应用基因组靶向编辑技术--CRISPR/Cas9研究杨树开花的分子调控机制

Tradditional researches on regulation mechanism of poplar flowering at molecular level and functional genomics of forest trees are facing problems that there are no methods for creating high-throughput mutants and no highly-efficient methods for identification of gene functions. To this end, genomic DNA editing technology—CRISPR/Cas9 system will be employed in this study to design, synthesize sgRNAs of floral genes of poplar, and to construct CRISPR/Cas9-sgRNA library. With the aid of anther haploid induction system which have been build in our Lab., genetic transformation in poplar will be carried out using Agrobectirum-mediated method to build a high-throughput mutant library, to analysis gene function, gene interaction relationship, and to explore the establishment of a high-throughput research system for creating poplar mutants and more efficient identification of poplar gene function. Meanwhile, it is likely to obtain Haploid mutant, Double Haploid mutant, and even new polyploid germplasm. This study will not only help us for understanding the molecular regulation mechanism of poplar flowering in theoretical value, but also for creating variation and breeding of new varieties in practical value. It will be a bold attempt to germplasm innovation theory and practice in poplar, also is a reference for other woody plants.

本项目针对当前林木功能基因组学研究中存在的缺乏高通量创造突变体和高效鉴定基因功能的技术体系，拟采用基因组定点编辑技术--CRISPR/Cas9，设计合成杨树开花关键基因的sgRNA，构建CRISPR/Cas9-sgRNA表达载体库，利用本实验室已建立的杨树花药诱导单倍体技术体系，采用农杆菌介导的遗传转化法，构建杨树花发育基因的突变体库，开展杨树花发育调控基因功能鉴定和基因互作关系的深入研究，旨在揭示杨树成花起始及花器官发育相关基因的功能及基因互作关系，探索建立一套高通量创建杨树突变体和高效鉴定杨树基因功能的研究体系。同时可能获得单倍型突变体、双单倍型突变体、甚至多倍体等新种质。该研究不仅对于阐明杨树开花调控的分子机制具有重要的理论意义，而且对于创造变异、选育新品种具有重要的实践价值，对杨树种质创新理论和实践将是一次大胆的尝试，对其它物种的研究具有较高的借鉴价值。

本研究以毛白杨为试材，采用基因编辑技术，开展了杨树开花调控与花器官发育关键基因sgRNAs设计、CRISPR/Cas9-sgRNAs载体构建，通过在杨树遗传转化、分子检测获得了基因编辑的转化株系；根据毛果杨基因序列，设计构建了杨树MYB基因家族以及花青素合成途径基因的sgRNA文库，通过文库转化、分子检测获得41个阳性株系，其中8个多sgRNA靶点转化株系，33个单靶点转化株系；通过杨树花芽发育过程样本转录组分析，在全基因组水平分析控制毛白杨花发育的关键基因和基因家族，联合使用RNA-seq和实时荧光定量PCR技术对关键基因的表达规律进行深入解析，筛选与毛白杨花分生组织和花器官发育相关的基因，构建权重共表达基因调控网络，挖掘关键节点基因；初步建立了毛白杨单倍体诱导技术体系，通过细胞学、SSR分子标记鉴定获得了单倍体植株，并进行了加倍试验，单倍体材料为进一步进行基因编辑提供了理想材料。在杨树中初步建立了一套高通量创造杨树突变体及高效鉴定基因功能的研究体系。