miR159调控柑橘果实成熟的功能解析

In our country, citrus is given priority to with fresh fruit. However, the maturation stage of citrus is concentration and it is hard to meet the demand of continuously supplying fresh fruit in the whole year. Deeply exploring the molecular regulation mechanism of citrus fruit development and ripening can promote us to realize the formation mechanism of citrus fruit maturation stage. In our previous study, “Fengjie 72-1” navel orange and its late-ripening mutant “Fengwan” were used as research material. A small RNA (csi-miR159) related to the development and ripening of citrus fruit, and its target genes, GAMYBs, were identified by small RNA and degradome sequencing. In this project, we propose to verify the gene function of csi-miR159 during citrus fruit ripening by over-expression and suppressing expression of csi-miR159; the tobacco transient expression system will be used to validate the key target gene CsGAMYB which is cleaved by csi-miR159, and then over express mutated CsGAMYB resistant to the cleavage of miR159, to illuminate the role of CsGAMYB during fruit ripening and identify the key “miR159-GAMYB” module regulating citrus fruit ripening. This project will provide new knowledge on regulatory function of csi-miR159 during citrus fruit ripening and provide theoretical basis and good gene for citrus maturation molecular breeding.

我国柑橘以鲜食为主，但是成熟期过于集中，不能满足周年鲜果供应的需求，深入研究柑橘果实发育和成熟调控的分子机制，可加深对柑橘果实成熟期形成机制的认识。申请人前期以‘奉节72-1’脐橙及其晚熟芽变‘奉节晚橙’为材料，通过小RNA组和降解组测序鉴定了柑橘果实发育和成熟过程相关小RNA（csi-miR159）及其靶基因GAMYBs。本项目拟通过超量和抑制表达CsMIR159基因，验证csi-miR159在柑橘果实成熟过程中的功能；利用烟草瞬时表达系统验证受csi-miR159剪切的关键靶基因CsGAMYB，进而超量表达对miR159剪切不敏感的CsGAMYB基因，阐明CsGAMYB在果实成熟中的作用，最终揭示在柑橘果实成熟中起关键作用的“miR159-GAMYB”组合。本项目将明确csi-miR159在柑橘果实成熟过程中的调控作用，为柑橘熟期分子育种提供理论依据并储备优良基因资源。

柑橘果实的成熟时期、果实大小和无核是柑橘育种的重要目标。深入研究非跃变型柑橘果实发育及成熟的分子调控机制，可为定向分子改良柑橘品种提供理论依据和基因储备。多项研究表明“miRNA-靶基因”模型能够参与调节植物许多重要的性状。本研究前期以晚熟芽变‘奉节晚橙’及其母本‘奉节72-1’脐橙为材料，通过小RNA组和降解组测序鉴定出参与柑橘果实发育和成熟过程的“miR159a-GAMYBs”组合。本研究以micro-Tom番茄和山金柑为遗传转化材料，通过5’RACE技术、超表达miR159a、超表达对miR159a剪切不敏感的关键靶基因、构建CRISPR/Cas9体系敲除关键靶基因等分子生物学技术手段，多方面验证“miR159a-靶基因”组合在柑橘果实发育与成熟过程中的作用。主要实验结果如下:.1、RLM-RACE扩增出MIR159家族成员基因全长，确定其多肽序列，人工合成多肽用于外源处理柑橘；5’RACE技术验证了miR159a的靶基因是DUO1、GAMYB、SPL，亚细胞定位结果显示CsDUO1和CsSPL定位在细胞核中。.2、在番茄中超量表达csi-miR159a产生了无籽或少籽果实性状，初步验证了miR159a的功能；在山金柑中超量表达miR159a可产生早熟无核果实，同时极大的改变了果实的果形指数，进一步明确了miR159a在柑橘果实发育和成熟过程中的作用。.3、构建miR159a靶基因STTM载体以及miR159a靶基因CRISPR基因编辑载体，并转化山金柑，用于验证各个靶基因的功能，明确miR159a下游调控网络。.4、利用酵母单杂验证了FhDUO1和FhMPK20的互作关系，表明MPK20参与了miR159a-DUO1调控模式并且在DUO1的下游起作用，进一步明确了下游调控网络。. “miR159a-靶基因”组合参与了柑橘小孢子发育的调控，进而影响果实的发育和成熟过程，本研究发现miR159a可以极大的改变柑橘果实种子数量、果实成熟期等果实性状，其在柑橘果实发育和成熟过程中起着非常重要的作用，其在柑橘分子育种中将具有非常大的应用价值，同时在柑橘果实发育研究中具有重要的科学价值。