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Highly Sensitive Fluorescent-labeled Probes and Glass Slide Hybridization for the Detection of Plant RNA Viruses and a Viroid

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Acta Biochimica et Biophysica Sinica 2007年第5期39卷 326-334页

作者： zhiyou du~(1,2), bo jin~3, wenhong liu~4, liang chen~1, and jishuang chen~(2,1*) 1 college of life sciences, zhejiang university, hangzhou 310029, china 2 Institute of Bioengineering, zhejiang Sci-Tech university, hangzhou 310018, china 3 college of life sciences, zhejiang Chinese Medical university, hangzhou 310053, china 4 college of Bioengineering, zhejiang Chinese Medical university, hangzhou 310053, china college of life sciences Zhejiang UniversityHangzhou 310029 China Institute of Bioengineering Zhejiang Sci-Tech UniversityHangzhou 310018 China college of life sciences Zhejiang Chinese Medical UniversityHangzhou 310053 China college of Bioengineering Zhejiang Chinese Medical UniversityHangzhou 310053 China

In this study, a modified method of the conventional RNA dot-blot hybridization was established, by replacing32p labels with CY5 labels and replacing nylon membranes with positive-charged glass slides, for detecting plant RNA viruses and a viroid. The modified RNA dot-blot hybridization method was named glass slide hybridization. The optimum efficiency of RNA binding onto the surfaces of activated glass slide was achieved using aminosilane-coated glass slide as a solid matrix and 5×saline sodium citrate （SSC） as a spotting solution. Using a CY5-labeled DNA probe prepared through PCR amplification, the optimized glass slide hybridization could detect as little as 1.71 pg of tobacco mosaic virus （TMV） RNA. The sensitivity of the modified method was four times that of dot-blot hybridization on nylon membrane with a32P-labeled probe. The absence of false positive within the genus Potyvirus [potato virus A, potato virus Y （PVY） and zucchini yellow mosaic virus] showed that this method was highly specific. Furthermore, potato spindle tuber viroid （PSTVd） was also detected specifically. A test of 40 field potato samples showed that this method was equivalent to the conventional dot-blot hybridization for detecting PVY and PSTVd. To our knowledge, this is the first report of using dot-blot hybridization on glass slides with fluorescent-labeled probes for detecting plant RNA viruses and a viroid.

关键词： glass slide hybridization fluorescent labeling dot-blot hybridization virus viroid

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Satellite RNA-mediated Reduction of Cucumber Mosaic Virus Genomic RNAs Accumulation in Nicotiana tabacum

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Acta Biochimica et Biophysica Sinica 2007年第3期39卷 217-223页

作者： Qiansheng LIAO~(1,2) Liping ZHU~2 zhiyou du~2 Rong ZENG~(2,3) Junli FENG~2 jishuang chen~(1,2*) 1 college of life sciences,zhejiang university,Hangzhon 310029,china 2 Institute of Bioengineering,zhejiang Sci-Tech university,hangzhou 310018,china 3 college of Biological Safety Technology Hunan Agricultural university,Changsha 410128,china college of life sciences Zhejiang UniversityHangzhou 310029 China Institute of Bioengineering Zhejiang Sci-Tech UniversityHangzhou 310018 China college of Biological Safety Technology Hunan Agricultural UniversityChangsha 410128 China

Satellite RNAs(satRNAs)are molecular parasites that interfere with the pathogenesis of thehelper *** this study,the relative accumulation of cucumber mosaic virus(CMV)-Fny genomicRNAs with or without satRNAs were quantitatively analyzed by real-time *** results showed thatsatRs apparently attenuated the symptoms of CMV-Fny on Nicotiana tabacum by depressing the accumu-lation of CMV-Fny genomic RNAs,tested as open reading *** accumulation of CMV-Fny 1a,2a,2b,3a,and CP genes was much higher than that of CMV-Fny with satRs added(CMV-Fsat),at differentinoculation ***-FnyΔ2b,in which the complete 2b gene and 41 amino acids at the C-terminal of the2a gene were deleted,caused only a slight mosaic effect on *** seedlings,similar to that of CMV-Fsat,but the addition of satRs to CMV-FnyΔ2b showed further decrease in the accumulation of CMV-FnyΔ2b genomic *** results indicated that the attenuation of CMV,by adding satRs or deleting the2b gene,was due to the low accumulation of CMV genomic RNAs,and that satRNA-mediated reduction ofCMV genomic RNAs accumulation in *** was possibly related to the 2b gene.

关键词： cucumber mosaic virus satellite RNA deletion of 2b gene relative accumulation

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