miR396应答新型细胞分裂素PBU调控尾巨桉体胚高频发生的分子机制研究

桉树是世界三大速生经济林树种之一。中国桉树人工林种植面积已有368万公顷，木材产量占国内纸浆用材的50%以上。作为遗传转化的良好受体，胚性愈伤组织及体胚诱导一直深受研究者重视。申请人在前期研究中，应用新型细胞分裂素PBU成功诱导了尾巨桉体胚高频发生（专利号：ZL201310125000.X），并发现miR396在尾巨桉体胚发生过程中表达水平显著上调，且与PBU的诱导作用表现出紧密的相关性。本项目在前期建立的尾巨桉高效遗传转化体系基础上，通过CRISPR/Cas9基因组靶向定点编辑技术编辑miR396目标基因及其上游激素响应元件，探讨miR396基因家族在尾巨桉体胚高频发生过程中的生物学功能以及与PBU应答机制，为解析尾巨桉体胚高频发生的分子机理提供参考，促进速生经济林树种体胚离体再生技术的建立。

Eucalyptus is one of the three fast-growing economic forest tree species in the world and is the most important forest tree in China, covering more than 3.68 million hectares of commercial plantations. More than 50% pulp is provided by Eucalyptus. Embryonic somatic embryogenesis induction has always been focused as a superior recipient system of gene transfer. Embryonic callus and somatic embryogenesis in Eucalyptus urophylla x Eucalyptus grandis were induced by PBU (patent number: ZL201310125000.X). Our previous studies found that the expression level of miR396 family genes increased significantly in the process of Eucalyptus somatic embryogenesis, and showed a close correlation to PBU. In this proposal, our studies are based on our efficient transformation system of Eucalyptus urophylla × Eucalyptus grandis established in the early stage. We will study the response mechanism of the upstream hormone response element of miR396 to PBU by Site-directed gene-editing CRISPR/Cas9 techniques , and find out the biological function of miR396 gene in the process of somatic embryos occurred in Eucalyptus urophylla× Eucalyptus grandis. With this work, we are to lay the foundation for the molecular mechanism of somatic embryogenesis of Eucalyptus urophylla× Eucalyptus grandis and to further promote the application of PBU in Eucalyptus micropropagation and somatic embryogenesis.