蛋白/酶量水平、活性变化规律及亚细胞定位的状态；实时监测内源H2S信号的消长和乙烯释放的时序变化；构建H2S/乙烯信号的动态消长、乙烯生物合成与LOX基因/蛋白/活性调节的时空模式图，揭示LOX响应H2S与乙烯信号介导果实成熟衰老的生理机制。研究结果为探索H2S的新功能、充实LOX?乙烯信号途径，而且为采后果实的保鲜机理研究提供新的理论依据。

气体信号分子硫化氢（H2S）参与植物生长发育的诸多过程，其在果实成熟衰老进程中的作用机制尚不清楚。本项目以呼吸跃变型果实（番茄和苹果）、非呼吸跃变型果实（草莓）及采后蔬菜（西兰花）为材料，研究了H2S与乙烯信号调节采后果蔬成熟衰老进程的生理功效及二者的互作关系，分析了受H2S与乙烯调节的LOX的活性变化、基因表达规律，及其对调控果实成熟衰老进程的生理贡献。证明了H2S通过阻抑LOX途径下游的膜脂过氧化和活性氧的过量产生，缓解细胞内的氧化损伤，进而行使其延迟果实的衰老进程的生理功能。利用RT-PCR技术，发现H2S能够显著降低乙烯合成相关基因的表达水平，证明H2S在转录水平上拮抗乙烯的生物合成，阐明了H2S对乙烯生物合成通路的调控模式及作用机制。建立了一种快速灵敏的H2S信号的检测方法，查明了果实成熟衰老进程中H2S信号的消长规律与衰老进程的关系。整体构建了“H2S↔LOX↔ROS↔乙烯合成与跃变↔成熟与衰老”的果实成熟衰老时空模式图，揭示了H2S拮抗乙烯调控果实成熟衰老的生理机制。此外，本项目尝试采用CRISPR/Cas9系统，构建了番茄中H2S合成关键基因亚硫酸还原酶（SR）和半胱氨酸脱硫酶（LCD）基因的敲除载体，转化番茄子叶，以获得基因敲除植株，试图从分子与遗传的角度研究内源H2S代谢异常对番茄果实衰老进程的影响。通过本项目，培养研究生5人，发表基金标注的SCI论文12篇，较好的完成了项目的既定目标。