The Schizosaccharmyces pombe pac\|1 gene product is a kind of dsRNA dependent ribonuclease,which has potential to degrade the dsRNA viral genome, the replication form of ssRNA viral genome and viroid genome.Therefore,...The Schizosaccharmyces pombe pac\|1 gene product is a kind of dsRNA dependent ribonuclease,which has potential to degrade the dsRNA viral genome, the replication form of ssRNA viral genome and viroid genome.Therefore,to introduce the pac\|1 gene into plants conferring them resistance to viruses is a new method of establishing the anti\|virus transgenic plant.The pac\|1 gene from the S.pmobe genome DNA isolated from China was cloned by means of PCR amplification.The pac\|1 gene was inserted into the cloning vector pGEM\|7Zf(+) by using restriction endonuclease Kpn Ⅰ/Bam HI.Sequencing analysis shows that it is a complete gene with 1095 necleotides.Compared to the reported pac\|1 gene,its homology is significant,but with 5 nucleotides differences,leading to only one amino acid difference. Pac\|1 gene was inserted into the prodaryotic expression vector pET\|21(a) by using the restriction endonuclase Nde Ⅰ/Bam HI.It was induced by the IPTG in E.coli BL21 harbouring the recombinant vector pET\| pac\|1 .The pac\|1 gene product is analyzed by the SDS\|PAGE.The result shows the product of pac\|1 gene exists in the supernatant part as soluble form and in the precipitant part as inclusion bodies after the cells were lysed by ultrasonic wave.The supernatant was applied to detect the enzyme activity of pac\|1 gene product.We conclued that pac\|1 gene has the biological activity of degrading the CMV\|dsRNA.展开更多
文摘The Schizosaccharmyces pombe pac\|1 gene product is a kind of dsRNA dependent ribonuclease,which has potential to degrade the dsRNA viral genome, the replication form of ssRNA viral genome and viroid genome.Therefore,to introduce the pac\|1 gene into plants conferring them resistance to viruses is a new method of establishing the anti\|virus transgenic plant.The pac\|1 gene from the S.pmobe genome DNA isolated from China was cloned by means of PCR amplification.The pac\|1 gene was inserted into the cloning vector pGEM\|7Zf(+) by using restriction endonuclease Kpn Ⅰ/Bam HI.Sequencing analysis shows that it is a complete gene with 1095 necleotides.Compared to the reported pac\|1 gene,its homology is significant,but with 5 nucleotides differences,leading to only one amino acid difference. Pac\|1 gene was inserted into the prodaryotic expression vector pET\|21(a) by using the restriction endonuclase Nde Ⅰ/Bam HI.It was induced by the IPTG in E.coli BL21 harbouring the recombinant vector pET\| pac\|1 .The pac\|1 gene product is analyzed by the SDS\|PAGE.The result shows the product of pac\|1 gene exists in the supernatant part as soluble form and in the precipitant part as inclusion bodies after the cells were lysed by ultrasonic wave.The supernatant was applied to detect the enzyme activity of pac\|1 gene product.We conclued that pac\|1 gene has the biological activity of degrading the CMV\|dsRNA.
