李坏死环斑病毒诱导的桃PpPDS沉默体系的优化及验证  

Optimization and Validation of PpPDS Gene Silencing Induced by Prunus Necrotic Ring Spot Virus in Prunus persica

作　　者：潘佳佳 张东梅 孟建 高苏南 朱凯杰 刘军伟 李国怀[1] PAN Jiajia;ZHANG Dongmei;MENG Jian;GAO Sunan;ZHU Kaijie;LIU Junwei;LI Guohuai(National Key Lab for Germplasm Innovation and Utilization of Horticultural Crops,College of Horticulture and Forestry Sciences,Huazhong Agricultural University,Wuhan 430070,China)

机构地区：[1]华中农业大学园艺林学学院,果蔬园艺作物种质创新与利用全国重点实验室,武汉430070

出　　处：《园艺学报》2023年第7期1587-1600,共14页Acta Horticulturae Sinica

基　　金：国家重点研发计划项目(2019YFD1000100);国家现代农业产业技术体系建设专项资金项目(CARS-30)。

摘　　要：病毒诱导的基因沉默(virus-induced gene silencing,VIGS)是在桃(Prunus persica)上进行基因沉默的主要手段,现有VIGS体系效率较低,亟待开发针对桃的高效沉默体系。以桃八氢番茄红素脱氢酶(Phytoene desaturase)基因PpPDS为指示基因,探究不同侵染方式、病毒载体、温度、接种苗龄和菌液浓度对桃叶片VIGS效率的影响。结果表明,在侵染方式上,叶片注射是有效的方式;李坏死环斑病毒(Prunus necrotic ring spot virus,PNRSV)载体(pCaRNA1/2和pCaRNA3)优于烟草脆裂病毒(Tobacco rattle virus,TRV)改造后的TRV载体(pTRV1和pTRV2);温度为22℃左右PpPDS的沉默效率较高;苗龄为5~6片完全展开叶的桃苗沉默效率较高,达到85%;菌液浓度为OD600=1.0时沉默效率较高。以优化好的VIGS体系参数,选择桃叶绿素合成基因(PpGluTR和PpPOR)为目标基因进行验证,结果显示PpGluTR和PpPOR的表达均被显著抑制,叶片颜色变化明显。这些结果表明优化的VIGS体系稳定性好且沉默效率高,可用于桃基因功能的研究。At present,VIGS still works as a main method for gene silencing studies in peach.However,the current VIGS efficiency is low that is urgent to develop a more efficient VIGS system in peach.Using PpPDS(Phytoene desaturase gene)as the indicator gene,the effects of infection mode,viral vector,temperature,age of inoculated seedling and bacterial concentration on the VIGS efficiency in peach leaves,were comprehensively investigated.The results showed that leaf agroinfiltration was an effective way of infection;Prunus necrosis ring spot virus(PNRSV)vectors(pCaRNA1/2 and pCaRNA3)were better than the modified TRV vector(s pTRV1 and pTRV2)of tobacco rattle virus;the infection temperature was better at 22℃;peach seedlings with 5-6 fully expanded leaves were higher,reaching 85% of gene silencing efficiency;the bacterial concentration at OD600=1.0 was better.Based on the optimized parameters of VIGS system,two peach chlorophyll biosynthetic genes(PpGluTR and PpPOR)were selected as target to verify the optimized VIGS system.The results showed that the expressions of PpGluTR and PpPOR were significantly repressed,and the change of leaf color was evident.The studies suggest that the optimized VIGS system has good stability and high silencing efficiency,which can be used for gene silencing studies in peach.

关 键 词：桃 病毒诱导的基因沉默 李坏死环斑病毒 八氢番茄红素脱氢酶 叶绿素合成基因 

分 类 号：S662.1[农业科学—果树学]