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 Hainan 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 sgRNAand1 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 papaya varieties agatnst PRSV.%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.
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