The Construction Of PRSV-HN2Infectious Clones Using Yeast Homologous Recombination System And It's Infectivity Analysis

Papaya is an important cash crop in subtropical and tropical regions. FRSV(Papaya ringspot virus) is the major pathogen of papaya, and the incidence rate can be more than90%. It limist the development of papaya industry.Currently, it can not prevent and control PRSV effectivly by chemical methods. Breeding new disease-resistant varieties by gene technology is an effective way of preventing Papaya RingSpot virus disease. Domestic and foreign researchers have successively transfered coat protein gene and replicase gene of PRSV into papaya and obtained the transgenic papaya of anti-PRSV. But the instability of resistance for transgenic papaya were reported due to the different PRSV strains from different regions,.In order to study the papaya ringspot virus pathogenesis at the molecular level, find a more broad-spectrum and effective antiviral strategy, and cultivate new varieties with resistant promising, the technology of RT-PCR and rapid-amplification of cDNA ends (RACE) were used to get the complete genomic sequence of papaya ringspot virus isolated from Hainan island in China and the sequence was determined (GenBank accession number:KF791028). The full-length cDNA contains10326nt (not including the3'end of the poly (A)), which encodes3346amino acids. This strain of PRSV has a similarity of81%to91%of the nucleotide sequence compared with that of Taiwan, Thailand, India, Bazil, Mexico, France, and Hawaii, also an amino acid similarity of88%-94%. When constructing a large RNA viral full-length cDNA clone in E. coli. it often showed the instability. So the PRSV-HN2full-length genome were divided into four large fragments cloned, and clones of pG-A, pG-B, pG-C, pG-D were obtained by yeast homologous recombination system to construct PRSV full-length sequence for avoid this instability. Five kinds of PRSV-HN2full-length cDNA clone and four infectious clone were successfully constructed, containing one cloning vector of full-length PRSV-HN2named as PB-P (pBS70T-PRSV-HN2), one expression vector of full-length PRSV-HN2named as PPH (pBIN61-PRSV-HN2), and another three clones of PRSV-HN2full length with green fluorescent protein (GFP) gne inserted after protein PI, P3and Nib, named as PPHP1G (pBIN61-PRSV-HN2-P1-GFP), PPHP3G (pBIN61-PRSV-HN2-P3-GFP) and PPHNIBG (pBIN61-PRSV-HN2-NIB-GFP) respectively.The Infection rates of PPH, PPHP1G, PPHP3G, PPHNIBG were30%to60%with vaccinating zucchini and papaya by Agrobacterium tumefaciens infects. Systemic infection was observed after40days, but it was a week later than the native viral infection.,The interaction between the virus and the host is an important research in the field of virology, and the latest developments in the field are largely dependent on the infectious clones. The construction of PRSV full-length cDNA infectious clones laid the theoretical foundation for the study of the PRSV infection, genetic variation and pathogenesis at the molecular level. Furthermore the method of yeast homologous recombination system provides a new way for rapid construction infectious clone of other potyvirus.