The Cloning And Sequence Analysis Of The Gene Papaya Proteinase IV And The Study On The Feasibility Of Its Use In Plant Virus-resistance

There are affluent cysteine proteinases in papaya latex. Based on the known result, the cDNA of papain was tried to cloned from total RNA of papaya in this study. Though the primers were designed according to the cDNA of papain, the cDNA of PPIV was attained, which was homologous to that of papin. When compared with the papain gene in EMBL Database, the cDNA of PPIV reachs 98.7% in homology. Hovever, the gene encoding the mature papain peptide was amplified using PCR from genomic DNA extracted mid-development Carica papaya fruit. About 98.8% of cDNA nucleotide sequence reported in literature were homologous to the responding sequences of our study. There are three introns in the gene, in which the content of A and T is 86.0%,79.5% and 90.6 % respectively.With protokaryotic over-expression, the products of PPIV were identified by SDS-PAGE analysis. The signal sequence inhibits the expression in E.coli.In this study, a new approach of plant virus-resistance was expored by making up the cDNA of PPIV. Nib gene was amplified from plasmid PYSR, which is plant expression vector containing potyvirus Y Nib gene . The Nib gene was fused with the fragment encoding the mature part of papaya proteinase IV. A conservative motif, recognized by proteinases of potato virus Y, was inserted between Nib and PPIV , which will release functional PPIV from the fused protein after infection by potato virus Y. Then, plant expression vector pNPA was constructed by ligating the fusing gene and pBI121,which is knocked out GUS gene. The fusing gene was transformed into leaf tissue of tobacco cultivar NC89. More than 100 plants were obtained. Transgenes were confirmed to be inserted into tobacco genome of R0 generation by PCR and dot-blotting analysis. However, we didn't get any virus-resistant strains.