青花菜侧枝调控基因BoBRC1的克隆及功能分析  

Cloning and Functional Analysis of Lateral Branching Regulatory Gene BoBRC1 in Broccoli

作　　者：刘宇香 韩风庆 赵鑫雨 刘玉梅[2] 李占省[2] 方智远 LIU Yuxiang;HAN Fengqing;ZHAO Xinyu;LIU Yumei;LI Zhansheng;FANG Zhiyuan(College of Horticulture,Hunan Agricultural University,Changsha 410125,China;State Key Laboratory of Vegetable Biobreeding,Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[1]湖南农业大学园艺学院,长沙410125 [2]中国农业科学院蔬菜花卉研究所,蔬菜生物育种全国重点实验室,北京100081

出　　处：《园艺学报》2024年第9期1997-2007,共11页Acta Horticulturae Sinica

基　　金：国家重点研发计划项目(2022YFF1003000);国家自然科学基金项目(32302544,32172580);中国农业科学院科技创新工程项目(CAAS-ASTIP-IVFCAAS);现代农业产业技术体系建设专项资助(CARS-23);内蒙古自治区科技重大专项(2021ZD0001);乌兰察布市揭榜挂帅项目(2022JB006)。

摘　　要：从青花菜优良自交系L461中克隆了BoBRC1,表达模式分析表明其在腋芽组织中表达量最高,并随腋芽生长动态变化。根据其基因序列,在第1个外显子上设计2个靶标,构建CRISPR-Cas9基因编辑载体,通过农杆菌介导的遗传转化法导入自交系L461中,获得18个阳性株系。测序结果显示,有3个株系在至少1个靶标处发生了基因突变,编辑效率为16%。其中L461-3株系在靶标1和靶标2均发生编辑事件,L461-1和L461-11株系仅在靶标2处发生编辑事件。与野生型相比,T1代纯合突变体侧枝数明显增加,侧枝长度变长。对bobrc1纯合突变体中侧枝发育基因BoBRC1下游关键基因进行qRT-PCR分析发现,BoHB21、BoHB53、BoNCED3基因表达量显著降低,BoHB40基因表达量显著增高。以上研究结果证实了BoBRC1基因参与青花菜侧枝发育调控,敲除该基因可促进青花菜侧枝生长。In this study,the BoBRC1 gene was cloned from the elite broccoli inbred line L461.Expression pattern analysis showed that BoBRC1 had the highest expression level in axillary bud tissues,and changed dynamically with axillary bud growth.According to the sequence of BoBRC1,two targets were designed on the first exon,and a CRISPR/Cas9 gene editing vector was constructed.The construct was introduced into L461 by Agrobacterium-mediated genetic transformation,and a total of 18 plants were confirmed as positive transgenic.The results showed that three plants harbored mutations in at least one of the targeted regions corresponding to an editing efficiency of 16%.Among them L461-3 was edited in both Target 1 and Target 2 while L461-1 and L461-11 were edited in Target 2.Compared with the wild type,the homologous editing mutants showed a significant increase in the number and length of lateral branches.In the bobrc1 mutant,qRT-PCR analysis of key genes downstream of lateral branch gene BoBRC1 showed a significant decrease in the expression of the BoHB21,BoHB53 and BoNCED3 genes while the expression of the BoHB40 significantly increased.This study verified the function of BoBRC1 gene in the regulation of lateral branch development in broccoli,and knocking out can promote the growth of broccoli lateral branches.

关 键 词：青花菜 CRISPR/Cas9 基因克隆 功能分析 BoBRC1 侧枝调控 

分 类 号：S635.9[农业科学—蔬菜学]