用Golden Gate法构建广谱抗番木瓜环斑病毒海南株系的CRISPR/FnCas9植物表达载体  

Construction of CRISPR/FnCas9 Plant Expression Vectors with Broad Spectrum Resistance to Hainan Papaya Ringspot Virus by Golden Gate

作　　者：王绪朋 赵辉[2] 贾瑞宗[2] 孔华[2] 郭运玲[2] 郭安平 WANG Xupeng;ZHAO Hui;JIA Ruizong;KONG Hua;GUO Yunling;GUO Anping(Institute of Tropical Agricultural and Forestry, Hainan University, Haikou, Hainan 570228, China;Institute of Tropical Bioscienee and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, 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

出　　处：《热带作物学报》2018年第5期955-962,共8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资金(No.1630052017012)

摘　　要：分子生物学育种是目前防治番木瓜环斑病毒(PRSV)最有效的方法。早期转基因番木瓜抗病毒的策略是根据"致病菌衍生的抗病性"(PDR)原理,将病毒来源的基因全长序列,转入番木瓜并获得抗性。PDR策略最主要特点依赖于靶目标病毒基因序列相似性,不能有效解决海南的番木瓜环斑病毒遗传多样性复杂的株系。本研究着眼于创制广谱的抗PRSV新策略,用最新的CRISPR/FnCas9基因编辑技术和Golden Gate载体构建系统,依据不同PRSV株系的保守序列分析结果,将5个sgRNA(2个CP sgRNA,2个Nib sgRNA和1个HC-Pro sgRNA)串联在一个载体上,实现一个载体可识别剪切5个位点,从而达到广谱抗PRSV的效果,应对海南地区PRSV株系复杂的问题。目前载体已经进行了测序验证,并在番木瓜上进行了叶片局部瞬时表达,初步表现出对PRSV的抑制效果,这为获得广谱抗番木瓜环斑病毒的番木瓜品种奠定良好基础。Molecular breeding is effective approach to develop resistance papaya against Papaya ring spot virus （PRSV）. Pathogen-derived resistance （PDR） was employed in the development of transgenic papaya. Fundamentally, PDR technology is sequence homology-dependent resistance, however, the genetic biodiversity of PRSV in Haiuan is higher than other region in the world. To solve the PRSV problem in Hainan, we used the latest genomic editing technology CRISPR/ FnCas9 assisted with Golden Gate vector system to construct 5 concatenated sgRNA （2 CP sgRNA, 2 Nib sgRNAandl HC-Pro sgRNA） on single vector, which can achieve the broad-spectrum PRSV resistance. In the later period, we can easily adjust or replace sgRNA according to the actual disease resistance, and deal with the complex problem of the papaya ring spot virus strain in Hainan. The vector has been sequenced and verified. The vector transiently expressed on leaves of papaya and showed a preliminary inhibitory effect on PRSV. This will lay a good foundation for obtaining broad spectrum resistance Danava varieties agatnst PRSV.

关 键 词：番木瓜 CRISPR 番木瓜环斑病毒 广谱抗病 基因组编辑 

分 类 号：S668.2[农业科学—果树学]