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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 *** specific to each CMV ORF were designed and *** were thenamplified with length varying from 61 to 153 *** 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 *** 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:*** results were confirmed by Lab-on-a-Chip and Northern blot hybridization *** 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

Microarray-based identification of tomato microRNAs and time course analysis of their response to cucumber mosaic virus infection

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2011年第2期12卷 116-125页

作者： Qiu-lei LANG Xiao-chuan ZHOU Xiao-lin ZHANG Rafal DRABEK Zhi-xiang ZUO Yong-liang REN Tong-bin LI Ji-shuang CHEN Xiao-lian GAO College of Life Sciences Zhejiang University Hangzhou 310058 China Institute of Bioengineering Zhejiang Sci- Tech University Hangzhou 310018 China LC Sciences 2575 W. Bellfort Suite 270 Houston ZY 77054 USA Department of Biology and Biochemistry University of Houston Houston TX 77004 USA

A large number of plant microRNAs (miRNAs) are now documented in the miRBase, among which only 30 are for Solanum lycopersicum (tomato). Clearly, there is a far-reaching need to identify and profile the expression of miRNAs in this important crop under various physiological and pathological conditions. In this study, we used an in situ synthesized custom microarray of plant miRNAs to examine the expression and temporal presence of miRNAs in the leaves of tomato plants infected with cucumber mosaic virus (CMV). Following computational sequence homology search and hairpin structure prediction, we identified three novel tomato miRNA precursor genes. Our results also show that, in accordance with the phenotype of the developing leaves, the tomato miRNAs are differentially expressed at different stages of plant development and that CMV infection can induce or suppress the expression of miRNAs as well as up-regulate some star miRNAs (miRNA*s) which are normally present at much lower levels. The results indicate that developmental anomalies elicited by virus infection may be caused by more complex biological processes.

关键词： Solanum lycopersicum Plant miRNA cucumber mosaic virus miRNA array miRNA response

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virus Movement Protein Gene Mediated Resistance Against cucumber mosaic virus Infection

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Acta Botanica Sinica 1999年第6期41卷 585-590页

作者：张振臣李大伟张力于嘉林刘仪中国农业大学农业生物技术国家重点实验室新加坡国立大学农业分子生物学研究所

Tobacco ( Nicotiana tabacum L.) “NC89” plants were transformed with deletion mutant of cucumber mosaic virus (CMV) movement protein (MP) gene and full_length CMV MP gene, respectively. The transformed plants were analyzed with polymerase chain reaction (PCR), PCR_Southern, Southern and Western blots. R 0 generation of the transgenic plants were inoculated with CMV. Five out of 10 lines of tobacco plants (BMPK) transformed with CMV MP deletion mutant gene showed high resistance to CMV infection and remained symptomless for up to 50 days post_inoculation. In contrast, tobacco plants (BMPR) transformed with full_length CMV MP gene did not show resistance to CMV infection. However, most of the infected full_length CMV MP gene transgenic plants recovered by showing none or very mild mosaic symptoms in 40 days post_inoculation. The results of R 1 generation of the BMPK transgenic plants tested under field conditions showed that all 5 lines of transgenic plants could delay the virus disease development.

关键词： cucumber mosaic virus Movement protein gene Transgenic plants Resistance

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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 Institute of Biotechnology Zhejiang University Institute of Biotechnology Zhejiang University Hangzhou 310029 China Hangzhou 310029 China Hangzhou 310029 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

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

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Journal of Zhejiang University Science 2003年第3期4卷 336-339页

作者： SandraPérezAlvarez 薛朝阳周雪平 InstituteofBiotechnology ZhejiangUniversityHangzhou310029China

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 Nsi I 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 expressing 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 replacement 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 *** 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 *** 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 *** 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 *** 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 *** 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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