Many plant viruses utilize subgenomic RNA as gene expression strategy, therefore mapping subgenomic promoter(SGP) is extremely important for constructing viral vectors. Although Cucumber green mottle mosaic virus(CGMM...Many plant viruses utilize subgenomic RNA as gene expression strategy, therefore mapping subgenomic promoter(SGP) is extremely important for constructing viral vectors. Although Cucumber green mottle mosaic virus(CGMMV)-based virus vectors have been constructed, SGP of the coat protein(CP) has not yet mapped. To this end, we firstly presumed 13 nucleotides upstream of the start codon as the transcription starting site(TSS) as previous study identified by random amplification of c DNA ends(RACE). Secondly, the region from nucleotides –110 to +175 is the putative CP SGP, as predicted, a long stem loop structure by the secondary structure of RNA covering movement protein(MP) and CP. To map the CGMMV CP SGP, we further constructed a series of deletion mutants according to RNA secondary structure prediction. The deletion of TSS upstream significantly enhanced CP transcription when 105 nucleotides were retained before the CP TSS. For the downstream of CP TSS, we analyzed the expression of enhanced green fluorescent protein(EGFP) in a series of vectors with partial deletion of the CGMMV CP and found that the nucleotides from +71 to +91 played a key role in the EGFP expression at the transcription level, while EGFP showed the highest expression level when 160 nucleotides were retained downstream of the CP TSS. To confirm these results, we applied online software MEME to predict the motifs and cis-acting elements in the 466 nucleotides covering the sequences of deletion analysis. Conserved motifs and relative acting elements were in regions in which transcription levels were the highest or enhanced. To our best knowledge, this is the first mapping of CGMMV SGP.展开更多
Cucumber green mottle mosaic virus (CGMMV), a member of the Tobamovirus genus, causes a severe disease of cucurbits. In the Moscow region of Russian Federation, the incidence of infection on cucumber plants in greenho...Cucumber green mottle mosaic virus (CGMMV), a member of the Tobamovirus genus, causes a severe disease of cucurbits. In the Moscow region of Russian Federation, the incidence of infection on cucumber plants in greenhouses is high;however, the virus is poorly studied. In this work, the full-length genomes of two pathogenic MC-1 and MC-2 strains of CGMMV isolated from cucumber plants grown in greenhouses in the Moscow region and the attenuated VIROG-43M strain were sequenced. Comparison of VIROG-43M nucleotide sequence with those of the pathogenic strains revealed three missense mutations. Their role in attenuation is discussed. For the first time, in a number of trials conducted under laboratory conditions and in commercial greenhouses, the efficiency of the attenuated VIROG-43M strain as a biocontrol agent for cucumber plant protection resulting in significant yield gain was demonstrated. Phylogenetic analysis with 83 full-length CGMMV coat protein genes isolated in 16 different countries showed that Russian strains are related to isolates from Spain, Greece, USA and Israel.展开更多
本研究旨在提高黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)介导的基因沉默(VIGS)在瓜类作物中应用效率。以黄瓜、甜瓜和西瓜优良种质为实验材料,采用不同的CGMMV病毒载体接种方式、并设置不同的培养温度和相对湿度...本研究旨在提高黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)介导的基因沉默(VIGS)在瓜类作物中应用效率。以黄瓜、甜瓜和西瓜优良种质为实验材料,采用不同的CGMMV病毒载体接种方式、并设置不同的培养温度和相对湿度,以测试基因沉默的有效性。结果表明,真空渗透和种子吸胀的接种方式能够在瓜类作物中产生最高的基因沉默率(FGS)。在22℃和25℃的培养环境下,黄瓜、甜瓜和西瓜PDS沉默有效性(EGSL)较高。黄瓜、甜瓜和西瓜生长1个月后,EGSL达到100%,显著高于30℃的培养环境下EGSL。生长3个月后,甜瓜和西瓜在22℃培养环境下EGSL分别为89.7%和95%;在25℃培养环境下EGSL分别为85.6%和86.1%,均显著性高于30℃下EGSL。在相对湿度为30%和50%环境下,黄瓜的EGSL分别为70%和72%,甜瓜的EGSL分别为76%和73%,显著高于相对湿度为80%的培养环境下EGSL。西瓜在相对湿度为30%的培养环境下的EGSL为69%,显著高于相对湿度为50%时的EGSL(38%)和相对湿度在80%时的EGSL(33%)。综上所述,通过优化了瓜类作物的CGMMV-VIGS的技术体系中接种方式和培养环境参数,提高了基因沉默率和有效性,并且能够快速获得整个植株基因沉默的种质资源,为作物优质和抗逆基因功能的研究提供有效途径。展开更多
Cucumber green mottle mosaic virus(CGMMV) is a member of the genus Tobamovirus, and is a serious pathogen of Cucurbitaceae crops. Virusderived small interfering RNAs(vsi RNAs), which are processed by Dicer-like and Ar...Cucumber green mottle mosaic virus(CGMMV) is a member of the genus Tobamovirus, and is a serious pathogen of Cucurbitaceae crops. Virusderived small interfering RNAs(vsi RNAs), which are processed by Dicer-like and Argonaute proteins as well as RNA-dependent RNA polymerase,mediate the silencing of viral genomic RNA and host transcripts. To identify the CGMMV derived vsi RNAs and reveal interactions between CGMMV and watermelon host plant, deep sequencing technology was used to identify and characterize the vsi RNAs derived from CGMMV in infected watermelon plants in present study. A total of 10 801 368 vsi RNA reads representing 71 583 unique s RNAs were predicted in CGMMVinoculated watermelon plants. The CGMMV vsi RNAs were mostly 21 or 22 nt long. The majority of the CGMMV vsi RNAs(i.e., 91.7%) originated from the viral sense strand. Additionally, uracil was the predominant 5′-terminal base of vsi RNAs. Furthermore, the putative targets and functions of some of the CGMMV vsi RNAs were predicted and investigated. The results enhance our understanding of the interaction between CGMMV and the host watermelon and provide molecular basis for CGMMV resistance improvement in watermelon and other Cucurbitaceae crops.展开更多
对黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)在辽宁省的分布、生物学特性及病毒侵染对西瓜产量和品质的影响进行了研究。结果表明:CGMMV在辽宁省的营口、鞍山、沈阳、丹东、大连、辽阳等地均有分布;在供试的5科21...对黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)在辽宁省的分布、生物学特性及病毒侵染对西瓜产量和品质的影响进行了研究。结果表明:CGMMV在辽宁省的营口、鞍山、沈阳、丹东、大连、辽阳等地均有分布;在供试的5科21种植物中,病毒仅可侵染葫芦科的西瓜、黄瓜、葫芦、南瓜、角瓜、甜瓜、瓠瓜7种植物,并表现系统花叶症状。该病毒致死温度95～100℃,稀释限点10-4～10-5,体外保毒期119d。电镜观察病毒粒子直棒状,长300nm,宽15nm。RT-PCR检测表明,盖州、台安、新民的西瓜花叶样品为黄瓜绿斑驳花叶病毒西瓜株系。利用春秋两季大棚栽培西瓜接种试验表明,西瓜感染CGMMV后,导致植株生长缓慢,结瓜稍小,自根西瓜产量减产幅度很大,秋季大棚减产38.7%～47.6%,春季大棚减产11.4%～19.4%;采用葫芦嫁接的西瓜产量损失变化较小,秋季大棚减产3.4%～17.5%,春季大棚减产2.5%～9.1%。另外,研究结果表明,CGMMV侵染直接导致西瓜倒瓤,使西瓜丧失食用价值,血清学检测倒瓤西瓜携带CGMMV。展开更多
以葫芦和南瓜种子为材料,比较了68、72、75、80℃4个干热处理温度条件及6℃和常温2种种子保存条件对种子质量的影响;通过田间栽培试验检测了种子干热处理对黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)的防治效果。...以葫芦和南瓜种子为材料,比较了68、72、75、80℃4个干热处理温度条件及6℃和常温2种种子保存条件对种子质量的影响;通过田间栽培试验检测了种子干热处理对黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)的防治效果。结果表明:68~80℃的温度处理种子72 h后,不会影响葫芦种子的发芽势和发芽率,但显著降低了南瓜种子的发芽势和发芽率。高温干热处理后,3个葫芦品种的种子分别在常温和6℃下保存15个月后,种子发芽势均显著下降;2个南瓜品种分别在常温和6℃下保存12个月后,除68℃和72℃处理的南瓜京欣砧2常温保存种子的发芽势以外,其他处理种子的发芽势和发芽率都显著下降。通过3 a 3种不同栽培模式的田间试验,不同带毒率的葫芦砧木种子经过72℃干热处理72 h后,嫁接西瓜在田间均未发生CGMMV。试验结果表明,不同葫芦科作物种类,甚至不同品种对温度的敏感性不一样;干热处理种子是防治CGMMV最有效的方法之一。展开更多
黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)是我国的检疫性有害生物,在广西、辽宁、河北、山东、广东和北京等地均发现疫情,已严重威胁着西瓜、甜瓜、黄瓜等作物的生产,因此加强该病毒的检测极为重要。目前黄瓜绿...黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)是我国的检疫性有害生物,在广西、辽宁、河北、山东、广东和北京等地均发现疫情,已严重威胁着西瓜、甜瓜、黄瓜等作物的生产,因此加强该病毒的检测极为重要。目前黄瓜绿斑驳花叶病毒病的检测主要有生物学检测、血清学检测、分子生物学检测及电镜显微观察等方法。笔者对有关黄瓜绿斑驳花叶病毒病的检测技术进行了综述,并分析了现有检测技术的优缺点和应解决的主要问题,探讨了该病毒检测技术今后发展的方向。展开更多
基金supported by the National Natural Science Foundation of China (31571247)the grants from the earmarked fund for the China Agriculture Research System (CARS-26-13)the Agricultural Science and Technology Innovation Program (ASTIP), Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2018-ZFRI-08)
文摘Many plant viruses utilize subgenomic RNA as gene expression strategy, therefore mapping subgenomic promoter(SGP) is extremely important for constructing viral vectors. Although Cucumber green mottle mosaic virus(CGMMV)-based virus vectors have been constructed, SGP of the coat protein(CP) has not yet mapped. To this end, we firstly presumed 13 nucleotides upstream of the start codon as the transcription starting site(TSS) as previous study identified by random amplification of c DNA ends(RACE). Secondly, the region from nucleotides –110 to +175 is the putative CP SGP, as predicted, a long stem loop structure by the secondary structure of RNA covering movement protein(MP) and CP. To map the CGMMV CP SGP, we further constructed a series of deletion mutants according to RNA secondary structure prediction. The deletion of TSS upstream significantly enhanced CP transcription when 105 nucleotides were retained before the CP TSS. For the downstream of CP TSS, we analyzed the expression of enhanced green fluorescent protein(EGFP) in a series of vectors with partial deletion of the CGMMV CP and found that the nucleotides from +71 to +91 played a key role in the EGFP expression at the transcription level, while EGFP showed the highest expression level when 160 nucleotides were retained downstream of the CP TSS. To confirm these results, we applied online software MEME to predict the motifs and cis-acting elements in the 466 nucleotides covering the sequences of deletion analysis. Conserved motifs and relative acting elements were in regions in which transcription levels were the highest or enhanced. To our best knowledge, this is the first mapping of CGMMV SGP.
文摘Cucumber green mottle mosaic virus (CGMMV), a member of the Tobamovirus genus, causes a severe disease of cucurbits. In the Moscow region of Russian Federation, the incidence of infection on cucumber plants in greenhouses is high;however, the virus is poorly studied. In this work, the full-length genomes of two pathogenic MC-1 and MC-2 strains of CGMMV isolated from cucumber plants grown in greenhouses in the Moscow region and the attenuated VIROG-43M strain were sequenced. Comparison of VIROG-43M nucleotide sequence with those of the pathogenic strains revealed three missense mutations. Their role in attenuation is discussed. For the first time, in a number of trials conducted under laboratory conditions and in commercial greenhouses, the efficiency of the attenuated VIROG-43M strain as a biocontrol agent for cucumber plant protection resulting in significant yield gain was demonstrated. Phylogenetic analysis with 83 full-length CGMMV coat protein genes isolated in 16 different countries showed that Russian strains are related to isolates from Spain, Greece, USA and Israel.
文摘本研究旨在提高黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)介导的基因沉默(VIGS)在瓜类作物中应用效率。以黄瓜、甜瓜和西瓜优良种质为实验材料,采用不同的CGMMV病毒载体接种方式、并设置不同的培养温度和相对湿度,以测试基因沉默的有效性。结果表明,真空渗透和种子吸胀的接种方式能够在瓜类作物中产生最高的基因沉默率(FGS)。在22℃和25℃的培养环境下,黄瓜、甜瓜和西瓜PDS沉默有效性(EGSL)较高。黄瓜、甜瓜和西瓜生长1个月后,EGSL达到100%,显著高于30℃的培养环境下EGSL。生长3个月后,甜瓜和西瓜在22℃培养环境下EGSL分别为89.7%和95%;在25℃培养环境下EGSL分别为85.6%和86.1%,均显著性高于30℃下EGSL。在相对湿度为30%和50%环境下,黄瓜的EGSL分别为70%和72%,甜瓜的EGSL分别为76%和73%,显著高于相对湿度为80%的培养环境下EGSL。西瓜在相对湿度为30%的培养环境下的EGSL为69%,显著高于相对湿度为50%时的EGSL(38%)和相对湿度在80%时的EGSL(33%)。综上所述,通过优化了瓜类作物的CGMMV-VIGS的技术体系中接种方式和培养环境参数,提高了基因沉默率和有效性,并且能够快速获得整个植株基因沉默的种质资源,为作物优质和抗逆基因功能的研究提供有效途径。
基金Project funded by China Postdoctoral Science Foundation (2016M601973)the National Natural Science Foundation of China (31572145 and 31272188)Zhejiang Major Agricultural Science and Technology Projects for New Varieties Breeding (2016C02051-4-2)
文摘Cucumber green mottle mosaic virus(CGMMV) is a member of the genus Tobamovirus, and is a serious pathogen of Cucurbitaceae crops. Virusderived small interfering RNAs(vsi RNAs), which are processed by Dicer-like and Argonaute proteins as well as RNA-dependent RNA polymerase,mediate the silencing of viral genomic RNA and host transcripts. To identify the CGMMV derived vsi RNAs and reveal interactions between CGMMV and watermelon host plant, deep sequencing technology was used to identify and characterize the vsi RNAs derived from CGMMV in infected watermelon plants in present study. A total of 10 801 368 vsi RNA reads representing 71 583 unique s RNAs were predicted in CGMMVinoculated watermelon plants. The CGMMV vsi RNAs were mostly 21 or 22 nt long. The majority of the CGMMV vsi RNAs(i.e., 91.7%) originated from the viral sense strand. Additionally, uracil was the predominant 5′-terminal base of vsi RNAs. Furthermore, the putative targets and functions of some of the CGMMV vsi RNAs were predicted and investigated. The results enhance our understanding of the interaction between CGMMV and the host watermelon and provide molecular basis for CGMMV resistance improvement in watermelon and other Cucurbitaceae crops.
文摘对黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)在辽宁省的分布、生物学特性及病毒侵染对西瓜产量和品质的影响进行了研究。结果表明:CGMMV在辽宁省的营口、鞍山、沈阳、丹东、大连、辽阳等地均有分布;在供试的5科21种植物中,病毒仅可侵染葫芦科的西瓜、黄瓜、葫芦、南瓜、角瓜、甜瓜、瓠瓜7种植物,并表现系统花叶症状。该病毒致死温度95～100℃,稀释限点10-4～10-5,体外保毒期119d。电镜观察病毒粒子直棒状,长300nm,宽15nm。RT-PCR检测表明,盖州、台安、新民的西瓜花叶样品为黄瓜绿斑驳花叶病毒西瓜株系。利用春秋两季大棚栽培西瓜接种试验表明,西瓜感染CGMMV后,导致植株生长缓慢,结瓜稍小,自根西瓜产量减产幅度很大,秋季大棚减产38.7%～47.6%,春季大棚减产11.4%～19.4%;采用葫芦嫁接的西瓜产量损失变化较小,秋季大棚减产3.4%～17.5%,春季大棚减产2.5%～9.1%。另外,研究结果表明,CGMMV侵染直接导致西瓜倒瓤,使西瓜丧失食用价值,血清学检测倒瓤西瓜携带CGMMV。
文摘以葫芦和南瓜种子为材料,比较了68、72、75、80℃4个干热处理温度条件及6℃和常温2种种子保存条件对种子质量的影响;通过田间栽培试验检测了种子干热处理对黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)的防治效果。结果表明:68~80℃的温度处理种子72 h后,不会影响葫芦种子的发芽势和发芽率,但显著降低了南瓜种子的发芽势和发芽率。高温干热处理后,3个葫芦品种的种子分别在常温和6℃下保存15个月后,种子发芽势均显著下降;2个南瓜品种分别在常温和6℃下保存12个月后,除68℃和72℃处理的南瓜京欣砧2常温保存种子的发芽势以外,其他处理种子的发芽势和发芽率都显著下降。通过3 a 3种不同栽培模式的田间试验,不同带毒率的葫芦砧木种子经过72℃干热处理72 h后,嫁接西瓜在田间均未发生CGMMV。试验结果表明,不同葫芦科作物种类,甚至不同品种对温度的敏感性不一样;干热处理种子是防治CGMMV最有效的方法之一。
文摘黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)是我国的检疫性有害生物,在广西、辽宁、河北、山东、广东和北京等地均发现疫情,已严重威胁着西瓜、甜瓜、黄瓜等作物的生产,因此加强该病毒的检测极为重要。目前黄瓜绿斑驳花叶病毒病的检测主要有生物学检测、血清学检测、分子生物学检测及电镜显微观察等方法。笔者对有关黄瓜绿斑驳花叶病毒病的检测技术进行了综述,并分析了现有检测技术的优缺点和应解决的主要问题,探讨了该病毒检测技术今后发展的方向。
