【作者】 李瑞雪； 王钰婷； 夏家凤； 汪泰初；

【Author】 Ruixue Li;Yuting Wang;Jiafeng Xia;Taichu Wang;The Sericultural Research Institute, An Hui Academy of Agricultural Sciences;

【机构】 安徽省农业科学院蚕桑研究所；

【摘要】 病毒诱导的基因沉默(VIGS)是近年来发现的一种转录后基因沉默(PTGS)现象,现已被开发为快速鉴定植物基因功能的一种反向遗传学新技术。建立桑树VIGS转化体系,为桑树功能基因研究提供有效简便的试验方法。文以丰驰桑(Morus alba)为材料,运用生物信息学方法和RT-PCR技术,选取同源性较高的片段设计八氢番茄红素脱氢酶(PDS)基因的特异性引物,克隆出片段长度为346bp的序列作为干扰片段,经XbaI和Bam HI双酶切后与烟草脆裂病毒(TRV2)连接构建VIGS重组载体p TRV2-MPDS,转化农杆菌(LBA4404)后,侵染桑树幼苗。利用构建的桑树PDS基因的重组载体pT RV2-MPDS侵染桑树幼苗,桑树内源PDS基因被沉默,叶片出现明显的白化表型,通过qR T-PCR定量检测显示,被侵染叶片的PDS基因显著降解,成功构建桑树VIGS转化体系。桑树VIGS转化体系的建立为桑树功能基因的研究及功能调控分子机制奠定了试验基础。更多还原

【Abstract】 Virus induced gene silencing(VIGS) is a post-transcriptional gene silencing(PTGS) phenomenon discovered in recent years and it has been developed to be a new technology of reverse genetics for rapid identification of plant gene function. The present study was conducted to establish mulberry VIGS transformation system in order to provide effective and simple identification method of mulberry functional genes. In this research, we used "Fengchi" mulberry(Morus alba) as experimental material, used bioinformatics method and RT-PCR technology to design gene-specific primers of eight hydrogen lycopene dehydrogenase(PDS) gene and cloned sequence fragment with the length of 346 bp as interference. After Xba I and BamH I double digestion, the recombinant vector p TRV2-MPDS was obtained by connection with tobacco rattle virus 2(TRV2) and transformed into "Fengchi" seedlings. The results showed that the VIGS recombinant vectors of mulberry PDS gene was successfully constructed and endogenous PDS gene in mulberry was inhibited and some leaves showed obvious photobleaching after being infected. q RT-PCR tests of the target gene further confirmed that PDS gene of infected leaves was significant degraded and the VIGS transformation system was successfully build in mulberry.In conclusion, mulberry VIGS transformation system can provide an experimental foundation for the research of mulberry functional genes and functional regulate molecular mechanism.更多还原