Virus-induced gene silencing (VIGS) technique, which is developed in recent years, is a rapid identification of plant gene function from reverse genetics. It is a manifestation of post-transcriptional gene silencing m...Virus-induced gene silencing (VIGS) technique, which is developed in recent years, is a rapid identification of plant gene function from reverse genetics. It is a manifestation of post-transcriptional gene silencing mechanism. Compared with the traditional transgenic technology, VIGS is a transient expression system, which can achieve good results in a short time. At present, it is widely used to study the function of plant genes, but most of them are model plants, and the experiments are carried out always in the indoor environment with controlled light and temperature conditions. In this study, we creatively provided a method to establish VIGS system using perennial Rosa plants as experimental materials under field conditions. The recombinant virus vector was constructed with RrGT1 gene as reporter gene and modified TRV-GFP virus as vector, and the perennial R. rugosa “Zizhi” and R. davurica were used as experimental verification materials. According to the growth conditions of Rosa plants, the natural environment in the field and the optimal conditions for the occurrence of VIGS, the technical problems such as the confirmation of the inoculation period, the preparation of the infective fluid, the inoculation technology of the virus vector and the light and temperature conditions of plant materials cultured after inoculation were solved one by one. When the RrGT1 gene was silenced, the Rosa plants showed a pale petal color phenotype. By detection, it was found that the expression of endogenous RrGT1 gene was significantly down-regulated, and the content of all anthocyanins also decreased significantly. Therefore, we believed that the attempt to establish VIGS system in perennial Rosa plants under field conditions was very successful.展开更多
VIGS(Virus-induced gene silencing),a method for posttranscriptional gene silencing,is an effective technique for investigating the activities of genes in plants.Since there is no report for available VIGS system in St...VIGS(Virus-induced gene silencing),a method for posttranscriptional gene silencing,is an effective technique for investigating the activities of genes in plants.Since there is no report for available VIGS system in Styrax japonicus,the application of a VIGS approach that results in a gene knockdown to study gene function is limited.In this study,we compared the characteristics that could affect the viability of VIGS in S.japonicus,including the acetosyringone(AS)concentration,the Agrobacterium’s optical density and the inoculation method.The stable reference genes of S.japonicus were selected to validate the gene’s knockdown by quantitative PCR.As a result,we successfully constructed 2 VIGS systems based on TRV virus:vacuum with AS concentration of 200μmol·L^(-1)and OD600of 0.5,and friction-osmosis with AS concentration of 200μmol·L^(-1)and OD600of 1.0,which silencing efficiency was 83.33%and 74.19%,respectively.The successfully applied VIGS method provides a rapid and effective reverse gene functional analysis approach in S.japonicus to identify unknown gene functions.展开更多
文摘Virus-induced gene silencing (VIGS) technique, which is developed in recent years, is a rapid identification of plant gene function from reverse genetics. It is a manifestation of post-transcriptional gene silencing mechanism. Compared with the traditional transgenic technology, VIGS is a transient expression system, which can achieve good results in a short time. At present, it is widely used to study the function of plant genes, but most of them are model plants, and the experiments are carried out always in the indoor environment with controlled light and temperature conditions. In this study, we creatively provided a method to establish VIGS system using perennial Rosa plants as experimental materials under field conditions. The recombinant virus vector was constructed with RrGT1 gene as reporter gene and modified TRV-GFP virus as vector, and the perennial R. rugosa “Zizhi” and R. davurica were used as experimental verification materials. According to the growth conditions of Rosa plants, the natural environment in the field and the optimal conditions for the occurrence of VIGS, the technical problems such as the confirmation of the inoculation period, the preparation of the infective fluid, the inoculation technology of the virus vector and the light and temperature conditions of plant materials cultured after inoculation were solved one by one. When the RrGT1 gene was silenced, the Rosa plants showed a pale petal color phenotype. By detection, it was found that the expression of endogenous RrGT1 gene was significantly down-regulated, and the content of all anthocyanins also decreased significantly. Therefore, we believed that the attempt to establish VIGS system in perennial Rosa plants under field conditions was very successful.
基金supported by the Subject of Key R&D Plan of Shandong Province(Major Scientific and Technological Innovation Project)“Mining and Accurate Identification of Forest Tree Germplasm Resources”(Grant Nos.2021LZGC02303)Science&Technology Specific Projects in Agricultural High-tech Industrial Demonstration Area of the Yellow River Delta(Grant No.022SZX16)。
文摘VIGS(Virus-induced gene silencing),a method for posttranscriptional gene silencing,is an effective technique for investigating the activities of genes in plants.Since there is no report for available VIGS system in Styrax japonicus,the application of a VIGS approach that results in a gene knockdown to study gene function is limited.In this study,we compared the characteristics that could affect the viability of VIGS in S.japonicus,including the acetosyringone(AS)concentration,the Agrobacterium’s optical density and the inoculation method.The stable reference genes of S.japonicus were selected to validate the gene’s knockdown by quantitative PCR.As a result,we successfully constructed 2 VIGS systems based on TRV virus:vacuum with AS concentration of 200μmol·L^(-1)and OD600of 0.5,and friction-osmosis with AS concentration of 200μmol·L^(-1)and OD600of 1.0,which silencing efficiency was 83.33%and 74.19%,respectively.The successfully applied VIGS method provides a rapid and effective reverse gene functional analysis approach in S.japonicus to identify unknown gene functions.
