Stress response at the protein level to viral infection in orchid plants has not been extensively investigated to date. To understand the proteomic basis of Phalaenopsis amabilis’s responses to Cymbidium Mosaic virus...Stress response at the protein level to viral infection in orchid plants has not been extensively investigated to date. To understand the proteomic basis of Phalaenopsis amabilis’s responses to Cymbidium Mosaic virus (CymMV), and/or Odontoglossum ring spot virus (ORSV), the total proteins were extracted from Phalaenopsis amabilis leaves infected with CymMV, ORSV, or both respectively. Differentially expressed proteins were identified by two-dimensional electrophoresis, and 27 of these proteins that had significant changes were further examined by mass spectrometry. Comparing CymMV-infected leaves with mock-inoculated ones, 2 proteins were significantly up-regulated, 9 were significantly down-regulated and 1 previously undetected protein was identified. 10 proteins were significantly up-regulated, 3 significantly down-regulated and 1 previously undetected protein was identified in ORSV-infected leaves. 6 proteins were significantly up-regulated and 9 significantly down-regulated proteins were found in co-infected leaves. These identified proteins are involved in disease resistance, stress response, transcriptional regulation, energy metabolism, protein modification and the previously unknown proteins were not involved with known protein pathways. Proteins significantly up-regulated were ATP sulfurylase, down-regulated proteins included glutamate decarboxylase isozyme 2, RNA polymerase alpha subunit and chloroplastic peptide deformylase 1A were proteins with similar alteration trend after all infection treatments. Significantly up-regulated were Thioredoxin H-type and down-regulated Cytosolic phosphoglycerate kinase I which were proteins that have been shown to be specifically regulated by the infection with CymMV. Significantly up-regulated were proteins like Rubisco large subunit, Triosephosphate isomerase, NADP-specific isocitrate dehydrogenase and Cinnamoyl CoA reductase CCR2 by the infection of ORSV. Protein regulation in coinfected leaves followed a pattern similar to that of any of the single virus infection results.展开更多
[Objectives]The paper was to explore the genetic information and evolution of CymMV,and to provide an important scientific basis for monitoring and early warning of orchid virus disease and anti-virus genetic engineer...[Objectives]The paper was to explore the genetic information and evolution of CymMV,and to provide an important scientific basis for monitoring and early warning of orchid virus disease and anti-virus genetic engineering of orchid in Guangdong Province.[Methods]RT-PCR and DASELISA were used to detect and identify CymMV from leaves with suspected virus disease of Cymbidium sinense collected from Guangzhou area.The genome sequence assembly,annotation,phylogeny and selection pressure analysis of CymMV isolates were performed with related molecular biology software.[Results]Two CymMV isolates(GZV013 and ZC29)were found in Guangdong Province for the first time in this study.The genome of both GZV013 and ZC29 were 6227 nt in length,encoding 5 functional proteins.The similarity analysis of the full sequence showed that the nucleotide sequence identity of GZV013 and Taiwan isolate M2 was 97.03% and that of ZC29 and Nanjing isolate NJ-1 was 97.11%.The complete genome sequence identity among CymMV isolates ranged from 86.85% to 98.31%,and the differentiation of diverse populations was closely related to host species and geographical isolation.Each region of CymMV genome was affected by negative selection and conformed to the neutral evolution model.The genes encoding RdRp,TGB1 and TGB2 had the highest mutation rates in the genome.[Conclusions]GZV013 was most closely related to Taiwan isolate M2,and ZC29 was most closely related to Nanjing isolate NJ-1,belonging to the same branch of a family.展开更多
The technique of SYBR Green-based quantitative real-time reverse transcription polymerase chain reaction(real-time RT-PCR)was applied to quantitative detect a 764 bp nucleotide sequence containing total coat protein(c...The technique of SYBR Green-based quantitative real-time reverse transcription polymerase chain reaction(real-time RT-PCR)was applied to quantitative detect a 764 bp nucleotide sequence containing total coat protein(cp)gene of Cymbidium mosaic virus(CyMV).The plasmid containing the target sequence was constructed to prepare the standard curve and detect the sensitivity.The standard curve was drawn based on the linear relationship between the logarithm(base 10)of the quantity of target sequence and cycle threshold[C(T)].While the concentration of plasmid DNA falling within the range of 2.6×10^(7)to 2.6×10^(2)copies per tube established a regression equation,y=-0.3583x+10.32,and related coefficient:r^(2)=0.995.The real-time RT-PCR assay for CyMV had a minimum detectable quantity of two copies per tube.The naturally infected samples of Phalaenopsis sp.and the artificially inoculated samples of Arachnis sp.with trace CyMV were quantitatively detected using this method.CyMVin the positive samples of Phalaenopsis sp.and Arachnis sp.was confirmed by DNA sequencing and cp gene homeology blast.The results showed that CyMV extracted from the leaves of orchid in Hangzhou,Zhejiang Province,China,could be derived from Kunming city(KM),Yunnan Province,China.This method characterized by high sensitivity,specificity,and precision is suitable for early diagnosis and quantitative detection of CyMV.展开更多
文摘Stress response at the protein level to viral infection in orchid plants has not been extensively investigated to date. To understand the proteomic basis of Phalaenopsis amabilis’s responses to Cymbidium Mosaic virus (CymMV), and/or Odontoglossum ring spot virus (ORSV), the total proteins were extracted from Phalaenopsis amabilis leaves infected with CymMV, ORSV, or both respectively. Differentially expressed proteins were identified by two-dimensional electrophoresis, and 27 of these proteins that had significant changes were further examined by mass spectrometry. Comparing CymMV-infected leaves with mock-inoculated ones, 2 proteins were significantly up-regulated, 9 were significantly down-regulated and 1 previously undetected protein was identified. 10 proteins were significantly up-regulated, 3 significantly down-regulated and 1 previously undetected protein was identified in ORSV-infected leaves. 6 proteins were significantly up-regulated and 9 significantly down-regulated proteins were found in co-infected leaves. These identified proteins are involved in disease resistance, stress response, transcriptional regulation, energy metabolism, protein modification and the previously unknown proteins were not involved with known protein pathways. Proteins significantly up-regulated were ATP sulfurylase, down-regulated proteins included glutamate decarboxylase isozyme 2, RNA polymerase alpha subunit and chloroplastic peptide deformylase 1A were proteins with similar alteration trend after all infection treatments. Significantly up-regulated were Thioredoxin H-type and down-regulated Cytosolic phosphoglycerate kinase I which were proteins that have been shown to be specifically regulated by the infection with CymMV. Significantly up-regulated were proteins like Rubisco large subunit, Triosephosphate isomerase, NADP-specific isocitrate dehydrogenase and Cinnamoyl CoA reductase CCR2 by the infection of ORSV. Protein regulation in coinfected leaves followed a pattern similar to that of any of the single virus infection results.
基金Supported by Science and Technology Project of Guangdong Province(2019B030316033,2021KJ121,C2024900075,C2024900210)Science and Technology Project of Guangzhou City(202102020809)。
文摘[Objectives]The paper was to explore the genetic information and evolution of CymMV,and to provide an important scientific basis for monitoring and early warning of orchid virus disease and anti-virus genetic engineering of orchid in Guangdong Province.[Methods]RT-PCR and DASELISA were used to detect and identify CymMV from leaves with suspected virus disease of Cymbidium sinense collected from Guangzhou area.The genome sequence assembly,annotation,phylogeny and selection pressure analysis of CymMV isolates were performed with related molecular biology software.[Results]Two CymMV isolates(GZV013 and ZC29)were found in Guangdong Province for the first time in this study.The genome of both GZV013 and ZC29 were 6227 nt in length,encoding 5 functional proteins.The similarity analysis of the full sequence showed that the nucleotide sequence identity of GZV013 and Taiwan isolate M2 was 97.03% and that of ZC29 and Nanjing isolate NJ-1 was 97.11%.The complete genome sequence identity among CymMV isolates ranged from 86.85% to 98.31%,and the differentiation of diverse populations was closely related to host species and geographical isolation.Each region of CymMV genome was affected by negative selection and conformed to the neutral evolution model.The genes encoding RdRp,TGB1 and TGB2 had the highest mutation rates in the genome.[Conclusions]GZV013 was most closely related to Taiwan isolate M2,and ZC29 was most closely related to Nanjing isolate NJ-1,belonging to the same branch of a family.
文摘The technique of SYBR Green-based quantitative real-time reverse transcription polymerase chain reaction(real-time RT-PCR)was applied to quantitative detect a 764 bp nucleotide sequence containing total coat protein(cp)gene of Cymbidium mosaic virus(CyMV).The plasmid containing the target sequence was constructed to prepare the standard curve and detect the sensitivity.The standard curve was drawn based on the linear relationship between the logarithm(base 10)of the quantity of target sequence and cycle threshold[C(T)].While the concentration of plasmid DNA falling within the range of 2.6×10^(7)to 2.6×10^(2)copies per tube established a regression equation,y=-0.3583x+10.32,and related coefficient:r^(2)=0.995.The real-time RT-PCR assay for CyMV had a minimum detectable quantity of two copies per tube.The naturally infected samples of Phalaenopsis sp.and the artificially inoculated samples of Arachnis sp.with trace CyMV were quantitatively detected using this method.CyMVin the positive samples of Phalaenopsis sp.and Arachnis sp.was confirmed by DNA sequencing and cp gene homeology blast.The results showed that CyMV extracted from the leaves of orchid in Hangzhou,Zhejiang Province,China,could be derived from Kunming city(KM),Yunnan Province,China.This method characterized by high sensitivity,specificity,and precision is suitable for early diagnosis and quantitative detection of CyMV.
