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 ...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 sequence with the two strains was almostthe same (93.3% and 91.8% respectively). This gene was cloned into the binary vector pBI121.1. We are transforming potato via Agrobacterium in order to obtain the transgenicplants which are resistant to PVY infection.展开更多
Potato virus Y (PVY) N coat protein (CP) coding sequence was cloned into a plant expression vector pMON316 under the CaMV 35S promoter. Leaf discs of potato (Solanum tuberosum) were used to Agrobacterium mediated gene...Potato virus Y (PVY) N coat protein (CP) coding sequence was cloned into a plant expression vector pMON316 under the CaMV 35S promoter. Leaf discs of potato (Solanum tuberosum) were used to Agrobacterium mediated gene transfer. A large number of regenerated putative transgenic plants were obtained based on kanamycin resistance. Using total DNA purified from transgenic plants as templates and two oligonucleotides synthesized from 5′ and 3′ of the PVY coat protein gene as primers, the authors carried out polymerase chain reaction(PCR) to check the presence of this gene and obtained a 0.8 kb specific DNA fragment after 35 cycles of amplification. Southern blot indicated that the PCR product was indeed PVY CP gene which had been integrated into the potato genome. Enzyme linked immunosorbent assay (ELISA) of our transgenic plants showed that CP gene was expressed in at least some transgenic potato plants.展开更多
基金This work was supported in part by ICSC World Laboratory, Switzerland.
文摘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 sequence with the two strains was almostthe same (93.3% and 91.8% respectively). This gene was cloned into the binary vector pBI121.1. We are transforming potato via Agrobacterium in order to obtain the transgenicplants which are resistant to PVY infection.
文摘Potato virus Y (PVY) N coat protein (CP) coding sequence was cloned into a plant expression vector pMON316 under the CaMV 35S promoter. Leaf discs of potato (Solanum tuberosum) were used to Agrobacterium mediated gene transfer. A large number of regenerated putative transgenic plants were obtained based on kanamycin resistance. Using total DNA purified from transgenic plants as templates and two oligonucleotides synthesized from 5′ and 3′ of the PVY coat protein gene as primers, the authors carried out polymerase chain reaction(PCR) to check the presence of this gene and obtained a 0.8 kb specific DNA fragment after 35 cycles of amplification. Southern blot indicated that the PCR product was indeed PVY CP gene which had been integrated into the potato genome. Enzyme linked immunosorbent assay (ELISA) of our transgenic plants showed that CP gene was expressed in at least some transgenic potato plants.
