E3泛素连接酶核心组件RhSKP1响应乙烯影响月季花瓣衰老的机制解析

Ethylene-induced petal senescence severely influences ornamental quality and economic value during production and postharvest handling of cut roses. Our previous work uncovered that RhNAC100, an ethylene responsive transcription factor, is a positive regulator of petal senescence in rose. We also obtained the downstream transcripts of RhNAC100 from RhNAC100-silenced petals by RNA-seq approach, and found that silencing of RhSKP1 significantly accelerated petal senescence among the DEGs (Differentially-expressed genes). RhSKP1 is the most important component of an E3 ubiquitin ligase, and its homolog plays an important role in ubiquitin-26S proteasome-dependent protein degradation in Arabidopsis. In this work, we will focus on the molecular mechanism of RhSKP1 in ethylene-induced petal senescence of rose. We will perform EMSA and ChIP-PCR to test the possible binding between RhNAC100 and the promoter of RhSKP1. And to understand the role of RhSKP1 in petal senescence, we will search its possible interaction proteins by IP-MS and identify their binding ability by BiFC and Co-IP. We will also perform temporal-spatial expression analysis of RhSKP1 and its interaction protein by real-time PCR and subcellular localization analysis by confocal. Finally, we will conduct functional analysis of RhSKP1 and its interaction protein by creating their own over expression and silenced-transgenic rose plants. Our goal is to provide novel evidents to understand ethylene-induced petal senescence, and to be valuable for the genetic improvement of longecity of rose, and to expand our understanding of the regulation mechanism of floral organ senescence.

在月季切花生产和流通中，乙烯诱导的花瓣衰老严重影响了月季的观赏品质及经济价值。申请者的前期工作发现，响应乙烯的转录因子RhNAC100是花瓣衰老的正调节子；通过测序获得了RhNAC100的下游基因群；基于此，对关键基因筛选发现沉默RhSKP1促进了花瓣衰老。SKP1作为E3泛素连接酶SCF的核心组件对花瓣衰老的调控尚无报道。本项目拟通过EMSA和ChIP-PCR确定RhSKP1是否为RhNAC100直接下游基因，通过IP-MS、BiFC和Co-IP筛选鉴定RhSKP1的互作蛋白，并对RhSKP1及互作蛋白进行时空表达、亚细胞定位以及基因功能综合鉴定。本项目的实施，将从蛋白质泛素化的转录后调控层面解析RhSKP1在乙烯诱导月季花瓣衰老中的作用机制，为了解花瓣这一非绿色器官的衰老提供新的认识；进而为延缓月季花瓣衰老的技术研发提供理论支撑，同时为通过遗传手段创制延缓花瓣早衰的新种质提供基因储备。

本研究对RhSKP1响应乙烯影响月季花瓣衰老的机制进行了解析。荧光定量PCR结果表明RhSKP1在月季花朵开放的5级时表达量最高，推测RhSKP1确实响应并参与了月季花朵的衰老调控。克隆了RhSKP1基因的ORF区全长，对其与其他物种中同源的SKP1类基因进行了比对和进化树分析。通过激光共聚焦观察发现RhSKP1定位在细胞核和细胞质上。通过月季花瓣（disc）进行VIGS，发现沉默RhSKP1延缓了月季花瓣的衰老。RhSKP1过表达拟南芥植株与野生型植株相比较，过表达植株叶片衰老程度较高，且其下胚轴长度、幼根长度和株高均短于野生型；使用100 µM ACC、1 mM SA对过表达植株进行处理，其叶片衰老明显高于野生型植株；说明RhSKP1以正调控的方式参与了月季花瓣衰老的调控及拟南芥叶片衰老的调控，此外，还以负调控的方式参与了拟南芥器官大小的调控。克隆了RhSKP1的启动子1500 bp，将其与RhNAC100基因共注射烟草进行启动子活性分析，初步分析表明RhNAC100抑制了RhSKP1启动子的活性。RhNAC100在3、4、5和6级的月季花瓣中高表达，说明RhNAC100可能参与了月季花朵的衰老调控。通过基因沉默实验证明，将RhNAC100沉默后可延缓月季花瓣衰老；说明RhNAC100以正调控的方式影响月季花瓣的衰老。通过EMSA体外分析证明了RhSKP1是RhNAC100的直接下游基因。综上所述，RhSKP1作为RhNAC100的直接下游基因调控了月季花朵的衰老，本研究发现了RhSKP1及SKP1类基因的新功能。. 此外，本项目通过代谢组和转录组解析了STS延迟月季花瓣脱落的机制，还开启了稀土元素对包括月季衰老在内的植物生长及遗传损伤机制的研究。基于项目的研究结果，已经发表SCI论文2篇，中文核心论文2篇，一般论文1篇；培养硕士研究生8名。