CLONING AND SEQUENCE ANALYSIS OF 3'-END REGION OF POTATO VIRUS Y GENOME

The entire coat protein (CP) gene and part of the 3'-noncoding sequence of the potatovirus Y (PVY, the Chinese isolate) genome were synthesized with polymerase chain reaction(PCR) using cDNA of its genomic RNA as a template. A restriction endonuclease site Ncoland the initiation codon AUG were included in primer Y5 while the SalI site was includedin primer Y3. After being double digested with Ncol and SalI enzymes, the PCR product wascloned into a pGEM derivative plasmid, and the CP gene in one of the clones, pPCY6, wassequenced. Several clones were selected from the cDNA library by using the CP gene frag-ment of pPCY6 as a probe and the sequences of these clones were determined. These se-quences included part of the NIb gene, entire CP gene and 3'-noncoding region, 1317 bp alltogether.Sequence analysis indicated that the nucleotide sequence homology of the CP geneof this strain with that of the 0 strain (94.2%) was a little higher than with that of the Nstrain (89.6%), but the homology of amino acid se

CLONING AND SEQUENCE ANALYSIS OF 3'-END REGION OF POTATO VIRUS Y GENOME

The entire coat protein (CP) gene and part of the 3'-noncoding sequence of the potato virus Y (PVY, the Chinese isolate) genome were synthesized with polymerase chain reaction (PCR) using cDNA of its genomic RNA as a template. A restriction endonuclease site Ncol and the initiation codon AUG were included in primer Y5 while the SalI site was included in primer Y3. After being double digested with Ncol and SalI enzymes, the PCR product was cloned into a pGEM derivative plasmid, and the CP gene in one of the clones, pPCY6, was sequenced. Several clones were selected from the cDNA library by using the CP gene frag- ment of pPCY6 as a probe and the sequences of these clones were determined. These se- quences included part of the NIb gene, entire CP gene and 3'-noncoding region, 1317 bp all together.Sequence analysis indicated that the nucleotide sequence homology of the CP gene of this strain with that of the 0 strain (94.2%) was a little higher than with that of the N strain (89.6%), but the homology of amino acid sequence with the two strains was almost the same (93.3% and 91.8% respectively). This gene was cloned into the binary vector pBI 121.1. We are transforming potato via Agrobacterium in order to obtain the transgenic plants which are resistant to PVY infection.