CsIPT1b和CsUGT85A2调控黄瓜超级子房形成的分子机制研究

The corolla opening of super ovaries delayed for 4~5 days, thus resulting fruits with flowers remaining on the tip, of seedless and high commodity. Our previous study showed that cytokinin plays a decisive role in super ovary development in cucumber. CsIPT1b is one of the key genes for cytokinin synthesis, and CsUGT85A2 acts as a glucuronosyltransferase that transforming the active cytokinin into inactive forms. The expression of CsIPT1b　and CsUGT85A2 correlates positively and negatively with super ovary formation, respectively, suggesting that CsIPT1b and CsUGT85A2 may regulate super ovary development by modulating cytokinin synthesis and/or metabolism, but the specific molecular mechanism remains elusive. Thus, in this study, we will use cucumber inbred line '6547' that producing super ovary spontaneously as material, to investigate the gene function of CsIPT1b and CsUGT85A2 in super ovary genesis through gene expression analyses, genetic transformation and phenotype analysis; to dissect the physiological mechanism of CsIPT1b and CsUGT85A2 regulating super ovary formation through hormone measurement, enzyme activity analysis, substrate and production analysis; to explore the putative methylation mechanism of CsIPT1b and CsUGT85A2 in the super ovary development through DNA bisulfite sequencing; and to uncover the upstream regulatory mechanism of CsIPT1b and CsUGT85A2 by cis-acting element analysis, yeast one hybrid and EMSA. The results obtained in this study will not only enrich the molecular mechanism of corolla opening regulation in plants, but also provide the theory basis for cucumber breeding with　excellent quality.

黄瓜超级子房花冠延迟开放4~5天，形成头顶鲜花的无籽商品瓜。前期研究已明确细胞分裂素是黄瓜超级子房形成的决定因子，细胞分裂素合成关键基因CsIPT1b和活性稳态关键基因CsUGT85A2的表达水平与黄瓜超级子房形成分别呈正、负相关，但其具体的分子调控机制尚不清晰。因此，本项目以黄瓜超级子房自交系‘6457’为材料，通过基因表达、遗传转化及表型观察等方法，明确CsIPT1b和CsUGT85A2调控黄瓜超级子房形成的基因功能；通过激素测定、酶活检测、作用底物及产物分析等方法，解析CsIPT1b和CsUGT85A2调控黄瓜超级子房形成的生理机制；利用DNA甲基化测序技术，探讨这两个基因是否通过DNA甲基化调控超级子房的形成；通过顺式作用元件分析，预测上游转录因子，通过酵母单杂交、EMSA等确定CsIPT1b和CsUGT85A2的上游调控机制。对丰富植物花冠开放分子机制，指导黄瓜优质育种有意义。

黄瓜超级子房花冠延迟开放4-5天，形成头顶鲜花的无籽商品瓜。前期研究已明确细胞分裂素是黄瓜超级子房形成的决定因子，细胞分裂素合成关键基因CsIPT1b和活性稳态关键基因CsUGT85A2的表达水平与黄瓜超级子房形成分别呈正、负相关，但CsIPT1b和CsUGT85A2及其上游调控因子具体如何调控黄瓜超级子房的形成尚不清晰。本研究通过分析CsIPT1b和CsUGT85A2的表达模式，发现这两个基因均在花器官中显著表达；通过转基因试验，明确了CsIPT1b正向调控黄瓜超级子房的形成，而CsUGT85A2负向调控超级子房的形成；细胞分裂素的测定结果表明CsIPT1b和CsUGT85A2是通过调控游离细胞分裂素的含量从而调控超级子房的产生，tZ、tZR和IPR是主要的游离细胞分裂素；通过酵母单杂交试验找到并初步验证了调控CsUGT85A2的转录因子CsZFP（XP_011659456）；正常子房与超级子房花冠发育不同时期鉴定到234个差异表达miRNAs (DEMs) 和 291个差异表达靶基因 (DE-target genes)，并预测到miRNA_157-CsCuRX、miRNA_411-CsGH3.6和miRNA_161/297/257-CsHD-Zip可能与黄瓜超级子房花冠开放有关。本项目阐明了CsIPT1b和CsUGT85A2调控黄瓜超级子房形成的分子机理，并为miRNA调控黄瓜超级子房形成提供了新的见解。