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
Virus-induced gene silencing(VIGS)and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas)systems are effective technologies for rapid and accurate gene function verification...Virus-induced gene silencing(VIGS)and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas)systems are effective technologies for rapid and accurate gene function verification in modern plant biotechnology.However,the investigation of gene silencing and editing in radish remains limited.In this study,a bleaching phenotype was generated through the knockdown of RsPDS using tobacco rattle virus(TRV)-and turnip yellow mosaic virus(TYMV)-mediated gene silencing vectors.The TYMV-mediated gene silencing efficiency was higher than the TRV-based VIGS system in radish.The expression level of RsPDS was significantly inhibited using VIGS in'NAU-067'radish leaves.The rootless seedlings of‘NAU-067'were infected with Agrobacterium rhizogenes using the 2300GN-Ubi-RsPDS-Cas9 vector with two target sequences.Nine adventitious roots were blue with GUs staining,and four of these adventitious roots were edited at target sequence 1 of the RsPDS gene as indicated by Sanger sequencing.Furthermore,albino lines were generated with A.tumefaciens-mediated transformation of radish cotyledons.Five base substitutions and three base deletions occurred at target sequence 2 in Line 1,and three base insertions and three base substitutions occurred at target sequence 1 in Line 2.This study shows that VIGS and CRISPR/Cas9 techniques can be employed to precisely verify the biological functions of genes in radish,which will facilitate the genetic improvement of vital horticultural traits in radish breeding programs.展开更多
Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis,yet their prolonged activation induces apoptosis and disrupts organismal health1-3.How stress responses are tur...Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis,yet their prolonged activation induces apoptosis and disrupts organismal health1-3.How stress responses are turned off at the right time and place remains poorly understood.Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress.展开更多
Virus-induced gene silencing(VIGS)is a natural defense mechanism of plants,which can cause sequence-specific degradation of viral RNA and is often used for studying the functional genome.At present,the VIGS system med...Virus-induced gene silencing(VIGS)is a natural defense mechanism of plants,which can cause sequence-specific degradation of viral RNA and is often used for studying the functional genome.At present,the VIGS system mediated by Tobacco rattle virus(TRV)has been successfully established in many plant species.However,the VIGS system in Chinese narcissus had not been reported yet.In this paper,the first use of the TRV-VIGS system in Chinese narcissus is described in detail.By injecting pTRV2-GFP into narcissus leaves,we confirmed that TRV could infect narcissus and move in narcissus plants.After the inoculation of pTRV2-Nt PDS by two methods,phenomena such as photo-bleaching appeared in narcissus leaves and qRT-PCR results proved that PDS gene of narcissus was successfully silenced.Besides,pTRV2-NtMYB3 was inoculated into basal plates of narcissus to study the function of NtMYB3;the results showed that NtMYB3 is an inhibitor of flavonoids biosynthesis,which can increase the content of proanthocyanidins in basal plates of narcissus by repressing the expression level of NtFLS.These results indicate that the TRV-VIGS system has been successfully established in Chinese narcissus and successfully applied to the functional study of NtMYB3.展开更多
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
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.
基金This work was supported by Jiangsu Seed Industry Revitalization Project,China[JBGS(2021)071]Fundamental Research Funds for the Central Universities,China(YDZX2023019)+3 种基金the National Natural Science Foundation of China(32172579)the earmarked fund for Jiangsu Agricultural Industry Technology System,China[JATS(2023)421]the Jiangsu Postgraduate Scientific Research Innovation Plan,China(KYCX21_0610-2021)the Project Founded by the Priority Academic Program Development of Jiangsu Higher Education Institutions,China(PAPD).
文摘Virus-induced gene silencing(VIGS)and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas)systems are effective technologies for rapid and accurate gene function verification in modern plant biotechnology.However,the investigation of gene silencing and editing in radish remains limited.In this study,a bleaching phenotype was generated through the knockdown of RsPDS using tobacco rattle virus(TRV)-and turnip yellow mosaic virus(TYMV)-mediated gene silencing vectors.The TYMV-mediated gene silencing efficiency was higher than the TRV-based VIGS system in radish.The expression level of RsPDS was significantly inhibited using VIGS in'NAU-067'radish leaves.The rootless seedlings of‘NAU-067'were infected with Agrobacterium rhizogenes using the 2300GN-Ubi-RsPDS-Cas9 vector with two target sequences.Nine adventitious roots were blue with GUs staining,and four of these adventitious roots were edited at target sequence 1 of the RsPDS gene as indicated by Sanger sequencing.Furthermore,albino lines were generated with A.tumefaciens-mediated transformation of radish cotyledons.Five base substitutions and three base deletions occurred at target sequence 2 in Line 1,and three base insertions and three base substitutions occurred at target sequence 1 in Line 2.This study shows that VIGS and CRISPR/Cas9 techniques can be employed to precisely verify the biological functions of genes in radish,which will facilitate the genetic improvement of vital horticultural traits in radish breeding programs.
文摘Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis,yet their prolonged activation induces apoptosis and disrupts organismal health1-3.How stress responses are turned off at the right time and place remains poorly understood.Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress.
基金the Science and technology innovation fund of Fujian Agriculture and Forestry University(Grant Nos.KFA17352A,KFA17602A)。
文摘Virus-induced gene silencing(VIGS)is a natural defense mechanism of plants,which can cause sequence-specific degradation of viral RNA and is often used for studying the functional genome.At present,the VIGS system mediated by Tobacco rattle virus(TRV)has been successfully established in many plant species.However,the VIGS system in Chinese narcissus had not been reported yet.In this paper,the first use of the TRV-VIGS system in Chinese narcissus is described in detail.By injecting pTRV2-GFP into narcissus leaves,we confirmed that TRV could infect narcissus and move in narcissus plants.After the inoculation of pTRV2-Nt PDS by two methods,phenomena such as photo-bleaching appeared in narcissus leaves and qRT-PCR results proved that PDS gene of narcissus was successfully silenced.Besides,pTRV2-NtMYB3 was inoculated into basal plates of narcissus to study the function of NtMYB3;the results showed that NtMYB3 is an inhibitor of flavonoids biosynthesis,which can increase the content of proanthocyanidins in basal plates of narcissus by repressing the expression level of NtFLS.These results indicate that the TRV-VIGS system has been successfully established in Chinese narcissus and successfully applied to the functional study of NtMYB3.
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.
