基于CRISPR-Cas9基因编辑技术创制大豆gmnark超结瘤突变体

Generation of gmnark Mutant with Supernodulation via CRISPR-Cas9 in Soybean

为研究豆科作物结瘤自我调节机制(autoregulation of nodulation, AON)的作用机理,运用CRISPR-Cas9基因编辑技术创制大豆品种华春6号超结瘤gmnark突变体,设计3条靶向目的基因GmNARK的特异sgRNA,构建CRISPR-Cas9敲除载体。通过毛根转化试验选取sgRNA-B和sgRNA-C两条编辑效率较高的sgRNA用于大豆稳定转化,在T1代筛选出8种不同突变类型的突变体,在T2代通过营养液水培试验证明其中5种突变体有超结瘤的表型。选取gmnark-A突变体作进一步表型分析,发现其具有超结瘤、植株矮小和叶片深绿的表型。本研究所创制的gmnark突变体是研究AON途径作用机制与根瘤发育的重要遗传材料,同时此新种质资源具有作为"绿肥"与其它作物进行间作或轮作的巨大潜力。

In order to study the mechanism of the autoregulation of nodulation(AON) in the legumes, the gene editing technology of CRISPR-Cas9 system was used to create the gmnark mutant of the soybean variety Huachun 6, and three specific sgRNAs were designed to locate the targeted gene GmNARK and constructed into a CRISPR-Cas9 knockout vector. Two sgRNAs with high editing efficiency, sgRNA-B and sgRNA-C, were selected for stable transformation through hairy root transformation experiments. Furthermore, eight mutants of different mutation types were screened in T1 generation and five of them had the phenotype of supernodulation which is verified by hydroponic experiment. In the further phenotypic analysis of gmnark-A mutant, it was found that gmnark-A owned a larger number of nodules, smaller shoot and root and greener leaves than WT. These data proved that the gmnark mutant created in the study is an important genetic material for studying the mechanism of AON pathway and the development of nodule. At the same time, this new germplasm resource has great potential in intercropping with other crops as ‘green manure’.