泛素结合酶E2在番茄花柄脱落过程中的作用及乙烯响应因子ERF52调控研究

Ubiquitin-proteasome pathway is a crucial regulatory mechanism of plant growth and development and response to environmental signals. However, information about its role in organ abscission is still lacking. In this study, tomato flower pedicel was used as experimental material. First, screen out ubiquitin-conjugating enzyme E2 which medicate protein degradation pathway based on differential protein expression analysis during different stages of abscission by iTRAQ labeling; Second, screen out the key E2 genes involved in abscission according to the effects of RNAi silencing and over-expressing candidate genes on abscission, and combine with differences in proteins ubiquitination level and sensibility to auxin and ethylene in transgenic plants, screen out members of auxin and ethylene signaling which display varying ubiquitination level via SWATH-MS, identify specific signal pathway regulated by ubiquitin protein degradation. Third, analyze the expression levels of selected E2 gene in the ERF52 antisense lines, and perform yeast one-hybrid system and EMSA (electrophoretic mobility shift assay) to clarify whether ERF52 regulate E2 expression directly and thereby mediate ubiquitin-proteasome pathway during abscission. Fourth, identify E2 which regulated directly by ERF52 using ChIP-qPCR, and detect corresponding E2 genes expression level taking advantage of RNA-seq and qRT-PCR, screen differential proteins based on quantitative determination of protein ubiquitination level by SWATH and ITRAQ assay, in order to provide a completely evalution for how ethylene impacts E2-regulated ubiquitin protein degradation pathway via ERF52 during tomato pedicel abscission. Altogether, above conclusion will contribute additional knowledge to mechanism mode of ubiquitin degradation and ERF52 during plant organ abscission.

泛素蛋白降解途径是植物生长发育和响应外界环境信号的重要调控机制，但其在器官脱落中的作用并不清楚。本研究以番茄花柄为试材，首先根据不同脱落阶段，iTRAQ筛选出差异表达的泛素结合酶E2；通过沉默和超表达候选基因研究其对脱落的影响，筛选调控脱落的关键E2基因，并结合其体内蛋白泛素化水平差异以及对生长素和乙烯的敏感性，SWATH-MS筛选出泛素化水平差异的生长素以及乙烯信号成员，明确泛素蛋白降解调控的具体信号路径；第三根据筛选出的关键E2基因，分析其在ERF52RNAi表达情况，并通过酵母单杂交技术和ChIP-qPCR实验，明确ERF52是否直接调控E2表达；最后，通过RNA-seq和qRT-PCR检测相应E2基因表达水平，SWATH定量检测蛋白泛素化水平结合iTRAQ筛选出差异蛋白，明确ERF52对于E2家族的调控，从而弄清脱落过程中乙烯通过ERF52影响E2调控的泛素蛋白降解途径。

泛素蛋白降解途径是植物生长发育和响应外界环境信号的重要调控机制，但其在器官脱落中的作用并不清楚。本研究以番茄花柄为试材，首先根据不同脱落阶段，筛选调控脱落的候选E2；通过VIGS技术研究其对脱落的影响，筛选出调控脱落的关键基因UBC15、16和34。以UBC34为诱饵，采用酵母双杂交筛选其互作的泛素连接酶E3或靶蛋白；通过构建互作蛋白的CRISPR/CAS9植株研究其对脱落的影响。结合EMSA和LUC试验，明确ERF52直接调控UBC34表达进而调控脱落过程中的泛素蛋白酶体途径。通过构建VIGS和超表达植株明确WUS调控脱落发生；以WUS作为诱饵筛选出互作的泛素连接酶E3，发现泛素连接酶E3通过影响WUS蛋白稳定性调控脱落。进一步研究发现，WUS直接抑制FUL2表达从而控制乙烯合成，延缓番茄花柄脱落。