INRA717-1B4杂交杨TAC基因CRISPR/Cas9敲除载体的构建及编辑效率检测

Construction and Efficiency Testing of TAC Gene CRISPR/Cas9 Knockout Vector in INRA717-1B4 Hybrid Poplars

TAC(tiller angle control)基因作为IGT [G?L(A/T)IGT]保守家族的一员,与植物体内各类激素相互作用,共同参与分枝角度的调控。为探究TAC基因的分子结构功能及其调节功能,利用CRISPR/Cas9基因编辑系统,以INRA717-1B4杂交杨为试验材料构建pDE-CAS9-NPTⅡ表达载体,通过农杆菌介导法侵染叶片进行遗传转化获得阳性植株,并通过测序检测基因编辑效率。最终获得基因编辑转基因株系12株,其中实现基因突变的2株,编辑效率为16.7%。本试验为CRISPR/Cas9系统在杨树TAC基因功能的验证提供了借鉴,为进一步在木本植物中的应用研究提供了技术支持。

As a member of the IGT(G?L(A/T)IGT) family, TAC(tiller angle control) gene interacts with various hormones in the plant and is involved in the regulation of branching angle. To explore the molecular structure function and regulatory function of TAC gene, the pDE-CAS9-NPTⅡ expression vector was successfully constructed using the CRISPR/Cas9 gene-editing system with the INRA717-1 B4 hybrid poplars as experimental materials. The leaves were infected by Agrobacterium-mediated method for genetic transformation, and positive plants were obtained. The gene editing efficiency was tested by sequencing. Finally, 12 transgenic lines were obtained, of which, gene mutation was realized in 2 lines, and the editing efficiency was 16.7%. This study could provide a reference for verifying the function of TAC gene in hybrid poplars with CRISPR/Cas9 system, and provide technical support for further application research in woody plants.