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Quantitative Determination of cucumber mosaic virus Genome RNAs in Virions by Real-Time Reverse Transcription-Polymerase Chain Reaction

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Acta Biochimica et Biophysica Sinica 2006年第10期38卷 669-676页

作者： Jun-Li FENG~1 Shao-Ning CHEN~1 Xiang-Shan TANG~1 Xian-Feng DING~1 Zhi-You DU~2 Ji-Shuang CHEN~(1,2*) 1 Institute of Bioengineering,Zhejiang Sci-Tech University,Hangzhou 310018,China 2 College of Life Sciences,Zhejiang University,Hangzhou 310029,China Institute of Bioengineering Zhejiang Sci-Tech UniversityHangzhou 310018 China College of Life Sciences Zhejiang UniversityHangzhou 310029 China

A real-time RT-PCR procedure using the green fluorescent dye SYBR Green Ⅰ was developedfor determining the absolute and relative copies of cucumber mosaic virus （CMV） genomic RNAs containedin purified virions.Primers specific to each CMV ORF were designed and selected.Sequences were thenamplified with length varying from 61 to 153 bp.Using dilution series of CMV genome RNAs prepared by invitro transcription as the standard samples,a good linear correlation was observed between their thresholdcycle （C_t） values and the logarithms of the initial template amounts.The copies of genomic RNA 1,RNA 2,RNA 3 and the subgenomic RNA 4 in CMV virions were quantified by this method,and the ratios were about1.00:1.17:3.58:5.81.These results were confirmed by Lab-on-a-Chip and Northern blot hybridization assays.Our work is the first report concerning the relative amounts of different RNA fragments in CMV virions asa virus with tripartite genome.

关键词： real-time reverse transcription-polymerase chain reaction cucumber mosaic virus genomic RNA quantitative determination

Synergy between cucumber mosaic virus and Zucchini Yellow mosaic virus on Cucurbitaceae Hosts Tested by Real-time Reverse Transcription-Polymerase Chain Reaction

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Acta Biochimica et Biophysica Sinica 2007年第6期39卷 431-437页

作者： Rong ZENG~(1,2#), Qiansheng LIAO~(2#), Junli FENG~2, Dingjun LI~1, and Jishuang CHEN~(2*) 1 College of Bio-Safety Science Technology, Hunan Agricultural University, Changsha 410128, China, 2 Institute of Bioengineering, Zhejiang Sci-Teeh University, Hangzhou 310018, China College of Bio-Safety Science Technology Human Agricultural UniversityChangsha 410128 China Institute of Bioengineering Zhejiang Sci-Tech UniversityHangzhou 310018 China

cucumber mosaic virus (CMV) and zucchini yellow mosaic virus (ZYMV) are two principal viruses infecting cucurbitaceous crops, and their synergy has been repeatedly observed. In our present work, a real-time reverse transcription-polymerase chain reaction procedure was established to study the accumulation kinetics of these two viruses in single and combined infections at the molecular level. The accumulations of open reading frames (ORFs) for 1a, 2a, 3a and coat protein (CP) of CMV and CP of ZYMV were tested. In the single infection, CMV-Fny ORFs accumulated to their maxima in cucumber or bottle gourd at 14 d post-inoculation (dpi), and gradually declined thereafter. ZYMV-SD CP ORF reached maximal accumulation at 14 and 28 dpi on cucumber and bottle gourd, respectively. However, when coinfected with CMV-Fny and ZYMV-SD, the maximal accumulation levels of all viral ORFs were delayed. CMV-Fny ORFs reached their maxima at 21 dpi on both hosts, and ZYMV-SD CP ORF reached maximal accumulation at 21 and 28 dpi on cucumber and bottle gourd, respectively. Generally, the accumulation levels of CMV-Fny ORFs in the co-infection were higher than those in the single infection, whereas the accumulation of ZYMV-SD CP ORF showed a reverse result.

关键词： cucumber mosaic virus genornic RNA real-time RT-PCR relative quantification synergy zucchini yellow mosaic virus

The RNA-binding domain of DCL3 is required for long-distance RNAi signaling

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aBIOTECH 2024年第1期5卷 17-28页

作者： Jie Li Bo-Sen Zhang Hua-Wei Wu Cheng-Lan Liu Hui-Shan Guo Jian-Hua Zhao State Key Laboratory of Plant Genomics Institute of MicrobiologyChinese Academy of SciencesBeijing 100101China CAS Center for Excellence in Biotic Interactions University of the Chinese Academy of SciencesBeijing 100049China Qilu Zhongke Academy of Modern Microbiology Technology Jinan 250022China

Small RNA(sRNA)-mediated RNA silencing(also known as RNA interference,or RNAi)is a conserved mechanism in eukaryotes that includes RNA degradation,DNA methylation,heterochromatin formation and protein translation repression.In plants,sRNAs can move either cell-to-cell or systemically,thereby acting as mobile silencing signals to trigger noncell autonomous silencing.However,whether and what proteins are also involved in noncell autonomous silencing have not been elucidated.In this study,we utilized a previously reported inducible RNAi plant,PDSi,which can induce systemic silencing of the endogenous PDS gene,and we demonstrated that DCL3 is involved in systemic PDS silencing through its RNA binding activity.We confirmed that the C-terminus of DCL3,including the predicted RNA-binding domain,is capable of binding short RNAs.Mutations affecting RNA binding,but not processing activity,reduced systemic PDS silencing,indicating that DCL3 binding to RNAs is required for the induction of systemic silencing.cucumber mosaic virus infection assays showed that the RNA-binding activity of DCL3 is required for antiviral RNAi in systemically noninoculated leaves.Our findings demonstrate that DCL3 acts as a signaling agent involved in noncell autonomous silencing and an antiviral effect in addition to its previously known function in the generation of 24-nucleotide sRNAs.

关键词： RNAi signaling DCL3 RBD RNAs cucumber mosaic virus

Emergence of a new satellite RNA from cucumber mosaic virus isolate P1

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Journal of Zhejiang University-Science A(Applied Physics & Engineering) 2003年第3期4卷 97-100页

作者： Sandra Pérez Alvarez 薛朝阳周雪平 Institute of Biotechnology Zhejiang University Hangzhou 310029 Zhejiang University China

The cucumber mosaic virus (CMV) isolate P1 caused very mild symptoms on many plant species. After serial passages by mechanical inoculation over five years, CMV P1 caused severe symptoms on several tobacco cultivars and tomato. A specific band of approximately 0.3 kb in length was amplified by RT PCR with primers synthesized based on reported CMV satellite RNA (satRNA) sequences. Sequence analysis showed there were two satRNAs (Sat P1 1 and Sat P1 2). Sat P1 1 contained 335 nucleotides, and Sat P1 2 contained 394 nucleotides. These two satRNAs shared 64% overall nucleotide sequence homology, and differences between the two satRNAs included mutations as well as deletions. Sat P1 1 was identical to a satRNA (Z96099) reported in 1995 in CMV P1. Based on differences in the sequence and secondary structure between these two satRNAs, we conclude that Sat P1 2 represents the emergence of a new satellite (necrotic satellite) from attenuated satRNA populations. The possible effect of the emergence of this new satRNA is discussed.

关键词： cucumber mosaic virus Satellite RNA Sequence

Recombination with coat protein transgene in a complementation system based on cucumber mosaic virus (CMV)

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Science China(Life Sciences) 2001年第3期44卷 263-273页

作者：雷万里方荣祥张国华陈晓英张晓勤 Laboratory of Plant Biotechnology Institute of Microbiology Chinese Academy of Sciences

In order to study the feasibility of cucumber mosaic virus (CMV) as an expression vector, the full-length cDNA of RNA 3 from strain SD was cloned and the sequence around the start codon of the coat protein (CP) gene was modified to create an NsiⅠ site for insertion of foreign genes. The CP gene was replaced by the green fluorescent protein (GFP) gene. The cDNAs of Fny RNAs 1 and 2 and the chimeric SD RNA 3 were cloned between the modified 35S promoter and terminator. Tobacco protoplasts were transfected with a mixture of the viral cDNAs containing 35S promoter and terminator as a replacement vector and expressed GFP. A complementation system was established when the replacement vector was inoculated onto the transgenic tobacco plants ex-pressing SD-CMV CP. GFP was detected in the inoculated leaves in 5 of 18 tested plants and in the first upper systemic leaf of one of the 5 plants ten days after inoculation. However, no GFP could be detected in all the plants one month after inoculation. Recombination between the CMV vector and the CP transgene was proved by retro-transcriptional polymerase chain reaction (RT-PCR) and verified by DNA sequencing. Our results argue against the feasibility of the CMV-based replace-ment vector trans-complemented by the CP transgene, and at the same time, enlighten ways to improve the CMV-based expression vector and the biosafety of CMV CP-mediated virus resistant transgenic plants.

关键词： cucumber mosaic virus complementation vector CP transgene recombination biosafety.

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Determination Suppressive Effect of Satellite 369 on Accumulation of cucumber mosaic virus by Real-time Reverse Transcript-Polymerase Chain Reaction

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Determination Suppressive Effect of Satellite 369 on Accumul...

A Real-time Reverse Transcription PCR Assay for Detection of cucumber mosaic virus in Individual Peach Aphid（Myzus persicae）

中国植物病理学会2007年学术年会

作者： Jun-Li Feng~(1,2) Rong Zeng~2 Shao-Ning Chen~2 Ji-Shuang Chen~(2,1*) 1.College of Life Sciences,Zhejiang University,Hangzhou 310029, 2.Institute of Bioengineering,Zhejiang Sci-Tech University,Hangzhou 310018

＜正＞Sat369 is a 369-nt cucumber mosaic virus satellite RNA（CMV-satRNA）,it can attenuate the symptom induced by its helper virus.To study the mechanism by which the symptom was altered, real-time reverse transcript-polymerase chain reaction（RT-PCR）was applied to examine the accumulation kinetics of viral genes at the absence or presence of sat369.In the infected tobacco plants, the amounts of viral genes accumulated to their highest levels in the second newly developed tobacco leaves,therefore these leaves were sampled at 3,7,14,21 and 28 days post inoculation（dpi）to study the influence of sat369.The results showed that sat369 depressed the accumulation of all viral genes but with different degrees,its effects on 2b,la and 2a genes were more remarkable than that of MP and CP genes.For all viral genes,the suppression effects were more considerable at early than later stage of infection,and the time point of maximum levels were delayed by 7 days or more in the presence of sat369.These results were confirmed by northern blot hybridization,and indicated that the attenuated symptom in the presence of sat369 might be the results of low accumulation levels of CMV-Fny genes, especially at early time post-inoculation,CMV-Fny la,2a and 2b genes were all involved in this complex interaction

关键词： real-time reverse transcript-polymerase chain reaction cucumber mosaic virus satellite RNA quantification virus genes

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A Real-time Reverse Transcription PCR Assay for Detection of...

Three-dimensional reconstruction and comparison of vacuolar membranes in response to viral infection

中国植物病理学会2009年学术年会

作者： YANG Jinguang~1 Xu Manlin~2 WANG Fenglong~(1*) SHEN Lili~1 QIAN Yumei~1 Chen Dexin~1 (1.Key Laboratory of Tobacco Quality Control,Ministry of Agriculture,the People''s Republic of China,Qingdao, Shandong 266101 2.HeZe University Resources and Environment Sci Department,Heze,Shandong 274000)

＜正＞cucumber mosaic virus（CMV） is transmitted in a nonpersistent manner by peach aphid （Myzus persicae）,leading to many crop diseases.The large number of viruliferous peach M.persicae is one of the major culprits for the rampant outbreak.A novel real-time PCR assay targeting CP gene was developed to detect CMV in individual M.persicae.The sensitivity of this method was 1000-fold higher than that of the conventional PCR.These primers,with a battery of reference virus isolates,including most isolates,showed high specificity for CMV.Additionally,this method yielded negative results on all other viruses with identical transmitting vector tested.Thus,the real time RT-PCR assay could be a valuable tool for rapid,sensitive,specific detection of CMV in M.persicae.It could also be used for determining a viruliferous M.persicae in natural M.persicae population.In turn,the information could help to forecast,and therefore prevent the tobacco virus disease by timely applying of pesticide to reduce the infected M.persicae population or altering planting time to avoid the insect’s migration period.

关键词： cucumber mosaic virus real-time RT-PCR detection aphid（Myzus persicae）

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Journal of Integrative Plant Biology 2021年第2期63卷 353-364页

作者： Xueting Wang Juncai Ma Xuejiao Jin Ning Yue Peng Gao Keith Ka Ki Mai Xian-Bing Wang Dawei Li Byung-Ho Kang Yongliang Zhang State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology College of Biological SciencesChina Agricultural UniversityBeijing 100193China State Key Laboratory of Agro-Biotechnology School of Life SciencesThe Chinese University of Hong KongHong Kong 999077China

The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three-dimensional(3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus(CMV) or tobacco necrosis virus A Chinese isolate(TNV-AC), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck-like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1 a and 2 a and TNV-AC auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast.Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant(+) RNA virus replication complexes.

关键词： comparison cucumber mosaic virus electron tomography spherules tobacco necrosis virus A vacuole viral replication

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Design, Synthesis, and Antiviral Activity of Novel Chalcone Derivatives Containing a Purine Moiety

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Chinese Journal of Chemistry 2017年第5期35卷 665-672页

作者： Xiuhai Gan Yanjiao Wang Deyu Hu Baoan Song State Key Laboratory Breeding Base of Green Pestieide and Agricultural Bioengineering Key Laboratory of Green Pesticide and Agrieultural Bioengineering Ministry of Education Center for Research and Development of Fine Chemicals Guizhou University Guiyang Guizhou 550025 China College of Chemistry and Life Science Guizhou Education University Guiyang Guizhou 550018 China

To find new antiviral agents,novel chalcone derivatives containing a purine moiety were designed and synthesized by combining bioactive substructures.The antiviral activities of the derivatives against tobacco mosaic virus （TMV） and cucumber mosaic virus （CMV） were evaluated.Results showed that most of the derivatives showed antiviral activities.Compounds 3n and 3p exhibited excellent curative,protective and inactivation activities against TMV,with the EC50values of 452.4,416.2,241.2 and 438.7,418.6,261.7 μg·mL-1,respectively,which were better than those of ribavirin （585.8,436.0 and 268.7 μg·mL-1）.Compounds 3n and 3p showed remarkable curative and protective activities against CMV.Compound 3n showed a moderate affinity to TMV coat protein,with binding constant Ka and Kd values of 1.5 × 104 L·mol-1 and 79.8 μmol·L-1,respectively.These findings provided an important structural insight for further designs of highly active chalcone derivatives and a basis for further study on their mechanism of action.

关键词： chalcone purine antiviral activity tobacco mosaic virus cucumber mosaic virus