Construction Of The Infectious Cdna Clone Of Potato Virus Y Liaoning Isolate And Studies On Its Pathogenic Molecular Mechanism

Potato virus Y(PVY) is one of the major plant viruses that affect tobacco production in China. PVY Liaoning isolate (PVY-LN) was isolated and purified from tobacco leaves of vein necrosis symptom in Liaoning province. PVY-LN genome was sequenced and phylogenetic analysed, infectious clone of PVY-LN was constructed and molecular determinants of PVY-LN induce tobacco vein necrotic symptom was studied. The main results had been already achieved as below.1. Specific primers based on genome sequences of PVY registered in GeneBank were designed and PVY-LN gemomic sequence was achieved using RT-PCR, RACE technology(NCBI accession number JQ971975). Genome sequences of PVY-LN was9714nucleotides in length, excluding the3’-terminal poly (A) tail. PVY-LN has a single long open reading frame (ORF) encode a polyprotein containing3061amino acid. No recombination can be predicted in PVY-LN sequences compared with that of the other PVY strains using Recombination Detection ProgrameV.4.16(RDP4). Complete genome sequence comparison and phylogenetic analysis indicated that PVY-LN is closely related to PVY necrosis strain (PVYN).2. PVY-LN infectious clone was constructed that can express by T7promoter and transctiptied in vitro and positive infectious clone was detected by ELISA, RT-PCR, infection rate was13.3%. Infectious clone was named pBlPVY-LN. The sequencing results showed that PVY-LN contains20PolyA construct on pBlueScript SK(+) vector. In this paper, PVY-LN clone was constructed also that can be expressed by35S promoter, but no positive infectious clone was detected.3. The result demonstrated that the region between Swa Ⅰ and Nco Ⅰ (5540nt to6491nt) could be the determinates of tobacco vein ncrosis symptom in PVY-LN sequence and protein blast analysis showed that these are two protein conservative domain in5540nt to6491nt, one conservative domain exist can induce vein ncrosis symptom in tobacco. First, three mutants were constructed. The were inoculated into Variety Yunyan87and test their infectivity. Mutant pB1PVY-LN-SwNc showed mosaic symptom. This result demonstrated that the region between Swa Ⅰ and Nco Ⅰ (5540nt to6491nt) could be the determinates of tobacco vein ncrosis symptom in PVY-LN sequence. Second, protein blast analysis showed that these are two protein conservative domain in5540nt to6491nt. Accoding to this result, three mutants pB1PVY-LN-R1,pB1PVY-LN-R2and pB1PVY-LN-R3were constructed to seprate these two conservative domain. All of the mutants showed vein ncrosis symptom in tobacco. This result demonstrated that one conservative domain exist can induce vein ncrosis symptom in tobacco.