基于CRISPR基因編輯系統抗番木瓜曲葉病毒的植物轉化載體的構建

龍歡 趙輝 王緒朋 賈瑞宗 賀萍萍 孔華 郭運玲 郭安平

1. 海南大學熱帶農林學院，海南海口??570228;2. 中國熱帶農業科學院熱帶生物技術研究所，海南海口 ?571101;3. 海南省南繁生物安全與分子育種重點實驗室，海南海口 ?571101

摘 ?要??雙生科病毒具有毀滅性強、寄主多樣、分布地域廣等特點，一直是困擾世界農作物生產的一大難題。用物理和化學方法防治該類病毒不僅花費成本高，見效低，而且還可能對環境造成負面影響，因此用分子手段培育抗病品種才是有效途徑之一。本研究結合最新的CRISPR/Cas9基因編輯技術和Golden Gate克隆技術，設計并構建了抗番木瓜曲葉病毒基因編輯串聯多切點表達載體。表達載體轉入農桿菌AGL1后經注射本氏煙草葉片進行瞬時表達，48 h后通過激光共聚焦顯微鏡能觀察到有綠色熒光的出現，定位的位置為細胞核與細胞質膜。本研究為番木瓜抗曲葉病毒研究提供理論基礎。

關鍵詞 ?基因編輯;曲葉病毒;番木瓜;瞬時表達中圖分類號??Q949.759.6??????文獻標識碼??A

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

LONG huan^1，2， ZHAO Hui^1，2*， WANG Xupeng²， JIA Ruizong^1，2， HE Pingping^1，2， KONG Hua^1，2，GUO Yunling^1，2， GUO Anping^1，2**

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

DOI10.3969/j.issn.1000-2561.2019.10.015

番木瓜（Carica papayaL.）是熱帶地區最重要的消費和出口熱帶水果之一。目前從番木瓜上發現的病毒大多以番木瓜環斑病毒（Papaya ring spot virus， PRSV）和番木瓜畸形花葉病毒（Papaya leaf-distortion mosaic virus， PLDMV）為主，均為RNA病毒^[1]，近期，項目組從海南地區番木瓜葉片上分離出番木瓜曲葉病毒（Papaya leaf curl virus， PaLCV），得病植株表現出植株矮小，葉柄卷曲，葉片皺縮失綠等現象，嚴重時植株絕產死亡。本研究利用最新的基因編輯手段，從病毒本身基因入手，力圖通過給番木瓜建立CRISPR免疫系統，來提高番木瓜對該類病毒的抗病性。……