CRISPR/Cas9介导的多基因编辑系统在大豆中的应用

自CRISPR/Cas9（clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9）基因 组编辑技术发现以来，迅速在作物中得到广泛应用。但是，CRISPR/Cas9多基因编辑系统在大豆中的研究尚待开 发。本文利用CRISPR/Cas9介导的多基因编辑系统，分别构建了两个载体，一个载体含6个靶点，编辑7个大豆基因 （4个Glycine max ASYMMETRIC LEAVES1（GmAS1）同源基因和3个GmAS2 同源基因），另一个载体含8个靶点，编辑 11个G. max AGAMOUS 家族同源基因（4个GmAG 同源基因，2个G. max SEEDSTICK（GmSTK）同源基因和5个G. max SHATTERPROOF1（GmSHP1/2）同源基因）。大豆遗传转化后，经表型鉴定和靶点检测发现，CRISPR/Cas9介导的多 基因编辑系统在大豆中成功实现了多基因编辑。当3个GmAS1 同源基因和3个GmAS2 同源基因同时突变时，导致 大豆叶片向远轴面弯曲、皱缩且叶柄变短的表型。当2个GmSHP1 同源基因和2个GmSTK 同源基因同时突变时，导 致豆荚停止发育的不育表型。

Application of CRISPR/Cas9-mediated multiplex genome editing system in soybean genome editing

To apply CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9)based genome editing system on soybean functional genomics and breeding,CRISPR/Cas9-mediated targeted mutagenesis were obtained for more than 5 genes in soybean.One CRISPR/Cas9 plasmid conferred 6 sgRNA expression cassettes targeting 4 Glycine max ASYMMETRIC LEAVES1(GmAS1)genes and 3 GmAS2 genes,and another plasmid conferred 8 sgRNA expression cassettes targeting 6 G.max AGAMOUS1(GmAG)genes and 5 G.max SHATTERPROOF1(GmSHP1/2)genes.From the double mutagenesis of 3 GmAS1 genes and 3 GmAS2 genes,a phenotype of upward curling leaves and shorten leaf petiole length was discovered.From the double mutagenesis of 2 GmSHP1/2 genes and 2 GmSTK genes,sterility phenotype was found.