半胱氨酸脱巯基酶LCD调控番茄果实成熟衰老的机制研究

植物内源信号分子硫化氢（H2S）可以延缓植物的成熟衰老进程，半胱氨酸脱巯基酶LCD是催化Cys生成H2S的关键酶，它是否参与调控果实成熟衰老尚不清楚。我们前期研究发现，番茄LCD基因缺失影响果实成熟进程，而且LCD具有明显的细胞核定位。本项目拟利用番茄LCD基因缺失植株和超表达植株，分析LCD在硫代谢和调控果实成熟衰老进程中的生理功能；进行表观遗传和转录组测序的联合分析，考察LCD对表观遗传修饰和转录组的协同调控作用；构建LCD截短突变/氨基酸位点突变与GFP融合的表达载体，分析参与LCD细胞核定位的关键氨基酸位点；利用酵母双杂交、双分子荧光互补和免疫共沉淀技术，筛选介导LCD入核的关键互作蛋白；利用酵母单杂交技术、凝胶迁移实验和双荧光素酶报告系统，筛选并鉴定调控LCD转录水平的上游转录因子。本项目将揭示H2S生成酶LCD调控番茄果实成熟衰老的生理机制，为果实发育研究提供新的理论依据。

Hydrogen sulfide (H2S), a plant endogenous signaling molecule, can delay the ripening and senescence of plant. The cysteine desulfhydrase LCD is a key enzyme that catalyzes the production of H2S by cysteine (Cys), and whether LCD participates in regulating fruit ripening and senescence is still unclear. Our previous study found that tomato LCD gene deletion affected tomato fruit ripening, and LCD protein had obvious nuclear localization. This project intends to use tomato LCD gene deletion mutant and overexpressing plant to analyze the physiological functions of LCD in sulfur metabolism and the regulation of fruit ripening and senescence. Joint analysis of epigenetics and transcriptome sequencing will be conducted to investigate the synergistic regulation of LCD on epigenetic modification and transcriptome. The LCD truncation mutations/amino acid site mutations will be fused to GFP expression vector, thereby analyzing the key amino acid sites involved in nuclear localization of LCD. The key proteins which interact and mediate the nuclear localization of LCD will be screened by yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (co-IP) techniques. The upstream transcription factors regulating LCD transcription level will be screened and confirmed by using yeast one-hybrid technique, electrophoretic mobility shift assay (EMSA) and dual luciferase reporter system. This project will reveal the physiological mechanism of H2S producing enzyme LCD in regulating tomato fruit ripening and senescence, and provide a new theoretical basis for fruit development research.