摘要
【目的】建立桑树的病毒诱导基因沉默(VIGS)转化体系,为桑树功能基因的分析研究提供技术支持。【方法】以丰驰桑为试验材料,克隆含Bam H I和Xba I酶切位点的八氢番茄红素脱氢酶(PDS)基因片段,经Xba I和Bam H I双酶切后与烟草脆裂病毒(TRV)连接构建重组病毒载体p TRV2-PDS,转化农杆菌GV3101后通过压迫注射方式侵染桑树幼苗叶片。【结果】克隆获得的桑树PDS基因干扰片段346 bp,与改造后的TRV质粒连接可成功构建携带桑树PDS基因的重组病毒载体p TRV2-PDS。桑树叶片被侵染15 d后,接种重组病毒载体p TRV2-PDS桑树的第二轮真叶开始出现叶片白化现象,侵染20 d后,白化现象沿叶脉向四周逐渐扩散,叶片呈现明显的白化表型。实时荧光定量PCR检测结果显示,重组病毒载体p TRV2-PDS侵染的桑叶PDS基因相对表达量仅为阴性对照(p TRV2)的58%,二者差异极显著(P<0.01),表明桑树PDS基因表达被有效抑制。【结论】利用TRV构建的桑树VIGS体系能有效沉默PDS基因,使桑树叶片呈现明显的白化症状,即可用于后续的桑树功能基因鉴定。
【Objective】The present study was conducted to establish mulberry virus-induced gene silencing(VIGS)transformation system and provide technical support for the analysis and research of the mulberry functional genes.【Method】In this research,Fengchi mulberry was used as experimental material,fragment of phytoene desaturase(PDS)gene with the Xba I and BamH I restriction enzyme sites were cloned.After Xba I and BamH I double digestion,the recombinant virus vector pTRV2-MPDS was obtained by ligating them into the tobacco rattle virus(TRV).Then pTRV2-PDS was transformed into Agrobacterium tumefaciens GV3101 and then transformed into mulberry leaves by pressure injection.【Result】The PDS interference fragment with the length of 346 bp was obtained by cloning and then the recombinant virus vector pTRV2-PDS containing mulberry gene PDS was obtained by connection with the modified TRV plasmid.After 15 d of infection,there were albino phenotypes in the second round of mulberry leaves that inoculated with recombinant virus vector pTRV2-PDS,and the albinism gradually spread along the veins when mulberry plants were infected 20 d,and the leaves showed obvious albino phenotypes.Real-time quantitative PCR detection results showed that the expression of gene PDS in leaves of recombinant virus vector pTRV2-PDS inoculated plant dropped to 58%compared with the negative control(recombinant virus vector),there was extremely significant difference between them(P<0.01),which showed that mulberry gene PDS was effectively silenced.【Conclusion】Gene PDS can be effectively silenced by using mulberry VIGS system constructed by TRV and then the mulberry leaves appeared obvious albino phenotypes.The VIGS system can be used for subsequent functional identification of mulberry genes.
作者
李瑞雪
王钰婷
胡飞
夏家凤
汪泰初
赵卫国
LI Rui-xue;WANG Yu-ting;HU Fei;XIA Jia-feng;WANG Tai-chu;ZHAO Wei-guo(Sericultural Research Institute,Anhui Academy of Agricultural Sciences,Hefei 230061,China;Jiangsu University of Science and Technology,Zhenjiang,Jiangsu 212018,China;Institute of Plant Protection and Agro-products Safety,Anhui Academy of Agricultural Sciences,Hefei 230061,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2018年第7期1432-1438,共7页
Journal of Southern Agriculture
基金
国家蚕桑产业技术体系建设项目(CARS-18-SYZ07)
安徽省农业科学院人才发展专项项目(17F0609)
安徽省农业科学院科技创新团队项目(18C0614)
安徽省重点研究和开发计划面上攻关项目(1804a07020140)
