CsCLE3调控黄瓜超级子房形成的分子机制研究

Cucumber fruit with 'flourishing corollas and spines' is an important attractive appearance for fresh market. The corolla opening of super ovaries was much larger and delayed for 4~5 days, thus resulting fruits with flowers remaining on the tip, of seedless and high commodity. Our previous study showed that integration of cytokinin and N, P nutritional plays a decisive role in super ovary development in cucumber. CsCLE3 acts as a peptide signaling molecule. The expression of CsCLE3 correlates negatively with super ovary formation, and contains the cis-acting elements for cytokinin and N, P in the promoter regions. Furthermore, exogenous application of cytokinin and N, P nutrition significantly induces the super ovary production and decrease the expression of CsCLE3, suggesting that CsCLE3 may regulate super ovary development by responding cytokinin and N, P nutrition, 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 CsCLE3 in super ovary genesis through gene expression, cucumber transformation, physiological index and phenotype analysis; to dissect the signal pathway and gene network of CsCLE3 regulating super ovary formation through response to hormones, RNA-Seq and gene expression analysis; to explore the putative methylation mechanism of CsCLE3 in the super ovary development through DNA bisulfite sequencing; and to uncover the upstream regulatory mechanism of CsCLE3 by cis-acting element analysis, yeast one hybrid and EMSA. The results obtained in this study will not only enrich the molecular mechanism of peptide signal molecule in fruit and flower development in plants, but also provide the theory basis for cucumber breeding with excellent quality and propagation practices.

‘顶花带刺’是鲜食黄瓜重要的外观品质性状，黄瓜超级子房花冠巨大且延迟开放4~5天，形成头顶鲜花的无籽果实。我们已明确细胞分裂素与N、P营养协同调控黄瓜超级子房的形成；多肽信号分子CsCLE3的表达与黄瓜超级子房形成负相关，其启动子区含有响应细胞分裂素和N、P营养的顺式作用元件；外源施加细胞分裂素和N、P营养能显著促进超级子房的形成，抑制CsCLE3的表达，但其具体的分子作用机制仍不清楚。因此，本项目拟通过基因时空表达、转基因、生理指标检测及表型分析等方法，明确CsCLE3调控黄瓜超级子房形成的基因功能；通过激素响应分析、干扰表达转基因材料RNA-Seq及信号转导基因表达分析等方法，解析CsCLE3可能的调控途径和机理；利用DNA甲基化分析、转录因子预测及酵母单杂交、EMSA等验证手段，确定CsCLE3的上游调控机制。对认识多肽激素调控植物果实及花发育分子机制，指导黄瓜育种和良种繁育有意义。

黄瓜是重要的世界性蔬菜。‘顶花带刺’是鲜食黄瓜重要的外观品质性状。有学者研究发现，黄瓜有些雌花开花时，子房更大、更长，称为超级子房。生产上，常采用氯吡脲等生长调节剂蘸花处理的方式获得，但费时费工。本项目前期发现黄瓜特异材料‘6457’不需生长调节剂处理，在自然生长条件下就能产生超级子房，花冠开放当天子房长度已达15.0～17.0 cm（正常子房5.0～7.0 cm），花冠更大且开放时间推后4~5天。花冠开放后1~2天，子房已达可采摘商品瓜大小，但花冠还未凋谢，因而形成头顶鲜花的商品瓜，深受消费者喜爱，市场竞争力强，具有重要的应用价值。.前期利用组学手段，发现黄瓜CsCLE3的表达与超级子房的形成负相关，外源施加细胞分裂素能显著促进超级子房的形成，显著抑制CsCLE3的表达。还发现，CsCLE3启动子区同时含有响应细胞分裂素和N、P营养的顺式作用元件。为进一步研究黄瓜CsCLE3与超级子房形成的关系，利用基因编辑技术、蛋白互作以及组学分析等技术手段，创制了黄瓜CsCLE3过量表达和敲除转基因株系；揭示了黄瓜CsCLE3通过影响雌花发育、超级子房发生率负调控超级子房形成的基因功能；开展了CsCLE3上游调控基因的筛选与验证等试验，但未发现相关互作蛋白；发现了细胞分裂素和N、P营养可能通过抑制CsCLE3的表达，进而正向调控黄瓜超级子房的形成；通过转基因株系与空载对照株系转录组分析，发现生长素响应因子、细胞分裂素代谢相关基因呈现出明显聚类，推测CsCLE3可能与细胞分裂素和生长素等激素存在其它未知相互作用，进而协同调控黄瓜超级子房的形成。.本研究丰富了黄瓜果实发育的理论，证实了CsCLE3在黄瓜超级子房形成过程中的负调控作用。通过基因编辑技术，实现了CsCLE3的有效基因编辑，提高了黄瓜超级子房率，为“顶花带刺”黄瓜新品种培育提供了育种材料和理论支撑，有望实现产业化应用。