Cloning And Analyzing Of The Complete Genomic Sequence Of Melon Necrotic Spot Virus From China

MNSV is a dangerous pathogen which can seriously affect the quality and yield of melon and cause significant economic losses in melon.MNSV can infect a variety of species of the family Cucurbitaceae,like:Cucumber,melon and loofah.MNSV can be easily transmitted by the chytrid fungus Olpidium bornovanus,seed and insect,so it poses a huge threat to China's fruits and vegetables industry.MNSV was reported the first time in China in 2007,only a few studies about MNSV were reported in China,So cloning and analyzing of the complete genomic sequence of Melon necrotic spot virus isolated in China is very important to know MNSV's pathogenic properties of molecular biology,analyze it's pathogenic mechanism and anti-Virus research using modern means of genetic engineering.In this study,we collected leaves of melon which was grown in plastic greenhouses and systematicly infected by MNSV,Sap extracts in 0.01 M phosphate buffer(pH 7.0)was mechanically inoculated on cotyledon of melon.MNSV and the RNA of MNSV was extracted and three medial fragments of MNSV genome were cloned using RT-PCR.On the basis of obtained three fragments,the 5' end and 3' end of MNSV were determined using RT-PCR.Analyzing and splicing the five fragments obtained using DNAMAN biology software,we fred that MNSV-China,is similar with MNSV isolated from other countries,which has a single stranded and positive sense RNA genome of 4271nt long,consisting of five open reading frames(ORFs) and two untranslated regions(UTRs).The first ORF locates in 88-894 nt,it has 807 nt; the second ORF locates in 88-2463 nt,it has 2376 nt;the third ORF locates in 2442-2639 nt,it has 197 nt;the Fourth ORF locates in 2643-2828nt,it has 186 nt;the Fifth ORF locates in 2815-3987nt,it has 1173 nt.These five ORFs code five proteins named p29,p89,p7A,p7B and p42,respectively.These proteins' molecular weight were 29 kDa,89 kDa,7 kDa,14kDa and 42kDa,respectively.5'UTR and 3'UTR were 87nt and 284nt in length,respectively.No 5'cap and 3'Poly(A) structure were found in MNSV-China.Based on the alignment of the complete genome sequence of MNSV-China and other countries,we deduced that the complete genomic sequence of MNSV-China was more similar to MNSVs collected from Spain and Israel,but different with MNSVs in Japan to some extent.Based on the alignment of the nucleotide sequence and amino acid sequence of five proteins named p29,p89,p7A,p7B and p42 of MNSV collected from China, Spain,Japan,British and Israel,respectively,we concluded that MNSV-China's nucleotide sequence of each protein was different a little bit with MNSV collected from other countries,but the amino acid sequence of each protein was more similar among these MNSV isolates from China,Spain,Japan,British and Israel.