Cloning, Prokaryotic Expression And Constructing Of The Plant Expression Vector For Pac-1 Gene Of Yeast

The pac-1 gene comes from the DNA of Schizosaccharomyces pombe, The product of which is a kind of dsRNA dependent ribonuclease, and has potential to degrade the dsRNA viral genome, the replication form of ssRNA viral genome and viroid genome (the replication form all are dsRNA). So if the pac-1 gene was introduced into plants with the technology of plant genetic engineering, it will not only do some technological preparation for development of transgenic wheat with resistance to multiple viruses, but also be in the hope of containing the transgenic plant with resistance to multiple viruses.Synthesizing the primers upstream and downstream according to the known pac-1 gene sequence, using DNA of S.pombe as template, we cloned the pac-1 gene by means of PCR amplification, which then was inserted into the clone vector pGEM-3zf(+) by cleavage with the restriction endonuclase KpnI/BamHI, ligation and transformation, and the recombinant vectors pGEM-pac-1^ (No. 2.1459) and pGEM-pac-f2 (No. 2.274) were contained. The result of sequence analysis showed this experiment got one full-length pac-1 gene with 1092 nucleotides, which had high homology with the gene sequence reported by Yuichi lino et. For pac-l^, the homology was 99.3% and 8 nucleotides were different, for pac-1 ~, the homology was 99.8% and 2 nucleotides, and only one encoded amino acid was different.The full-length pac-1 gene, which was cleaved with restriction endonuclase BamH/Ndel from the recombinant vectors pGEM-pac-7*1 and pGEM-pac-1*2, was inserted into the prokaryotic expression vector pET-5a and the expression vector was constructed. The pET-5a was transformed into the competent cell of BL21(DE3)pLysS. When the cells were induced by the IPTG, the production of the gene was high expressed and 43KD protein band generated in SDS-PAGE. After the cells were induced by IPTG ,lysed by ultrasonic wave and centrifuged, both the supernatant and precipitant parts were examined by SDS-PAGE. The result showed that the product of pac-1 gene existed both in the supernatant part as soluble form and in the precipitant part as inclusion bodies. The content of protein in the supernatant part was measured by the method of UV. absorption. It was also proved that the expression product of pac-1 gene has strong activity of degrading RDV-dsRNA.To get further transgenic plant with resistance to multiple viruses, we selected important crops: wheat and rice as the targets of transformation and constructed two kinds of plant expression vectors respectively used in the transformation of wheat and rice. By firstly synthesizing primer downstream and then amplifying pac-1 gene segment by PCR, which then were inserted into the plasmid pEmu-mcs-N with Emu promoter, we constructed a expression vector pEmu- pac-f1 for the transformation of wheat by pollen tube pathway. By the cleaving with Xmal/SacI, the pac-1 gene was lignated with the vector pBI121, which was used in the transformation of plant by transfection of Agrobaterium. The plant expression vector pBI-121-pac-#1 was also constructed.