Cloning, Prokaryotic Expression Of Pacl Gene And Detection Of Enzyme Activity Of Its Product

The pacI gene comes from Schizosaccharmyces pombe, which encodes a kind of double-strand RNA specific ribonuclease, and has a potential to degrade viral RNA with dsRNA genome and the replication form (RF) of plant virus with single strand RNA genome. Therefore, make pacI transgenic plant by means of modern-technique is a new strategy for broad-spectrum virus resistance. This research is part of works funded by National Natural Science Foundation of China (NSFC), in the title of "Cloning of dsRNA specific-nuclease and to produce transgenic pepper resistance against multi-plant virus infection". In this study, pacI gene was isolated from DNA of Schizosaccharmyces pombe by means of PCR and expressed in E.coli BL21 (DE3) pLysS. Its biological activity was then test. Antiserum was prepared against pacI and plant expression vector pBI121-pacI.was also constructed.All these laid the foundation for transgenic pepper against CMV and TMV. I pacI gene cloning and sequence analysis Synthesizing the primers upstream and downstream according to the known pacI gene sequence, using DNA of S.pombe as template, we cloned the pacI 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 recombinant vectors pGEM-pacI#1(No.2.1459) and pGEM-pacI#2(No.2.274) were contained. We sequenced inserted sequence bidirectionally from promoter T7/SP6 of pGEM-3Zf(+) by dioxigenation. Sequenced after selecting the recombinant by being cleavaged and the result was analyzed by software. The result of sequence analysis showed this experiment got one full-length pacI gene with 1092 nucleotides, which had high homology with the gene sequence reported by Yuichi Iino et. For pacI#1, 8 nucleotides were different and the homology was 99.3%;For pacI#2, 2 nucleotides were different and the homology was 99.8%,and only one encoded amino acid was different. Nucleotides sequence both changed from G430 to A430, i.e. that amino acid changed from Glu to Lys, though nucleotides changed in different positions. Selected two recombinant to sequence and the result showed their sequence were all the same and it is impossible that the change in nucleotides sequence resulted from PCR. II Prokaryotic expression of pacI gene and detection of enzyme activity of its product i Prokaryotic expressed of pacI gene in E.coli The full-length pacI gene, which was cleaved with restriction endonuclase BamHI/NdeI from the recombinant vectors pGEM-pacI#1 (No.2.1459) and pGEM-pacI#2(No.2.274), 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. 12%SDS-PAGE showed: induced by IPTG, The 43KD protein band in pET-pacI became thicker,while pET-pacI which was not induced and pET-5a induced by IPTG has no change. We conclude the product of the pacI gene was high expressed. ii Detection of the enzyme activity of pacI gene product Most protein are expressed in the precipitant part as inclusion bodies.After the cells were induced by IPTG, lysed by ultrasonic wave and centrifuged, both the supernatant and the precipitant parts were examined by SDS-PAGE. The result showed that the product of pacI gene existed both in the supernatant part as soluble form and in the precipitant part as inclusion bodies. The supernatant was applied to detect the enzyme activity. Ten fold diluted of the content of protein in the supernatant was measured by the method of UV. absorption. Double stranded RNA (dsRNA) of TMV and CMV were applied to test the enzyme activity of pacI gene product. As substrate, dsRNA-TMV and dsRNA-CMV was mixed with the supernatant which contained the same content of pET5a and pET5a-pacI respectively and put in 37℃for 60 minutes. The result showed that the dsRNA mixed with pacI induced by IPTG were both degraded, while the control has no change. It was proved that the expression product of pacI gene has strong activity of degrading TMV-dsRNA and CMV-dsRNA. iii Protein purification and antiserum preparation Preparation of antiserum would be applied to detect transgenic pepper later. Induced by IPTG, pacI was expressed largely. Protein were collected and examined by SDS-PAGE. The gelatin was put into 0.2mol/L KCl, the protein band of 43Kda became white and was cut into the dialytic bag and purged by gelose gelatin electrophoresis setting. Put protein into dialytic bag to purge in PBS for 12 hours and analyzed the purity by SDS-PAGE. We injected the houserabbit with the purified protein and prepare antiserum. III Constructing plant expression vectors pBI121-pacI Plant expression vectors pBI121-pacI was constructed by biology...