Detection And Infectious Clone Construction Of Cucurbit Aphid-borne Yellows Virus

Cucurbit aphid-borne yellows virus(CABYV)is a member of the genus Polerovirus,the family Luteoviridae.It has been reported wide spread in main growing areas in Europe and Asia,and causes serious yield decreasing.Currently CABYV is reported in most provinces of China,and may has a great threat to the safety production of cucurbit plant,such as melon and watermelon.To identify the viruses infecting watermelon and melon in Henan,Gansu and Xinjiang.134 samples with suspected virus-like symptoms were collected from those areas,and tested by Double Antibody Sandwich Enzyme Linked Immunosorbent Assay(DAS-ELISA)and Reverse transcript polymerase chain reaction(RT-PCR).The results showed that the detection rates of Zucchini yellow mosaic virus(ZYMV),Cucurbit aphid-borne yellows virus(CABYV),Watermelon mosaic virus(WMV),Cucumber mosaic virus(CMV),Melon aphid-borne yellows virus(MABYV)among the 134 detected samples were 31.3%,26.9%,26.1%,14.2%,8.2%,respectively,whereas Squash mosaic virus(SqMV)and Papaya ring spot virus-watermelon strain(PRSV-W)were not detected.It's the first time to find MABY in Henan and Xinjiang.Our study found that ZYMV,WMV,CABYV,MABYV occurred in Henan;ZYMV,CMV,CABYV in Gansu;ZYMV,WMV,CMV,CABYV,MABYV in Xinjiang.21.6%of the samples were mix-infected with more than two virus.In addition,we found that the virus dominant species were different in those areas,implying the distributions are associated with environmental condition and cultivar grown.Sixty three samples exhibiting yellowing symptom were collected from Shanshan of Xinjiang,Guazhou of Gansu,and Tongxu of Henan provinces in China.Among them,36 were positive to CABYV detected by RT-PCR.A 1.4kb fragment consisting of partial RNA-dependent RNA polymerase(Rd Rp),intergenic NCR and complete coat protein(CP)gene was amplified by RT-PCR from 25 positive samples.Blast analysis showed that the fragments have high similarities of 93.2%-100%with other CABYV isolates from GenBank.The nucleotide sequence identities of CP gene were99.2%-100% for Shanshan isolates,98.2%-100% for Guazhou isolates,98.8%-99.8% for Tongxu isolates.Phylogenetic trees based on partial RdRp,NCR and CP gene showed that the 25 isolates had a very close relationship with other isolates from China and other countries neighboring China including Thailand,Republic of Korea et al.,but a distant relationship with European isolates,suggesting that CABYV variation appeared to be associated with its geographical distribution.In preliminary work,we cloned full-length CP gene,and insert into the prokaryotic expression vector BL21(DE3),but no target protein was expressed.Sequence analysis showed that its nucleotide sequence contained high percent of rare codons.Nucleotide sequence that suppress prokaryotic expression of CP gene was optimized,the 5'-terminal 280 nucleotides sequence were mutated by artificial synthesis of DNA.The new recombinant plasmid was constructed by using optimized sequence to replace previous sequence.The engineered strain was induced with 1mmol/L IPTG at 37?for 4 h.SDS-PAGE analysis found a 40 k Da fusion protein.The result show that prokaryotic expression of the optimized CP gene was success.Infectious clone is the basis of viral gene function research and many other research.Full length genome of CABYV was amplified by RT-PCR and insert into binary expression vector p XT1 to construct a Agrobacturium-mediated infectious clone.When N.benthamiana agro-infiltration with infectious clone,full genome and subgenome can be detected 28 days post inoculation by RT-PCR and Northern-Blot,typical symptom of yellowing was observed 40 days post inoculation,all results show that infectious clone has the ability to infect N.benthamiana.