Maize chlorotic dwarf virus （MCDV） is a quarantine pest as approved by Chinese government. A rapid, sensitive and specific MCDV detection method using reverse transcription-loop-mediated isothermal amplification （R...Maize chlorotic dwarf virus （MCDV） is a quarantine pest as approved by Chinese government. A rapid, sensitive and specific MCDV detection method using reverse transcription-loop-mediated isothermal amplification （RT-LAMP） was estab- lished in this study. Based on the sequence of MCDV coat protein coding gene, specific primers were designed and similar sensitivities were observed between RT- LAMP and RT-PCR, except that RT-LAMP was quicker, and the reaction could be finished within 1 h. In addition, the presence or absence of the fluorescent display in daylight allows naked easy detection of the amplification of MCDV genomic RNA using calcein. The RT-LAMP assay was applied successfully to detect MCDV in maize seeds, and the result by the addition of calcein was consistent with the result detected by the real time turbidimeter.展开更多
Maize chlorotic mottle virus (MCMV) infects maize plants and causes significant losses in corn production worldwide. In this study, purified MCMV particles were used as the immunogen to produce monoclonal antibodies (...Maize chlorotic mottle virus (MCMV) infects maize plants and causes significant losses in corn production worldwide. In this study, purified MCMV particles were used as the immunogen to produce monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs). Four murine MAbs (4B8, 8C11, 6F4, and 9G1) against MCMV were obtained through the hybridoma technology. The triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-immunobinding assay (DIBA), and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) using the MAb 4B8 were then developed for sensitive, specific, and rapid detection of MCMV in fields. MCMV could be detected in infected leaf crude extracts at dilutions of 1:327 680, 1:64000, and 1:3276800 (w/v, g/ml) by TAS-ELISA, DIBA, and IC-RT-PCR, respectively. One hundred and sixty-one maize field samples showing virus-like symptoms and sixty-nine symptomless maize field samples from ten different provinces of China were collected and screened for the presence of MCMV using the established serological methods. A phylogenetic tree was constructed based on the full length CP genes and Chinese MCMV isolates formed one branch with Thailand isolates. The detection results demonstrated that MCMV is one of most prevalent viruses infecting maize in the Yunnan and Sichuan provinces of China.展开更多
In 2009, an emerging citrus viral disease caused by Citrus chlorotic dwarf-associated virus(CCDaV) was discovered in Yunnan Province of China. However, the occurrence and spread of CCDaV in other citrus-growing provin...In 2009, an emerging citrus viral disease caused by Citrus chlorotic dwarf-associated virus(CCDaV) was discovered in Yunnan Province of China. However, the occurrence and spread of CCDaV in other citrus-growing provinces in China is unknown to date. To better understand the distribution and molecular diversity of CCDaV in China, a total of 1 772 citrus samples were collected from 11 major citrus-growing provinces and were tested for CCDaV by PCR. Among these, 134 citrus samples from Guangxi, Yunnan and Guangdong were tested positive for CCDaV, demonstrating that the occurrence and spread of CCDaV are increasing in China. The complete genome sequences of 17 CCDaV isolates from different provinces and hosts were sequenced. Comparisons of the whole-genome sequences of the 17 CCDaV isolates as well as the 15 isolates available in GenBank revealed that the sequence identity was about 99–100%, showing that the CCDaV isolates were highly conserved. Phylogenetic studies showed that the 32 CCDaV isolates belonged to four different groups based on geographical origins and host species, and that CCDaV isolates from China and Turkey were clustered into different groups. The results provide important information for clarifying the distribution and genetic diversity of CCDaV in China.展开更多
Soybean chlorotic mottle virus(SbCMV)was first detected from soybean plants in Jiangxi Province of China by high throughput sequencing and was confirmed by PCR.The complete nucleotide sequence of NC113 was determined ...Soybean chlorotic mottle virus(SbCMV)was first detected from soybean plants in Jiangxi Province of China by high throughput sequencing and was confirmed by PCR.The complete nucleotide sequence of NC113 was determined to be 8210 nucleotides,and shared the highest similarity(91.7%)with sequences of SbCMV that was only reported in Japan.It encodes nine putative open reading frames(ORFs Ia,Ib and Ⅱ-Ⅷ),and contains a large intergenic region located at nucleotide 5976-6512 between ORFs VI and VII.Sequence analysis and phylogenetic tree indicated that NC113 is an isolate of SbCMV,and is more related to the soymoviruses Blueberry red ringspot virus(BRRSV),Peanut chlorotic streak virus(PCSV)and Cestrum yellow leaf curling virus(CmYLCV)than to other representative members in the Caulimoviridae family.Field survey of 472 legume plants from Jiangxi and Zhejiang provinces showed SbCMV was only detected from soybean in Nanchang City with a low incidence rate.This is the first report of Soybean chlorotic mottle virus identified in China.展开更多
Maize chlorotic mottle virus （MCMV） is a quarantine pest as approved by Chinese government： A rapid, sensitive and specific MCMV detection method using reverse transcription-loop-mediated isothermal amplification ...Maize chlorotic mottle virus （MCMV） is a quarantine pest as approved by Chinese government： A rapid, sensitive and specific MCMV detection method using reverse transcription-loop-mediated isothermal amplification （RT-LAMP） was established in this study. Based on the sequence of MCMV coat protein coding gene, 3 sets of primers were designed and specificity test showed that the second set of primers was specific to MCMV, Similar sensitivities were observed on RT-LAMP and RT-PCR, except that RT-LAMP was quicker, and the reaction could be finished within 1 h. In addition, the presence or absence of the fluorescence under daylight allows naked easy detection of the amplification of MCMV genomic RNA using calcein. The RT-LAMP assay was applied successfully to detect MCMV in maize seeds, and the result by the addition of calcein was consistent with the result detected by the real time turbidimeter. The method is rapid, specific, sensitive without the need for complicated equipment, and is suitable for rapid field detection of MCMV.展开更多
双生病毒(Geminivirus)是一类基因组为单链环状DNA的植物病毒,具有孪生的病毒粒子。国际病毒分类委员会(International Committee on Taxonomy of Viruses,ICTV)最新的分类报告将双生病毒分为9个属[1]。随着测序技术的发展,利用小RNA或...双生病毒(Geminivirus)是一类基因组为单链环状DNA的植物病毒,具有孪生的病毒粒子。国际病毒分类委员会(International Committee on Taxonomy of Viruses,ICTV)最新的分类报告将双生病毒分为9个属[1]。随着测序技术的发展,利用小RNA或转录组测序可鉴定植物中的已知和未知病毒,新的双生病毒也不断被发现和鉴定[2]。展开更多
Mature chloroplasts,as the main sites of photosynthesis,are essential for seedling growth in higher plants.Loss of function of genes involved in chloroplast development changes plant phenotype.We obtained a YELLOW COT...Mature chloroplasts,as the main sites of photosynthesis,are essential for seedling growth in higher plants.Loss of function of genes involved in chloroplast development changes plant phenotype.We obtained a YELLOW COTYLEDON (YCO) mutant in rapeseed (Brassica napus L.) using CRISPR-Cas9.Bn.YCO,a gene of unknown function,has two homologous copies (Bna A01.YCO and Bna C01.YCO) in B.napus.Homozygous mutation of these two homologs resulted in yellow cotyledons and chlorotic true leaves.Transmission electron microscopy revealed that the formation of thylakoid membranes was inhibited in yellow cotyledons.Sequence similarity search revealed that YCO was conserved in different species,and a subcellular location assay verified that Bn.YCO was located in the chloroplast.Bn.YCO was expressed in multiple tissues,most highly in cotyledons.Knockout of Bn.YCO blocked the transcription of plastid genes,especially those of photosystem genes transcribed by plastid-encoded polymerase.Transcriptome sequencing showed that the majority of genes involved in ribosome assembly and photosynthesis were down-regulated in Bn.yco mutants.These results suggested that loss of function of Bn.YCO affected plastid gene transcription,which influenced chloroplast biogenesis in rapeseed seedlings.展开更多
Maize(Zea mays),as a staple food and an important industrial raw material,has been widely cultivated for centuries especially by smallholder farmers.Maize lethal necrosis disease(MLND)is a serious disease infecting ma...Maize(Zea mays),as a staple food and an important industrial raw material,has been widely cultivated for centuries especially by smallholder farmers.Maize lethal necrosis disease(MLND)is a serious disease infecting maize,which caused devastating damage in the African region recently.MLND is induced by co-infection of maize chlorotic mottle virus and one of several cereal-infecting viruses in the Potyviridae family,with the symptoms ranging from chlorotic mottle to plant death at different infection stages.Integrated pest management for MLND needs strengthening detection,focusing on prevention and effective control.Early detection system of MLND has been successfully established by serological methods,nucleic acid-based methods,next-generation sequencing,etc.The practices,such as using certified seeds,sanitary measures,crop rotation,tolerant or resistant varieties etc.,have been considered as the effective,economical and eco-friendly way to prevent and control MLND.展开更多
Micro RNA-26a(miR-26a)has been verified to promote osteogenic differentiation of mesenchymal stem cells in recent years.The main obstacles to its application in bone regeneration are instability in the physiological e...Micro RNA-26a(miR-26a)has been verified to promote osteogenic differentiation of mesenchymal stem cells in recent years.The main obstacles to its application in bone regeneration are instability in the physiological environment and low efficiency of cellular membrane penetration.To overcome these problems,we constructed a novel plant virus gene delivery system based on Cowpea chlorotic mottle virus(CCMV).By encapsulating miR-26a with purified capsid protein(CP)dimers derived from CCMV,CPmiR-26a(CP26a)virus-like particles(VLPs)were obtained.CP26a retained a structure similar to the native CCMV and protected miR-26a from digestion with its exterior CP.Moreover,CP26a featured similar cellular uptake efficiency,osteogenesis promotion ability,and better biocompatibility compared with Lipofectamine2000-miR-26a(lipo26a),which indicated a promising prospect for CCMV as a novel gene delivery system.展开更多
文摘Maize chlorotic dwarf virus （MCDV） is a quarantine pest as approved by Chinese government. A rapid, sensitive and specific MCDV detection method using reverse transcription-loop-mediated isothermal amplification （RT-LAMP） was estab- lished in this study. Based on the sequence of MCDV coat protein coding gene, specific primers were designed and similar sensitivities were observed between RT- LAMP and RT-PCR, except that RT-LAMP was quicker, and the reaction could be finished within 1 h. In addition, the presence or absence of the fluorescent display in daylight allows naked easy detection of the amplification of MCDV genomic RNA using calcein. The RT-LAMP assay was applied successfully to detect MCDV in maize seeds, and the result by the addition of calcein was consistent with the result detected by the real time turbidimeter.
基金supported by the National Natural Science Foundation of China (No. 31272015)the Ministry of Education of China (No. 313052)the Zhejiang Provincial Natural Science Foundation of China (No. Z3090039)
文摘Maize chlorotic mottle virus (MCMV) infects maize plants and causes significant losses in corn production worldwide. In this study, purified MCMV particles were used as the immunogen to produce monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs). Four murine MAbs (4B8, 8C11, 6F4, and 9G1) against MCMV were obtained through the hybridoma technology. The triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-immunobinding assay (DIBA), and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) using the MAb 4B8 were then developed for sensitive, specific, and rapid detection of MCMV in fields. MCMV could be detected in infected leaf crude extracts at dilutions of 1:327 680, 1:64000, and 1:3276800 (w/v, g/ml) by TAS-ELISA, DIBA, and IC-RT-PCR, respectively. One hundred and sixty-one maize field samples showing virus-like symptoms and sixty-nine symptomless maize field samples from ten different provinces of China were collected and screened for the presence of MCMV using the established serological methods. A phylogenetic tree was constructed based on the full length CP genes and Chinese MCMV isolates formed one branch with Thailand isolates. The detection results demonstrated that MCMV is one of most prevalent viruses infecting maize in the Yunnan and Sichuan provinces of China.
基金supported by the National Key R&D Program of China(2019YFD1001800)the China Agriculture Research System,Overseas Expertise Introduction Project for Discipline Innovation(B18044)+2 种基金the China Agriculture Research System of MOF and MARA(CARS-26-05B)the Natural Science Foundation of Chongqing,China(cstc2019jcyj-msxmX0557)the Guangxi Natural Science Foundation,China(2018GXNSFBA050027)。
文摘In 2009, an emerging citrus viral disease caused by Citrus chlorotic dwarf-associated virus(CCDaV) was discovered in Yunnan Province of China. However, the occurrence and spread of CCDaV in other citrus-growing provinces in China is unknown to date. To better understand the distribution and molecular diversity of CCDaV in China, a total of 1 772 citrus samples were collected from 11 major citrus-growing provinces and were tested for CCDaV by PCR. Among these, 134 citrus samples from Guangxi, Yunnan and Guangdong were tested positive for CCDaV, demonstrating that the occurrence and spread of CCDaV are increasing in China. The complete genome sequences of 17 CCDaV isolates from different provinces and hosts were sequenced. Comparisons of the whole-genome sequences of the 17 CCDaV isolates as well as the 15 isolates available in GenBank revealed that the sequence identity was about 99–100%, showing that the CCDaV isolates were highly conserved. Phylogenetic studies showed that the 32 CCDaV isolates belonged to four different groups based on geographical origins and host species, and that CCDaV isolates from China and Turkey were clustered into different groups. The results provide important information for clarifying the distribution and genetic diversity of CCDaV in China.
基金supported by the Special Fund for Agro-Scientific Research in the Public Interest, China (201303028)the National Natural Science Foundation of China (31571977)
文摘Soybean chlorotic mottle virus(SbCMV)was first detected from soybean plants in Jiangxi Province of China by high throughput sequencing and was confirmed by PCR.The complete nucleotide sequence of NC113 was determined to be 8210 nucleotides,and shared the highest similarity(91.7%)with sequences of SbCMV that was only reported in Japan.It encodes nine putative open reading frames(ORFs Ia,Ib and Ⅱ-Ⅷ),and contains a large intergenic region located at nucleotide 5976-6512 between ORFs VI and VII.Sequence analysis and phylogenetic tree indicated that NC113 is an isolate of SbCMV,and is more related to the soymoviruses Blueberry red ringspot virus(BRRSV),Peanut chlorotic streak virus(PCSV)and Cestrum yellow leaf curling virus(CmYLCV)than to other representative members in the Caulimoviridae family.Field survey of 472 legume plants from Jiangxi and Zhejiang provinces showed SbCMV was only detected from soybean in Nanchang City with a low incidence rate.This is the first report of Soybean chlorotic mottle virus identified in China.
文摘Maize chlorotic mottle virus （MCMV） is a quarantine pest as approved by Chinese government： A rapid, sensitive and specific MCMV detection method using reverse transcription-loop-mediated isothermal amplification （RT-LAMP） was established in this study. Based on the sequence of MCMV coat protein coding gene, 3 sets of primers were designed and specificity test showed that the second set of primers was specific to MCMV, Similar sensitivities were observed on RT-LAMP and RT-PCR, except that RT-LAMP was quicker, and the reaction could be finished within 1 h. In addition, the presence or absence of the fluorescence under daylight allows naked easy detection of the amplification of MCMV genomic RNA using calcein. The RT-LAMP assay was applied successfully to detect MCMV in maize seeds, and the result by the addition of calcein was consistent with the result detected by the real time turbidimeter. The method is rapid, specific, sensitive without the need for complicated equipment, and is suitable for rapid field detection of MCMV.
文摘双生病毒(Geminivirus)是一类基因组为单链环状DNA的植物病毒,具有孪生的病毒粒子。国际病毒分类委员会(International Committee on Taxonomy of Viruses,ICTV)最新的分类报告将双生病毒分为9个属[1]。随着测序技术的发展,利用小RNA或转录组测序可鉴定植物中的已知和未知病毒,新的双生病毒也不断被发现和鉴定[2]。
基金financially supported by the National Natural Science Foundation of China (31871654, 31501340)National Key Research Development Program of China (2016YFD0101300)the China Agriculture Research System (CARS-12)。
文摘Mature chloroplasts,as the main sites of photosynthesis,are essential for seedling growth in higher plants.Loss of function of genes involved in chloroplast development changes plant phenotype.We obtained a YELLOW COTYLEDON (YCO) mutant in rapeseed (Brassica napus L.) using CRISPR-Cas9.Bn.YCO,a gene of unknown function,has two homologous copies (Bna A01.YCO and Bna C01.YCO) in B.napus.Homozygous mutation of these two homologs resulted in yellow cotyledons and chlorotic true leaves.Transmission electron microscopy revealed that the formation of thylakoid membranes was inhibited in yellow cotyledons.Sequence similarity search revealed that YCO was conserved in different species,and a subcellular location assay verified that Bn.YCO was located in the chloroplast.Bn.YCO was expressed in multiple tissues,most highly in cotyledons.Knockout of Bn.YCO blocked the transcription of plastid genes,especially those of photosystem genes transcribed by plastid-encoded polymerase.Transcriptome sequencing showed that the majority of genes involved in ribosome assembly and photosynthesis were down-regulated in Bn.yco mutants.These results suggested that loss of function of Bn.YCO affected plastid gene transcription,which influenced chloroplast biogenesis in rapeseed seedlings.
基金the National Natural Science Foundation of China(31930089)the Food and Agriculture Organization of the United Nations,the International Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences(CAASTIP)(CAASZDRW202108)the Central Public-interest Scientific Institution Basal Research Fund,China(Y2022GH05).
文摘Maize(Zea mays),as a staple food and an important industrial raw material,has been widely cultivated for centuries especially by smallholder farmers.Maize lethal necrosis disease(MLND)is a serious disease infecting maize,which caused devastating damage in the African region recently.MLND is induced by co-infection of maize chlorotic mottle virus and one of several cereal-infecting viruses in the Potyviridae family,with the symptoms ranging from chlorotic mottle to plant death at different infection stages.Integrated pest management for MLND needs strengthening detection,focusing on prevention and effective control.Early detection system of MLND has been successfully established by serological methods,nucleic acid-based methods,next-generation sequencing,etc.The practices,such as using certified seeds,sanitary measures,crop rotation,tolerant or resistant varieties etc.,have been considered as the effective,economical and eco-friendly way to prevent and control MLND.
基金supported by the research funding from West China School/Hospital of Stomatology,Sichuan University(Nos.RCDWJS2021-15,RD-03-202010,RD-02-202004)the research funding from the State Key Laboratory of Oral Diseases(No.SKLOD202111)the fellowship of China Postdoctoral Science Foundation(No.2020TQ0211)。
文摘Micro RNA-26a(miR-26a)has been verified to promote osteogenic differentiation of mesenchymal stem cells in recent years.The main obstacles to its application in bone regeneration are instability in the physiological environment and low efficiency of cellular membrane penetration.To overcome these problems,we constructed a novel plant virus gene delivery system based on Cowpea chlorotic mottle virus(CCMV).By encapsulating miR-26a with purified capsid protein(CP)dimers derived from CCMV,CPmiR-26a(CP26a)virus-like particles(VLPs)were obtained.CP26a retained a structure similar to the native CCMV and protected miR-26a from digestion with its exterior CP.Moreover,CP26a featured similar cellular uptake efficiency,osteogenesis promotion ability,and better biocompatibility compared with Lipofectamine2000-miR-26a(lipo26a),which indicated a promising prospect for CCMV as a novel gene delivery system.
