Apple stem pitting virus(ASPV)is an important causal agent of pear diseases.Nowadays,the infection status and molecular characteristics of the virus in old pear trees have never been investigated.In this study,we prov...Apple stem pitting virus(ASPV)is an important causal agent of pear diseases.Nowadays,the infection status and molecular characteristics of the virus in old pear trees have never been investigated.In this study,we provide the first complete genome sequence of an ASPV isolate LYC from an over 300-year-old tree of a local Pyrus bretschneideri cultivar‘Chili’specifically grown at Laiyang area in China.ASPV-LYC possesses a chimeric genome consisting of 9273 nucleotides excluding a poly(A)tail at its 3′end and harboring a recombination region in its open reading frame(ORF1)with Aurora-1 and KL9 identified as the major and minor parents.Western blot analysis with antisera against recombinant coat proteins(CPs)of three ASPV isolates from pear indicates that ASPV-LYC is serologically related to these ASPV isolates,but with differential activities.Further biological tests on indicator plants of Pyronia veitchii show that ASPV-LYC can induce serious leaf and stem symptoms as other ASPV isolates.The results provide an important information for understanding molecular evolution of ASPV and suggest a need to prevent dissemination of the isolate among pear trees.展开更多
To understand the distribution of Apple stem pitting virus （ASPV） in tissues of pear tree and provide directly theorical and technical support for shoot tips detoxication, leaves and shoot tips of Korla pear were us...To understand the distribution of Apple stem pitting virus （ASPV） in tissues of pear tree and provide directly theorical and technical support for shoot tips detoxication, leaves and shoot tips of Korla pear were used as the materials, cDNA probe for ASPV was synthesized through RT-PCR reaction system using the unradioactive digoxigenin-labeled probe, and the specificity and sensitivity of probe were verified by blot hybridization method. Paraffin slice for in situ PCR and in situ hybridization was made and the location and distribution of ASPV RNA were detected in paraffin slices using in situ reverse transcription polymerase chain reaction, and the important factors which influenced the experiment results were optimized. ASPV mainly distributed in palisade tissue of mesophyll cells, external cortex of the tip, and the corresponding newborn vascular bundles. 20 min was the suitable digestive time for proteinase K. For the better amplication, RT reaction system should be above 0.2 U μL^-1 and 0.4 mmol L^-1 for RNasin and dNTPs respectively, 0.1-1.3 U μ L^-1 SuperScript Ⅱ, 0.6- 0.8 μmol L^-1 primer concentration, and above 0.5 U 100 μL^-1 LA Taq DNA polymerase. The suitable annealing temperature in PCR reaction was 60℃ with 35 cycles. The apical meristem of 0.25 mm was the region of virus-free.展开更多
Using Kuala pear leaves and cortexes as materials, the total RNA was extracted using two methods and these two methods were compared. The most suited methods for Kuala pear were screened; and biotin-labeled cDNA probe...Using Kuala pear leaves and cortexes as materials, the total RNA was extracted using two methods and these two methods were compared. The most suited methods for Kuala pear were screened; and biotin-labeled cDNA probe was synthesized using RT-PCR. The main factors that affected the sensitivity of hybridization were studied. Studies indicated that the highest sensitivity was obtained under the following conditions： probe concentration 400 ng mL^-1, formamide concentration 45%, temperature 42℃, hybridization time 6 hours. The best hybridization results were obtained when the nitrocellulose membrane purchased from Gelman was used. Better blocking of hybridization was obtained using Tween 20 compared with albumin in bovine. The detection of the total RNA using different tissues and different extraction methods was compared. This study indicates that the total RNA of fresh leaf, old leaf, cortexes, and frozen leaf showed signs of hybridization using the two extraction methods.展开更多
Partial sequence(314 bp) of ASPV was cloned and used as a probe labelled with digoxigenin-11dUTP.The total RNA extracted from samples with Apple stem pitting virus and a series of dilutions of plasmid with ASPV-cDNA w...Partial sequence(314 bp) of ASPV was cloned and used as a probe labelled with digoxigenin-11dUTP.The total RNA extracted from samples with Apple stem pitting virus and a series of dilutions of plasmid with ASPV-cDNA were detected by dot blot hybridization.The results showed that the probe was sensitive and specific.The probe couldn’t hybridize with total RNA of Apple stem grooving virus,Apple mosaic virus and Apple chlorotic leaf spot virus samples as well as negative control,only hybridized with that extracted from dormant shoot infected with ASPV.The sensitivity for detection of plasmid contained ASPV-cDNA was 1.64 μg.展开更多
基金financially supported by the program for the Key International S&T Cooperation Projects (2017YFE0110900)the National Key Research and Developemnt Program of China (2018YFD0201400)+1 种基金the Fundamental Research Funds for the Central Universities, China (2662016PY107)the earmarked fund for the China Agriculture Research System (CARS-28-15)
文摘Apple stem pitting virus(ASPV)is an important causal agent of pear diseases.Nowadays,the infection status and molecular characteristics of the virus in old pear trees have never been investigated.In this study,we provide the first complete genome sequence of an ASPV isolate LYC from an over 300-year-old tree of a local Pyrus bretschneideri cultivar‘Chili’specifically grown at Laiyang area in China.ASPV-LYC possesses a chimeric genome consisting of 9273 nucleotides excluding a poly(A)tail at its 3′end and harboring a recombination region in its open reading frame(ORF1)with Aurora-1 and KL9 identified as the major and minor parents.Western blot analysis with antisera against recombinant coat proteins(CPs)of three ASPV isolates from pear indicates that ASPV-LYC is serologically related to these ASPV isolates,but with differential activities.Further biological tests on indicator plants of Pyronia veitchii show that ASPV-LYC can induce serious leaf and stem symptoms as other ASPV isolates.The results provide an important information for understanding molecular evolution of ASPV and suggest a need to prevent dissemination of the isolate among pear trees.
基金supported by the National Natural Science Foundation of China (30360066)the National Key Technologies R&D Program of China (2003BA546C)the Foundation Science and Technology Commission Xinjiang Production and Construction Corps,China(NKB02SDXNK01SW)
文摘To understand the distribution of Apple stem pitting virus （ASPV） in tissues of pear tree and provide directly theorical and technical support for shoot tips detoxication, leaves and shoot tips of Korla pear were used as the materials, cDNA probe for ASPV was synthesized through RT-PCR reaction system using the unradioactive digoxigenin-labeled probe, and the specificity and sensitivity of probe were verified by blot hybridization method. Paraffin slice for in situ PCR and in situ hybridization was made and the location and distribution of ASPV RNA were detected in paraffin slices using in situ reverse transcription polymerase chain reaction, and the important factors which influenced the experiment results were optimized. ASPV mainly distributed in palisade tissue of mesophyll cells, external cortex of the tip, and the corresponding newborn vascular bundles. 20 min was the suitable digestive time for proteinase K. For the better amplication, RT reaction system should be above 0.2 U μL^-1 and 0.4 mmol L^-1 for RNasin and dNTPs respectively, 0.1-1.3 U μ L^-1 SuperScript Ⅱ, 0.6- 0.8 μmol L^-1 primer concentration, and above 0.5 U 100 μL^-1 LA Taq DNA polymerase. The suitable annealing temperature in PCR reaction was 60℃ with 35 cycles. The apical meristem of 0.25 mm was the region of virus-free.
基金supported by the National Natural Science Foundation of China(30060053).
文摘Using Kuala pear leaves and cortexes as materials, the total RNA was extracted using two methods and these two methods were compared. The most suited methods for Kuala pear were screened; and biotin-labeled cDNA probe was synthesized using RT-PCR. The main factors that affected the sensitivity of hybridization were studied. Studies indicated that the highest sensitivity was obtained under the following conditions： probe concentration 400 ng mL^-1, formamide concentration 45%, temperature 42℃, hybridization time 6 hours. The best hybridization results were obtained when the nitrocellulose membrane purchased from Gelman was used. Better blocking of hybridization was obtained using Tween 20 compared with albumin in bovine. The detection of the total RNA using different tissues and different extraction methods was compared. This study indicates that the total RNA of fresh leaf, old leaf, cortexes, and frozen leaf showed signs of hybridization using the two extraction methods.
文摘Partial sequence(314 bp) of ASPV was cloned and used as a probe labelled with digoxigenin-11dUTP.The total RNA extracted from samples with Apple stem pitting virus and a series of dilutions of plasmid with ASPV-cDNA were detected by dot blot hybridization.The results showed that the probe was sensitive and specific.The probe couldn’t hybridize with total RNA of Apple stem grooving virus,Apple mosaic virus and Apple chlorotic leaf spot virus samples as well as negative control,only hybridized with that extracted from dormant shoot infected with ASPV.The sensitivity for detection of plasmid contained ASPV-cDNA was 1.64 μg.
