基于CRISPR基因编辑系统抗番木瓜曲叶病毒的植物转化载体的构建  

Construction of CRISPR Plant Expression Vectors Resistant to Papaya Leaf Curl Virus

作　　者：龙欢 赵辉 王绪朋[2] 贾瑞宗 贺萍萍 孔华[1,2] 郭运玲 郭安平[1,2] 无 LONG huan;ZHAO Hui;WANG Xupeng;JIA Ruizong;HE Pingping;KONG Hua;GUO Yunling;GUO Anping(Institute of Tropical Agricultural and Forestry, Hainan University, Haikou, Hainan 570228, China;Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China;Hainan Key Laboratory for Biosafety Monitoring and Molecular Breeding in Off-Season Reproduction Regions, Haikou, Hainan 571101, China)

机构地区：[1]海南大学热带农林学院,海南海口570228 [2]中国热带农业科学院热带生物技术研究所,海南海口571101 [3]海南省南繁生物安全与分子育种重点实验室,海南海口571101

出　　处：《热带作物学报》2019年第10期2022-2028,共7页Chinese Journal of Tropical Crops

基　　金：海南省重点研发项目(No.ZDYF2017017);中国热带农业科学院基本科研业务费专项基金(No.1630052017012)

摘　　要：双生科病毒具有毁灭性强、寄主多样、分布地域广等特点,一直是困扰世界农作物生产的一大难题。用物理和化学方法防治该类病毒不仅花费成本高,见效低,而且还可能对环境造成负面影响,因此用分子手段培育抗病品种才是有效途径之一。本研究结合最新的CRISPR/Cas9基因编辑技术和Golden Gate克隆技术,设计并构建了抗番木瓜曲叶病毒基因编辑串联多切点表达载体。表达载体转入农杆菌AGL1后经注射本氏烟草叶片进行瞬时表达,48 h后通过激光共聚焦显微镜能观察到有绿色荧光的出现,定位的位置为细胞核与细胞质膜。本研究为番木瓜抗曲叶病毒研究提供理论基础。The Geminivirus are characterized by strong destructiveness, diverse hosts, and wide geographical distribution. They have always been a major problem in the crop production of the world . The use of physical and chemical methods to control them is not only costly, but also has low efficacy, and may also have a negative impact on the environment. Therefore, it is one of the effective ways to cultivate disease-resistant varieties by molecular breeding technology. The CRISPR/Cas9 gene editing technology and Golden Gate cloning technology were used to design and construct a gene editing multi-cut expression vector resistant to papaya leaf curl virus. The vector was transferred into Agrobacterium tumefaciens AGL1 and injected into the tobacco leaves for transient expression. After 48 h, the presence of green fluorescence was observed by a laser confocal microscopy. It was located in the nucleus and cytoplasmic membrane. The work would provide a theoretical basis for the study of papaya leaf curl virus.

关 键 词：基因编辑 曲叶病毒 番木瓜 瞬时表达 

分 类 号：Q949.759.6[生物学—植物学]