转录因子CsERF025调控黄瓜果实弯曲的机理研究

The fruit bending of cucumber leads to loss of vegetable commodity value, which always leads to great economic losses to farmers. In the previous study, cell senescence was found in the abdomen of cucumber bending fruit, which is the main reason of the abdomen of fruit grown slowly. Transcriptome of the bending cucumber was analyzed using Illumina technology. We found that the expression pattern of CsERF025 was similar with the ethylene biosynthesis gene CsACS2. Moreover, the relative expression level of CsERF025 in the abdomen of fruit was 70 times higher than that in the ridge using real-time quantitative RT-PCR. It was hypothesized that CsERF025 played a major role in fruit bending of cucumber by regulating ethylene biosynthesis. In order to test this hypothesis, the biological function of CsERF025 will be studied by gene overexpression and RNAi, the expression characteristics of CsERF025 protein will be analyzed at the tissue expression level and subcellular localization level, the downstream genes regulated by CsERF025 will be tested by yeast one-hybrid method. Taken together, the aim of this project is to elucidate the mechanism of cucumber fruit bending regulated by CsERF025, and the results will build the basis for breeding the new varieties of straight cucumber fruit and molecular breeding. It will also provide theoretical foundation for genetic improvements and germ plasma innovation of other cucurbit varieties, and regulatory mechanisms of fruit development.

在生产中黄瓜果实弯曲畸形导致商品性丧失，给农民造成很大的经济损失。本实验室前期研究表明弯曲果实腹部细胞呈现衰老趋势，导致腹部生长缓慢。进而利用Illumina技术对转录组进行测序分析，经qRT-PCR检测，确定转录因子CsERF025和乙烯合成关键基因CsACS2的表达存在一致性，并且CsERF025在黄瓜果实腹部上调表达量是脊部的70倍，表明其在黄瓜果实弯曲形成过程中发挥着主要作用，推测CsERF025调控乙烯生物合成影响黄瓜果实弯曲。本项目拟在前期分离获得CsERF025的基础上，采用过量表达和RNAi技术以及promoter-GUS、亚细胞定位和酵母单杂交技术等，对CsERF025的功能进行分析，以期探明CsERF025调控黄瓜果实弯曲的机理，从而为顺直的黄瓜新品种选育及分子育种奠定基础，同时也将会为其它葫芦科作物品种的遗传改良与种质创新，以及果实发育的分子调控机理研究提供理论依据。

果实作为黄瓜重要的营养和食用器官，其外观商品品质决定其商品价值，而弯曲的果实严重影响黄瓜的外观品质。因此，探索黄瓜果实弯曲发育机理对改善黄瓜外观品质和培育出黄瓜顺直的新品种具有重大意义。本项目选用华北型易弯曲黄瓜品系“L18”，利用转录组和PCR技术，筛选、分离获得黄瓜果实弯曲相关基因CsERF025的基础上，采用过量表达和RNAi以及亚细胞定位技术等对CsERF025进行功能分析，以期探明了CsERF025对黄瓜弯曲果实形成的调控机理，从而为顺直的黄瓜新品种选育及分子育种奠定基础。重要研究结果如下：. （1）确定了不同发育时期、不同部位乙烯相关基因 EIN3和乙烯响应因子CsERF025基因的表达模式。CsERF025、EIN3在果实弯曲角度最大保持期（4d），凸面表达极显著高于凹面，在不同弯曲率黄瓜品种中得到相同结果，且在顺直果实两侧的表达没有显著的差异。.（2）克隆获得了黄瓜CsERF025基因，同时构建了CsERF025基因的正义和反义表达载体；亚细胞定位发现CsERF025蛋白定位在细胞核中；通过转黄瓜验证CsERF025基因的存在，CsERF025过表达株系显著增加果实弯曲角度和弯曲比率，反义表达株系降低果实弯曲角度和弯曲率。.（3）揭示了黄瓜弯曲果实凸面和凹面两侧存在差异乙烯信号，且凸面乙烯信号强于凹面；CsERF025能够促进乙烯信号转导，且能增强弯曲果实两侧差异乙烯信号（凸面乙烯信号强于凹面）；CsERF025很可能通过增加乙烯合成相关基因的表达进而促进乙烯的合成。.（4）明确了CsERF025是调控黄瓜果实弯曲很重要的功能基因。ACC能够促进果实的弯曲，且随着ACC施用剂量的增加，果实弯曲角度增加，0.1μm ACC能有效提高果实的弯曲角度；反义表达果实施用0.1μm ACC，果实弯曲角度没有显著的增加；CsERF025基因的表达影响乙烯对弯曲果实的诱导的作用。