[Cloning, sequence analysis and high-level expression in Escherichia coli and activity assay of pac-1 gene from Schizosaccharmyces pombe].
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/BamHI. 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/BamHI. 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 concluded that pac-1 gene has the biological activity of degrading the CMV-dsRNA.
Bu
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Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
Sun
J L
JL
Yang
X C
XC
D003001:Cloning, Molecular; D004722:Endoribonucleases / Q000032:analysis / Q000235:genetics* / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D005656:Fungal Proteins; D012330:RNA, Double-Stranded / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins
D005656:Fungal Proteins; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D029702:Schizosaccharomyces pombe Proteins; D004722:Endoribonucleases; C499540:pac1 protein, S pombe