落叶松miR156b调控体细胞胚发育的功能解析

Given the diverse functions of the miR156/SPL module with in plant growth and development, it has important implications for the genetic improvement of plants. miR156-mediated repression of AtSPL10/11 have been implicated in cell differentiation during early embryogenesis in Arabidopsis thaliana. Overexpression miR156b of Larix leptolepis significantly increased the rate of embryo formation and rooting in preliminary study, which can overcome a number of challenges in conifer somatic embryogenesis, such as difficulties with in vitro synchronization and a low root frequency. This project will continue to identify miR156b-targeted genes in Larix leptolepis by using nucleic acid sequences of larch and other gymnosperms, and study their regulatory networks. One aim of this project was to identify the major target genes of miR156b during somatic embryogenesis and its biological function through analysis of molecular and phenotypic characterization of overexpression miR156b. The other aim of this project was to create knockout mutants using CRISPR/Cas9 system and elucidate the function of miR156b during somatic embryogenesis of Larix leptolepis. A better understanding of the function of miR156/SPL in gymnosperms will certainly help the precise design of tools for improving agronomic traits.

miR156/SPL系统在植物生长发育中具有多种生物学功能，对植物的遗传改良具有重要意义。拟南芥miR156通过调控AtSPL10/11参与了早期胚胎的细胞分化。我们前期研究表明，落叶松过表达miR156b可显著提高体细胞胚成胚率和生根率，这为解决针叶树体细胞胚同步化发育、生根率低等难题带来了曙光。本项目将利用落叶松及其他裸子植物核酸数据，进一步鉴定落叶松miR156b靶基因，研究miR156b的表达调控规律，揭示其靶标互作调控网络；重点研究：(1)过表达miR156b转基因细胞系的表型及分子特征，明确miR156b调控体细胞胚发育的主效靶基因及其生物学功能；(2)利用CRISPR/Cas9系统创制miR156b的定向敲除突变体，阐释miR156b在体细胞胚发育过程中的调控机制。该研究可以拓展人们对裸子植物miR156/SPL调控系统的认识，为其在植物中精确改良目标农艺性状奠定基础。

本项目围绕落叶松miR156b的功能研究，搜集现有落叶松核酸数据，构建非冗余数据库；克隆了LamiR156b前体序列及靶基因LaSPLs序列，并进行了生物信息学分析、切割位点验证及亚细胞定位研究；获得了LamiR156b及靶基因LaSPLs启动子序列，分析表明其可能与激素、抗性和光应答相关；检测了LamiR156b及靶基因LaSPLs在激素和体细胞胚发育中的表达模式；成功实施了LamiR156b promoter（-2937bp）：GUS的转化，研究了其在体细胞胚不同发育时期的空间定位；检测了转基因细胞系中premiR156b及靶基因LaSPLs的表达，发现它们呈负调控关系；RNA-seq分析表明过表达LamiR156b调控类黄酮生物合成相关基因的表达；上述关于LamiR156b的研究工作，揭示了miR156b在体细胞胚发育中的调控机制。