甜瓜乙烯应答因子CmERFI-15基因促进果实成熟的分子机理研究

The ethylene response factor (ERF), a downstream component of the ethylene signal transduction pathway, is a large group of transcription factors that determine the specificity of the ethylene response. In our previous work, the function of melon ERF-subfamily genes in fruit development was systematically studied. Overexpression and genome editing techniques conducted on melon ERF-subfamily genes showed that a gene CmERFI-15 can significantly promote melon fruit ripening. In this study, we intend to apply chromatin immunoprecipitation coupled to sequencing（ChIP-seq）to identify target genes of CmERFI-15 transcription factor, and further validate its target genes using yeast one-hybrid system and electrophoresis mobility-shift assay (EMSA). Furthermore, the yeast two-hybrid method will be used to screen and identify proteins that interact with the CmERFI-15 transcription factor. Meanwhile, the overexpressed and genome-edited T1 transgenic plants of CmERFI-15 gene will be cultivated and self-pollinated to obtain homozygous lines, which will further confirm the target genes of CmERFI-15 by transcriptome sequencing and qPCR method. The results are expected to reveal the molecular mechanism of CmERFI-15 gene promoting fruit ripening and provide new understanding for the molecular mechanism of ethylene-regulated melon fruit development. Our study is of great scientific significance and application value in genetic engineering of fruit ripening related traits of melon.

乙烯应答因子（ERF）是乙烯信号转导途径的最下游组分，是一个大的转录因子家族，它决定乙烯应答反应的特异性。本课题组前期对甜瓜ERF转录因子亚家族基因在果实发育中的功能进行了较系统的研究，通过基因超表达和基因组编辑技术发现一个ERF转录因子亚家族基因CmERFⅠ-15具有显著促进果实成熟的作用。本项目拟采用染色质免疫共沉淀测序技术，筛选鉴定CmERFⅠ-15转录因子的靶基因，应用酵母单杂交和凝胶阻滞实验进一步验证靶基因。用酵母双杂交方法筛选鉴定与CmERFⅠ-15转录因子互作的蛋白。同时，对已获得的CmERFⅠ-15基因的超表达和基因组编辑T1代转基因植株进行加代，获得纯合株系，进行转录组测序和qPCR验证靶基因。研究结果将揭示CmERFⅠ-15基因促进果实成熟的分子机理，为阐明乙烯调控甜瓜果实发育的分子机理提供新的认识，具有重要科学意义，对甜瓜果实成熟相关性状改良的基因工程具有应用价值。

乙烯应答因子（Ethylene response factor，ERF）是乙烯信号转导途径的下游组分，是一个大的转录因子家族，本课题组前期研究发现甜瓜ERF家族基因CmERFⅠ-15具有显著促进果实成熟的作用。本项目采用染色质免疫共沉淀测序技术，筛选鉴定了CmERFⅠ-15转录因子的靶基因，应用酵母单杂交和凝胶阻滞实验进一步验证了靶基因，结果表明CmERFⅠ-15转录因子的靶基因为CmACO7。用酵母双杂交方法筛选鉴定与CmERFⅠ-15转录因子的互作蛋白，但未发现强相互作用的互作蛋白。采用转基因技术获得了CmERFⅠ-15基因的超表达和基因编辑T3代转基因纯合株系，转录组测序和RT-qPCR验证结果表明CmERFⅠ-15基因正调控CmACO7表达。总之，研究结果揭示了CmERFⅠ-15基因促进甜瓜果实成熟的功能及其分子机理，进一步加深了乙烯调控甜瓜果实发育的分子机理的认识，具有重要科学意义，对甜瓜果实成熟性状改良的育种方面具有应用价值。