转录因子CrNAC036调控柑橘成熟果实细胞壁代谢的机制解析

柑橘是我国南方最重要的水果之一。近年来与细胞壁代谢相关的贮藏性能（果皮硬度）和决定消费者购买意愿的感官品质（果肉化渣性）正成为柑橘绿色保鲜技术创新和鲜果市场竞争力提升的重要内容，但目前对柑橘成熟果实细胞壁代谢调控机制的认识还十分有限。申请人前期研究发现青瓯柑在成熟及贮藏期果皮硬度显著高于瓯柑果实；转录组分析表明细胞壁代谢相关基因表达在青瓯柑中显著下调，并与CrNAC036表达呈显著负相关趋势，但其具体调控机制尚不清楚。本项目拟超表达并利用CRISPR技术敲除CrNAC036基因，研究其对柑橘果皮细胞壁代谢及相关基因表达的影响；通过染色质免疫共沉淀、双荧光素酶和酵母双杂等研究，解析CrNAC036及其互作蛋白对果皮细胞壁代谢相关基因的调控机制，并评价CrNAC036对早红脐橙果肉优良化渣性形成的影响。项目实施将为改良柑橘果实贮运性能及提升消费者购买意愿提供理论依据及调控新思路。

Citrus is one of the most important fruits in south area of China. Recently, the storage quality (pericarp firmness) and fruit quality (such as mastication trait) which are closely related to cell wall metabolism, are the hot research spots of the innovation of postharvest treatment and the improvement of the fresh fruit quality. However, the regulatory research about the citrus cell wall metabolism is rare. In previous studies, the applicant has found that compared with that of Ougan, the firmness of its bud mutant Green Ougan was significantly higher during the ripening and storage period. The transcriptome data indicated that the expression of genes (such as CrPEL and CrExpansins) involved in cell wall metabolism was significantly down-regulated in Green Ougan while the expression of CrNAC036 was remarkably higher in Green Ougan than that of in Ougan. However, the specific regulatory mechanism of CrNAC036 remains elusive. In the present project, the overexpression vector of CrNAC036 will be constructed and transformed into Hongkong kumquat, and CrNAC036 of Hongkong kumquat will be knocked out by CRISPR method. Functional analysis of CrNAC036 will be performed by the observation of cell wall metabolism phenotypes of transgenic fruits. The detailed regulatory effect of CrNAC036 on cell wall related genes will be analyzed by chromatin immunoprecipitation assay, electrophoretic mobility shift assay and dual luciferase experiment. Moreover, yeast-two-hybrid, fluorescence resonance energy transfer and bimolecular fluorescence complementary experiment will be used to identify the proteins that interact with CrNAC036. Besides, the effect of the protein-protein interactions on the regulation of downstream genes will also been analyzed. Furthermore, the present project will also evaluate the regulation of CrNAC036 on the mastication trait of Zaohong navel orange. In summary, the present project is expected to thoroughly dissect the regulatory effect of CrNAC036 on citrus fruit cell wall metabolism, and accelerate the innovation of postharvest treatment about the exact regulation of citrus fruit firmness and mastication trait.