[ Objective] This study aimed to screen and identify tobacco mutants resistant to potato virus Y （PVY）, thus laying the foundation for obtaining PVY resistance genes. [ Method ] At seedling stage, tobacco mutant mat...[ Objective] This study aimed to screen and identify tobacco mutants resistant to potato virus Y （PVY）, thus laying the foundation for obtaining PVY resistance genes. [ Method ] At seedling stage, tobacco mutant materials were inoculated with PVY virus and preliminarily screened by naked-eye observation. Enzyme-linked immunosorbent assay （ELISA）, real-time fluorescence quantitative PCR and test strip assay were performed to further identify the pre-screened PVY- resistant seedlings. [ Result ] In 2011, two highly PVY-resistant tobacco mutants （MZE2-15 and MZE2-16） and three PVY-tolerant mutants （MZE2-70, MZE2- 207 and MZE2-228） were obtained, which were further screened and identified in 2012. According to the results, tobacco mutant materials MZE2-407 and MZE2- 428 were susceptible to PVY; mutant materials MZE2-16 and MZE2-15 were resistant to PVY. [ Conclusion] This study provide theoretical basis for the control of tobacco PVY disease.展开更多
Potato virus Y(PVY)is a non-persistent virus that is transmitted by many aphid species and causes significant damage to potato production.We constructed a spatially-explicit model simulating PVY spread in a potato fie...Potato virus Y(PVY)is a non-persistent virus that is transmitted by many aphid species and causes significant damage to potato production.We constructed a spatially-explicit model simulating PVY spread in a potato field and used it to investigate possible effects of transmission efficiency,initial inoculum levels,vector behavior,vector abundance,and timing of peak vector activity on PVY incidence at the end of a simulated growing season.Lower PVY incidence in planted seed resulted in lower virus infection at the end of the season.However,when populations of efficient PVY vectors were high,significant PVY spread occurred even when initial virus inoculum was low.Non-colonizing aphids were more important for PVY spread compared to colonizing aphids,particularly at high densities.An early-season peak in the numbers of noncolonizing aphids resulted in the highest number of infected plants in the end of the season,while mid-and late-season peaks caused relatively little virus spread.Our results highlight the importance of integrating different techniques to prevent the number of PVY-infected plants from exceeding economically acceptable levels instead of trying to control aphids within potato fields.Such management plans should be implemented very early in a growing season.展开更多
The viruses titer and the ultrastructure of infected cells in tobacco host (Nicotiana tabacum cv. Samsun), which doubly infected with potato virus Y necrosis strain (PVYN) and potato virus X (PVX), were studied under ...The viruses titer and the ultrastructure of infected cells in tobacco host (Nicotiana tabacum cv. Samsun), which doubly infected with potato virus Y necrosis strain (PVYN) and potato virus X (PVX), were studied under greenhouse conditions. The results indicated that PVYN and PVX interacted synergistically. and tobacco plants which doubly infected with PVX and PVYN could greatly increase symptom severity as compared with that induced by the single virus. As determined by triple antibody sandwich enzyme-linked immunosor-bent assay (TAS-ELISA), the titer of PVX in the tobacco leaves infected with both PVYN and PVX was up to 9.10 times higher than the plants infected with PVX only. No significant differences in PVYN titer were detected between singly and doubly infected plants. The enhancement of PVX titer in doubly infected plants was evident in greenhouse and was independent of the virus strains, tested seasons, intervals between PVYN and PVX inoculation. When sections of doubly infected leaves were examined with an electron microscope, it could be frequently found that cells contained pinwheels and large masses of PVX-like particles, pinwheels and laminate inclusions, or all three structures, most of them were swollen, and their chloroplast and other organella were destructed heavily. This suggested that doubly infected cells were involved in the enhancement phenomenon, which seemed to be the result of an increase in the amount of PVX synthesized in them.展开更多
RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust an...RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion.In our study,a 480bp fragment of the capsid protein gene of potato virus Y(CP-PVY) was used as a target to downregulate PVY mRNA expression in-vitro,as the CP gene interferes with viral uncoating,translation and replication.A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells.CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs.Six biological replicates were performed in this study.In our findings,one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.展开更多
A cDNA library was constructed in λgt11 vectors, complementary to the mRNA isolated from a mouse hybridoma raised against potato virus Y(PVY). Thirty cDNA clones were selected from the cDNA library by in situ immunoh...A cDNA library was constructed in λgt11 vectors, complementary to the mRNA isolated from a mouse hybridoma raised against potato virus Y(PVY). Thirty cDNA clones were selected from the cDNA library by in situ immunohybridization with goat anti-mouse kappa-chain-specific antibody conjugated to alkaline phosphatase, from which one clone, k6, having the largest insert was characterized by sequence analysis. The result shows that the immunoglobulin messenger RNA corresponding to k6 is 956 nucleotides in length excluding the poly(A) region, among which 31 bases code for the 5’ non-coding region, 57 for the leader sequence of the protein, 657 for the mature protein and 211 for the 3’ non-coding region. Comparison of deduced amino acid sequences of the protein and other kappa light chains shows that they share a 100% identity in their constant regions(CL) and 93.7% identity in their variable regions(VL). The kappa light chain encoded by k6 is considered to be specific to PVY since only one type of light chain is expressed in the hybridoma.展开更多
[Objective]The aim was to establish the multiplex PCR method for three virus of potato：PVA（potato virus A）,TMV（Tobacco mosaic virus）and PVY（potato virus Y）.[Method]According to the PVA,TMV and PVY sequences ava...[Objective]The aim was to establish the multiplex PCR method for three virus of potato：PVA（potato virus A）,TMV（Tobacco mosaic virus）and PVY（potato virus Y）.[Method]According to the PVA,TMV and PVY sequences available in GenBank,pairs of primer were designed for establishing a multiplex PCR method,and constructing recombinant plasmid of target genes by PCR amplified of three viruses as reference standard simple to be used in sensitivity test;PVX（Potato virus X）,PVM（Potato virus M）,PVS（Potato virus S）,PVV（Potato virus V）and CMV（Cucumber mosaic virus）were used to carry out the specificity test and detection of 11 samples which were suspected of virus infected.[Result]The detection limit for PVA,TMV and PVY was 14,14 and 14 copies/ml,respectively.No cross-reactivity was observed with other viruses.Seven of 11 samples were infected by three viruses.[Conclusion]The multiplex PCR for PVA,TMV,PVY three viruses of potato was established successfully,which had provided basis for the detection technology of potato virus.展开更多
马铃薯Y病毒(Potato virus Y,PVY)是危害烟草的重要病害,种植抗病品种是经济有效的防治措施。分子标记辅助选择可提高抗病育种效率。来源于X-射线诱变的Virgin A Mutante(VAM)的隐性抗PVY基因位点(va)被广泛应用于烟草抗病育种。为了提...马铃薯Y病毒(Potato virus Y,PVY)是危害烟草的重要病害,种植抗病品种是经济有效的防治措施。分子标记辅助选择可提高抗病育种效率。来源于X-射线诱变的Virgin A Mutante(VAM)的隐性抗PVY基因位点(va)被广泛应用于烟草抗病育种。为了提高va位点的育种利用效率,根据烟草隐性抗PVY基因(感病基因)e IF4E-1基因序列,设计特异扩增的引物CF2GR11,开发e IF4E-1基因的分子标记,并检测了该标记与抗性的遗传距离和在常见烟草资源中的适用性。CF2GR11在云烟87、红花大金元和K326等感PVY品种可扩增出500 bp产物,在NC102、NC55和K326PVY等抗病品种无扩增条带。以抗、感PVY亲本构建的101个F2单株为定位群体,遗传连锁分析表明,CF2GR11标记与烟草PVY感病基因的遗传距离为0.99 c M。对46份PVY抗性明确的栽培烟草资源的检测表明,供试资源的标记检测结果与抗性的吻合度为100%,表明CF2GR11标记适用性高。该目的基因标记可用于抗PVY育种的辅助选择和抗PVY资源的鉴定。展开更多
基金Supported by Major Mutant Program of National Tobacco Genome Project of China[110201201004(JY-04)]
文摘[ Objective] This study aimed to screen and identify tobacco mutants resistant to potato virus Y （PVY）, thus laying the foundation for obtaining PVY resistance genes. [ Method ] At seedling stage, tobacco mutant materials were inoculated with PVY virus and preliminarily screened by naked-eye observation. Enzyme-linked immunosorbent assay （ELISA）, real-time fluorescence quantitative PCR and test strip assay were performed to further identify the pre-screened PVY- resistant seedlings. [ Result ] In 2011, two highly PVY-resistant tobacco mutants （MZE2-15 and MZE2-16） and three PVY-tolerant mutants （MZE2-70, MZE2- 207 and MZE2-228） were obtained, which were further screened and identified in 2012. According to the results, tobacco mutant materials MZE2-407 and MZE2- 428 were susceptible to PVY; mutant materials MZE2-16 and MZE2-15 were resistant to PVY. [ Conclusion] This study provide theoretical basis for the control of tobacco PVY disease.
基金supported in part by the United States Department of Agriculture National institute of Food and Agriculture Special Crops Research initiative (Award # 2014-51181-22373)Funding for Hongchun Qu’s stay at the University of Maine was received from the National Natural Science Foundation of China (Award # 61871061)
文摘Potato virus Y(PVY)is a non-persistent virus that is transmitted by many aphid species and causes significant damage to potato production.We constructed a spatially-explicit model simulating PVY spread in a potato field and used it to investigate possible effects of transmission efficiency,initial inoculum levels,vector behavior,vector abundance,and timing of peak vector activity on PVY incidence at the end of a simulated growing season.Lower PVY incidence in planted seed resulted in lower virus infection at the end of the season.However,when populations of efficient PVY vectors were high,significant PVY spread occurred even when initial virus inoculum was low.Non-colonizing aphids were more important for PVY spread compared to colonizing aphids,particularly at high densities.An early-season peak in the numbers of noncolonizing aphids resulted in the highest number of infected plants in the end of the season,while mid-and late-season peaks caused relatively little virus spread.Our results highlight the importance of integrating different techniques to prevent the number of PVY-infected plants from exceeding economically acceptable levels instead of trying to control aphids within potato fields.Such management plans should be implemented very early in a growing season.
基金This project was supported by the National Natu ral Science Foundation of China(30100117)China Postdoctoral Science Foundation(2002031168)the Key Task of Shandong Provincial Scientific Foundation and Technological Department(981020171),China.
文摘The viruses titer and the ultrastructure of infected cells in tobacco host (Nicotiana tabacum cv. Samsun), which doubly infected with potato virus Y necrosis strain (PVYN) and potato virus X (PVX), were studied under greenhouse conditions. The results indicated that PVYN and PVX interacted synergistically. and tobacco plants which doubly infected with PVX and PVYN could greatly increase symptom severity as compared with that induced by the single virus. As determined by triple antibody sandwich enzyme-linked immunosor-bent assay (TAS-ELISA), the titer of PVX in the tobacco leaves infected with both PVYN and PVX was up to 9.10 times higher than the plants infected with PVX only. No significant differences in PVYN titer were detected between singly and doubly infected plants. The enhancement of PVX titer in doubly infected plants was evident in greenhouse and was independent of the virus strains, tested seasons, intervals between PVYN and PVX inoculation. When sections of doubly infected leaves were examined with an electron microscope, it could be frequently found that cells contained pinwheels and large masses of PVX-like particles, pinwheels and laminate inclusions, or all three structures, most of them were swollen, and their chloroplast and other organella were destructed heavily. This suggested that doubly infected cells were involved in the enhancement phenomenon, which seemed to be the result of an increase in the amount of PVX synthesized in them.
文摘RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion.In our study,a 480bp fragment of the capsid protein gene of potato virus Y(CP-PVY) was used as a target to downregulate PVY mRNA expression in-vitro,as the CP gene interferes with viral uncoating,translation and replication.A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells.CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs.Six biological replicates were performed in this study.In our findings,one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.
文摘A cDNA library was constructed in λgt11 vectors, complementary to the mRNA isolated from a mouse hybridoma raised against potato virus Y(PVY). Thirty cDNA clones were selected from the cDNA library by in situ immunohybridization with goat anti-mouse kappa-chain-specific antibody conjugated to alkaline phosphatase, from which one clone, k6, having the largest insert was characterized by sequence analysis. The result shows that the immunoglobulin messenger RNA corresponding to k6 is 956 nucleotides in length excluding the poly(A) region, among which 31 bases code for the 5’ non-coding region, 57 for the leader sequence of the protein, 657 for the mature protein and 211 for the 3’ non-coding region. Comparison of deduced amino acid sequences of the protein and other kappa light chains shows that they share a 100% identity in their constant regions(CL) and 93.7% identity in their variable regions(VL). The kappa light chain encoded by k6 is considered to be specific to PVY since only one type of light chain is expressed in the hybridoma.
文摘[Objective]The aim was to establish the multiplex PCR method for three virus of potato：PVA（potato virus A）,TMV（Tobacco mosaic virus）and PVY（potato virus Y）.[Method]According to the PVA,TMV and PVY sequences available in GenBank,pairs of primer were designed for establishing a multiplex PCR method,and constructing recombinant plasmid of target genes by PCR amplified of three viruses as reference standard simple to be used in sensitivity test;PVX（Potato virus X）,PVM（Potato virus M）,PVS（Potato virus S）,PVV（Potato virus V）and CMV（Cucumber mosaic virus）were used to carry out the specificity test and detection of 11 samples which were suspected of virus infected.[Result]The detection limit for PVA,TMV and PVY was 14,14 and 14 copies/ml,respectively.No cross-reactivity was observed with other viruses.Seven of 11 samples were infected by three viruses.[Conclusion]The multiplex PCR for PVA,TMV,PVY three viruses of potato was established successfully,which had provided basis for the detection technology of potato virus.
文摘马铃薯Y病毒(Potato virus Y,PVY)是危害烟草的重要病害,种植抗病品种是经济有效的防治措施。分子标记辅助选择可提高抗病育种效率。来源于X-射线诱变的Virgin A Mutante(VAM)的隐性抗PVY基因位点(va)被广泛应用于烟草抗病育种。为了提高va位点的育种利用效率,根据烟草隐性抗PVY基因(感病基因)e IF4E-1基因序列,设计特异扩增的引物CF2GR11,开发e IF4E-1基因的分子标记,并检测了该标记与抗性的遗传距离和在常见烟草资源中的适用性。CF2GR11在云烟87、红花大金元和K326等感PVY品种可扩增出500 bp产物,在NC102、NC55和K326PVY等抗病品种无扩增条带。以抗、感PVY亲本构建的101个F2单株为定位群体,遗传连锁分析表明,CF2GR11标记与烟草PVY感病基因的遗传距离为0.99 c M。对46份PVY抗性明确的栽培烟草资源的检测表明,供试资源的标记检测结果与抗性的吻合度为100%,表明CF2GR11标记适用性高。该目的基因标记可用于抗PVY育种的辅助选择和抗PVY资源的鉴定。
