Construction Of Infectious CDNA Clone Of Cucumber Mosaic Virus Strain M And 3a Gene Expressed In Escherichia Coli

Total RNA was extracted from Nicotiana tabacum cv. White Burley which wasinfected by CMV (Cucumber mosaic virus) M strain and then applied to complementaryDNA synthesis by reverse transcription. The full-length genome sequences included RNA1,RAN2 and RNA3 were amplified by polymerase chain reaction. Recombinant plasmid ofpUC-1,pUC-2 and pUC-3, three full-length of cDNA clones of CMV-M driven individuallyby T7 RNA polymerase promoter, were constructed for in vitro transcription of the viralRNA. The analysis of sequence shows that, the full-length sequence of CMV-M RNA1 has3361 nucleotides (nt) and one open reading frames (ORF1) are nested in it, encoding 112 kula protein. 5'NTR is 93nt, 3'NTR is 222nt; the full-length of CMV-M RNA2 has 3037nucleotides (nt) and two open reading fi'ames (ORF2, 3) are nested in it, encoding 96.7ku 2aprotein and 13.1ku 2b protein. 5'NTR is 87nt, 3'NTR is 299nt; the full-length of CMV-MRNA3 has 2215 nucleotides(nt) and two open reading frames(ORF4, 5) are nested in it,encoding 31ku 3a protein and 24ku CP protein. 5'NTR is 120nt, 3'NTR is 289nt.The total length was cloned into pUC and templates which were used for in vitrotranscription were got by digesting the recombinants (pUC-1,pUC-2 and pUC-3) with Sph I.By mechanical inoculation, the in vitro transcripts of pUC-1,pUC-2 and pUC-3 wereinfection to Nicotiana tabacum cv.White Burley, causing mosaic symptom on the leavessame as that of wild type CMV-M. Also, the virus amplication could be detected by westernblotting.In order to understand the role of CMV-M 3a gene in viral pathogenicity, MP protein(which was encoded by 3a) was constructed based on pET22b(+), With IPTG induction, theMP was overexpressed in Escherichia coli BL21.