The Identification Of Watermelon Mosaic Disease In Suzhou

cucumber mosaic virus (CMV)is the representative of Cucumovirus in Bromoviridae and is the one of the main plant virus to imperil Solanaceae, Leguminosae and Cucurbitaceae. It can be transmitted by seventy five kinds of aphides or by seeds and infect more than one thousand plants. CMV is a single strand, including RNA1, RNA2 and RNA3. Some have the satellite RNA. RNA1 and RNA2 encode 1a and 2a protein which are related with the replicase. RNA2 also encode 2b protein which is related with plant gene expression. RNA3 encode move protein and coat protein. One isolate, named CMV-As was isolated from infected watermelon leaves in Suzhou. 17 plant species in 6 genera could be experimentally infected by this isolate. The thermal inactivation point(TIP) of the isolate was between 55℃ and 60℃ , the dilution end-point ranged from 10-4 to 10-5, and the longevity in vitro was 2～3d. The purified virus particles observed by electronic microscope were typical globosity with the diameter of about 20～30nm. The ratio of the A260 to A280 of the isolate was 0.69, and the RNA content was about 11.3%. The isolate showed positive reaction with standard CMV and CMV-As antiserum. All this showed that the isolate resembles the reported CMV. CMV-As was confirmed to belong to Cucumovirus by the biological properties detection, but because of the high similarity of the subgroup I and subgroup II, only the biological properties check can't ensure the kind of CMV-As. So the molecular biology search was needed. We synthesized the upstream and downstream primers according to the known CMV coat protein gene. A DNA fragment with 657bp was obtained by reverse transcription PCR. After the double digestion with the Kpn I and BamH I, the PCR product was inserted into the vector pGEM-3Zf(+). The recombinant vector was transformed into competent E.Coli DH5a. Positive recombinant plasmid pGEM-cp extracted by alkaline lysis was screened by PCR amplification and double digestion. 3 positive clones were sequenced. The result of sequence showed that the 3 sequences were identical, which conclude there was no mismatch base in PCR amplification. And the right CP gene had obtained. Analysis of the nucleotides and amino acid sequences, the CP gene was 657bp and encodes 218 amino acids. 12 CMV isolates, which represents subgroup I or subgroup II, were selected from GenBank. After analysis, CMV-As shared the identities of 91.5%～97.4% with subgroup I and of 76.3%～77% with subgroup II in nucleotide sequence. The identities is 94.4%～98.1% with subgroup I and 76.4%～78.2% with subgroup II in amino acids. The result showed CMV-As was closer to subgroup I and belong to CMV I. The CMV-As showed high identity with isolate(GenBank D10538). The identity of nucleotide sequence is 97.4% and amino acid sequence is 98.1%. The full-length cp gene, which was got from the recombinant vector pGEM-cp cleaved with restriction endonuclase Nde I/BamH, was inserted into the prokaryotic expression vector pET-5a, recombinant prokaryotic expression vector pET-cp was constructed. The recombinant vector was transformed into competent E.Coli DH5a. Positive recombinant plasmid pET-cp extracted by alkaline lysis was screened by PCR amplification and double digestion. The positive plasmid was transformed into competent E.Coli BL21 (DE3) pLysS. After induction of IPTG, the production of the CP gene was high expressed by SDS-PAGE detection. The production of the CP gene can be used to prepare antiserum, which settles the base to detect the virus and tap the reagent of diagnosis. Based on the above results, it is concluded that this isolate belongs to CMV I.