利用CRISPR/Cas9系统敲除葡萄中VviEDR2提高对白粉病的抗性  

CRISPR/Cas9-mediated Mutagenesis of VviEDR2 Results in Enhanced Resistance to Powdery Mildew in Grapevine(Vitis vinifera)

作　　者：杨禄山 郭晔 胡洋 文颖强[1] YANG Lushan;GUO Ye;HU Yang;WEN Yingqiang(College of Horticulture,Northwest A&F University,Key Laboratory of Horticulture Plant Biology and Germplasm Innovation in Northwest China,Ministry of Agriculture,State Key Laboratory of Crop Stress Biology for Arid Areas,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学园艺学院,农业农村部西北地区园艺作物生物学与种质创制重点实验室,旱区作物逆境生物学国家重点实验室,陕西杨凌712100

出　　处：《园艺学报》2020年第4期623-634,共12页Acta Horticulturae Sinica

基　　金：国家重点研发计划项目(2018YFD1000300);国家自然科学基金项目(31772264)。

摘　　要：利用CRISPR/Cas9系统定点编辑葡萄白粉病感病基因VviEDR2(Enhanced disease resistance 2),在VviEDR2的DUF1336结构域设计靶位点VviEDR2-T1,构建CRISPR/Cas9敲除载体,通过农杆菌介导法转化‘无核白’葡萄胚性愈伤组织。对PCR阳性植株进行靶位点扩增测序,结果表明,共有8个转基因植株在靶位点处发生不同类型的双等位基因突变,编辑效率为32%;突变体植株生长势较弱,叶片较小,茎秆丛生、细弱。进一步对突变体植株进行抗病检测,结果表明,接种葡萄白粉菌(Erysiphe necator Schw.)5 d后,突变体植株叶片上白粉菌孢子仅能萌发出少量较短初级菌丝,表皮细胞产生大量明显的H2O2,而野生型叶片中白粉菌萌发出大量初级菌丝、次级菌丝和吸器,无明显H2O2产生。这些结果表明,可以利用CRISPR/Cas9技术编辑葡萄感病基因VviEDR2,提高葡萄白粉菌抗性。Grapevine(Vitis vinifera L.)incurs significant yield and quality losses from powdery mildew(Erysiphe necator Schw.).Here,we used the CRISPR/Cas9 system to precisely edit the susceptibility gene VviEDR2(Enhanced disease resistance 2)and provide reference for disease resistance breeding.We designed a target site in DUF1336 domain of the VviEDR2 gene for the CRISPR/Cas9 vector,and obtained transgenic plants via Agrobacterium-mediated transformation,using somatic embryos of the Thompson Seedless cultivar.Sequencing revealed that 8 out of 25 transgenic plants carried different type of biallelic mutations in target sites and the editing efficiency is 32%.These mutants exhibited reduced growth compared to wild type,such as smaller leaves,thinner and fasciculate stems.Inoculation assays with an adapted grapevine powdery mildew isolate En NAFU1 showed that hyphal growth is significantly restricted on mutant grapevine leaves with obvious H2O2 accumulation in penetrated epidermal cells at 5 days post inoculation.Conversely,massive hyphae and haustoria,but no obvious accumulation of H2O2,were observed in the leaves of wild type.These results indicated that CRISPR/Cas9 technology could be utilized to knockout susceptibility gene VviEDR2 in grapevine,which could improve powdery mildew resistance.

关 键 词：葡萄 CRISPR/Cas9 基因编辑 VviEDR2 白粉菌 

分 类 号：S663.1[农业科学—果树学]