乙烯响应因子调控柿果实脱涩后软化机制研究

Most of persimmon (Diospyros kaki) cultivars belong to astringent type, which require artificial postharvest treatments to remove the astringency. Moreover, the undesirable rapid softening usually consequently happened after astringency removal. Thus, both deastringency and firmness maintain are critical for persimmon industry. We previously studied various de-astringency treatments and identified several DkERF genes that involved in deastringency. The present project will focus on the regulatory mechanism of DkERF genes on deastringency associated softening of persimmon fruit. In order to investigated the roles of DkERF genes on deastringency associated softening of persimmon fruit, different treatments will be applied to different persimmon cultivars; persimmon softening associated cell wall related genes will be isolated and characterized, using RNA-seq and QPCR; deastringent treatments responsive DkERF genes will obtained and their trans-activation effects on cell wall related genes will be investigated. Based on ERF-target gene analyses, the association between different DkERF genes will also be included. Moreover, using the established persimmon transient over-expression system and tomato transgenic system, functions of DkERF genes will be verified. The results of the present project will provide new insight for de-astringency associated persimmon fruit softening, and will also benefit for the improvements of persimmon fruit postharvest treatments.

柿是我国特色浆果，目前栽培品种以涩柿为主，需经采后脱涩方可食用，但脱涩处理后的柿果实易软化，严重限制柿果实的货架寿命，影响产业增效，因此采后脱涩保脆始终是柿研究的重要主题之一。本项目在前期研究的基础上，围绕柿果实脱涩后易软化这一现实问题，以乙烯响应因子（DkERF）为核心，开展柿果实采后脱涩诱导软化的转录调控机制研究。研究不同采后处理对柿果实脱涩及软化的影响；利用RNA-seq结合QPCR技术，筛选与柿果实脱涩后软化相关的细胞壁相关结构基因，并利用脱涩保脆处理和脱涩后不易软化的特色品种进行相关验证；获得柿果实软化相关的DkERF转录因子，并利用双荧光素酶体系、酵母杂交技术等研究DkERF转录因子对柿果实软化相关细胞壁基因的调控效应及机制；利用双荧光素酶体系、BiFC等技术，分析不同DkERF间协同调控软化的效应；通过柿瞬时表达体系和番茄转基因体系，开展柿果实软化相关DkERF基因的同源功能

目前主栽柿品种以涩柿为主，成熟的涩柿需经采后脱涩方可食用，然而脱涩后的柿果实极易软化，耐贮性下降甚至丧失，易造成较大损耗。本项目研究了ERF参与柿果实脱涩后软化的机制。研究了不同采后处理（CO2、CO2结合1-MCP等）对不同品种柿果实采后脱涩（可溶性单宁含量测定、印迹分析）和软化（硬度）的影响；利用转录组测序测序，比较分析了不同处理中差异表达基因（含转录因子和细胞壁代谢基因）；并利用CO2处理脱涩后不易软化的‘刀根早生’果实进行了基因表达与软化关联性验证；利用双荧光素酶体系、EMSA体系、启动子点突变、BiFC、LCI等体系，发现了参与柿果实脱涩后软化调控的ERF成员。项目研究结果显示，ERF分别通过两条途径参与柿果实脱涩后软化，其一为DkERF8/16/19通过识别DRE元件，调控DkXTH9（半纤维素降解途径）启动子；另一途径为DkERF8/16与DkNAC9形成转录因子复合体，通过DkNAC9结合DkEGase1（纤维素降解途径）启动子，促进柿果实脱涩后软化。项目研究结果，解析了柿果实脱涩后以软化这一常见产业现象的分子机制，明确的关键调控基因及其作用通路。项目研究成果已在Plant Biotechnol J、J Exp Bot、Plant Physiol等刊物发表SCI论文8篇（其中IF>5.0论文5篇），培养研究生8名，已按项目合同完成各项任务指标。