不同Cas9启动子对大豆CRISPR/Cas9系统效率的作用分析  

Function analysis of different Cas9 promoters on the efficiency of CRISPR/Cas9 system in soybean

作　　者：牛志远 秦超 刘军[1] 王海泽[2] 李宏宇[1] NIU Zhi-Yuan;QIN Chao;LIU Jun;WANG Hai-Ze;LI Hong-Yu(Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China;College of Agriculture,Heilongjiang Bayi Agricultural University,Daqing 163319,Heilongjiang,China)

机构地区：[1]中国农业科学院作物科学研究所,北京100081 [2]黑龙江八一农垦大学农学院,黑龙江大庆163319

出　　处：《作物学报》2023年第12期3227-3238,共12页Acta Agronomica Sinica

基　　金：中国农业科学院科技创新工程-作物生物信息学及应用项目(2060302-2-20);大豆藏粮于技项目(CAAS-ZDRW202003)资助。

摘　　要：CRISPR/Cas9系统作为高效基因编辑系统,已被广泛应用于动植物中。多种Cas9基因启动子,如RPS5A、YAO等被报道用于提高CRISPR/Cas9系统的基因编辑效率。但在大豆中,不同Cas9启动子对CRISPR/Cas9基因编辑系统效率的影响还没有被阐明。本研究选择了6个已知功能的高效Cas9启动子(p35S、pGmRPS5Ab、pGmRPS5Ac、pAtRPS5A、pGmYAO、pZmUbiquitin)和1个大豆内源未知功能的启动子(pGmHE),构建CRISPR/Cas9基因敲除载体。通过农杆菌介导的大豆发根系统,检测这些Cas9启动子对大豆内源基因GmSPA1a和GmEID1的编辑效率,结果显示大豆内源启动子pGmRPS5Ab对靶基因的编辑效率最高,pAtRPS5A、p35S、pZmUbiquitin对下游基因的编辑效率高于pGmYAO和pGmRPS5Ac。进一步对靶位点测序峰图的分析发现,使用了pGmRPS5Ab和pAtRPS5A启动子的测序峰图中,高峰占比较大,分别为64.0%和58.6%;而使用p35S的测序峰图中,低峰占比较高,为63.3%。这表明pGmRP5SAb和pAtRPS5A启动子不但编辑效率高,而且编辑效果好,更有利于在下一代中分离出纯合突变体植株。这些结果将为构建高效大豆基因编辑载体提供参考,为提高大豆基因编辑效率提供依据。The CRISPR/Cas9 system has been widely used in plants and animals as an efficient gene editing system.Several Cas9 promoters,such as RPS5A and YAO,have been reported to improve the efficiency of gene editing in CRISPR/Cas9 system.In soybean,the influence of different Cas9 promoters on the efficiency of the CRISPR/Cas9 gene-editing system has not been elucidated.In this study,six efficient promoters with known functions(p35S,pGmRPS5Ab,pGmRPS5Ac,pAtRPS5A,pGmYAO,and pZmUbiquitin)and one endogenous soybean promoter with unknown function(pGmHE)were selected to construct the CRISPR/Cas9 knockout vectors.The editing efficiency of the Cas9 promoters on the endogenous soybean genes GmSPA1a and GmEID1 was tested by Agrobacterium tumefaciens mediated hair roots system,which indicated that soybean endogenous promoter pGmRPS5Ab had the highest editing efficiency.The editing efficiency of pAtRPS5A,p35S,and pZmUbiquitin were higher than that of pGmYAO and pGmRPS5Ac.Further analysis of the sequencing maps of target sites showed that the high peak maps accounted for 64.0%and 58.6%in the sequencing maps of pGmRPS5Ab and pAtRPS5A promoters,respectively,while in the sequencing maps of p35S-driven hair roots,the low peak accounted for a higher proportion(63.3%).These above results indicated that pGmRP5SAb and pAtRPS5A promoters not only had high editing efficiency,but also had better editing effect,and were more conducive to the isolation of homozygous mutants in the next generation.In conclusion,this study provide the reference for the construction of efficient soybean CRISPR/Cas9 vectors and help to improve the efficiency of soybean gene editing.

关 键 词：基因编辑 CRISPR/Cas9 发根系统 启动子 大豆 

分 类 号：S565.1[农业科学—作物学]