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
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，定位的位置為细胞核与细胞质膜。本研究为番木瓜抗曲叶病毒研究提供理论基础。
关键词 基因编辑;曲叶病毒;番木瓜;瞬时表达中图分类号 Q949.759.6 文献标识码 A
Construction of CRISPR Plant Expression Vectors Resistant to Papaya Leaf Curl Virus
LONG huan1，2， ZHAO Hui1，2*， WANG Xupeng2， JIA Ruizong1，2， HE Pingping1，2， KONG Hua1，2，GUO Yunling1，2， GUO Anping1，2**
1. Institute of Tropical Agricultural and Forestry， Hainan University， Haikou， Hainan 570228， China; 2. Institute of Tropical Bioscience and Biotechnology， Chinese Academy of Tropical Agricultural Sciences， Haikou， Hainan 571101， China; 3. Hainan Key Laboratory for Biosafety Monitoring and Molecular Breeding in Off-Season Reproduction Regions， Haikou， Hainan 571101， China
Abstract 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.
Keywords genome editing; leaf curl virus; papaya; transient expression
DOI 10.3969/j.issn.1000-2561.2019.10.015
番木瓜（Carica papaya L.）是热带地区最重要的消费和出口热带水果之一。目前从番木瓜上发现的病毒大多以番木瓜环斑病毒（Papaya ring spot virus， PRSV）和番木瓜畸形花叶病毒（Papaya leaf-distortion mosaic virus， PLDMV）为主，均为RNA病毒[1]，近期，项目组从海南地区番木瓜叶片上分离出番木瓜曲叶病毒（Papaya leaf curl virus， PaLCV），得病植株表现出植株矮小，叶柄卷曲，叶片皱缩失绿等现象，严重时植株绝产死亡。本研究利用最新的基因编辑手段，从病毒本身基因入手，力图通过给番木瓜建立CRISPR免疫系统，来提高番木瓜对该类病毒的抗病性。
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