The Action Mechanism Of Cytosinpeptidemycin On The Replication Protein Gene Expression Of Tobacco Mosaic Virus

Cytosinpeptidemycin(CytPM)is a new antiviral agent,which is the secondary metabolites from Streptomyces ahygroscopicus var.liaoningensisdeveloped by the Plant Virus Laboratory of Shenyang Agricultural University.The antiviral effect of this agent is high specific and efficient,but this agents molecular antiviral mechanism is still remains to be clarify.In this paper,the prokaryotic expression vector pET28a-HEL was constructed and the purified protein was expressed as an antigen.In this study,specific polyclonal antibody was prepared and the detection system of polyclonal antibody was established.Finally,Northern blot and Western blot were used to analyze the agentson the TMV p126 protein has an inhibitory effect.The main results and conclusions of this study were as follows:1.The recombinant plasmids pET28a-HEL,pET32a-HEL,pET28a-MET and pET32a-MET were successfully constructed.The recombinant plasmids were transformed into the competent cells BL21,and the prokaryotic expressed recombinant proteins were induced by 0.5mM IPTG.The size of the recombinant plasmids was about 25kDa,39kDa,30kDa and 45kDa,respectively.Size of the recombinant protein was consistent with the expected protein size.It was found that the recombinant protein pET32a-MET and pET32a-HEL were centrifuged by ultrasound,and a large amount of protein bands appeared in the precipitate.And the pET28a-HEL protein fragment was clear and single,so the pET28a-HEL was selected for subsequent purification experiments.The successful construction of the prokaryotic expression vector laid the foundation for the preparation of polyclonal antibody.2.The recombinant plasmid pET28a-HEL was purified and the p126 antibody was prepared by immunizing New Zealand white rabbits with high purity.The Dot-ELISA was used to detect the antibody availability.It was found that the tobacco leaves infected with TMV had specific spots after exposure to the NC membrane with CDP-Star color,while the tobacco leaves infected with PMMoV had no spots with healthy tobacco leaves.The tobacco leaves infected with TMV on the NC film of NBT and BCIP showed a discoloration reaction.No specific reaction between tobacco leaf infected with PMMoV and healthy tobacco leaves showed that the antibody could bind to the antigen,indicating that the antibody was available.Western blot was used to detect the specificity of the antibody.It was found that in the TMV tobacco leaf and BY-2 protoplast samples,a bright band appeared near 130 kDa,and the replicative protein p126 was identified as TMV.There is a suspected p183 band;healthy tobacco leaf samples as a control,no band.The immunoreactive PVDF membrane background was clean and clear without bandage,indicating that antibody specificity was strong and could be used for the detection of TMV p126.The p126 polyclonal antibody to the successful preparation of TMV-LN for the detailed translation mechanism to provide an important means of research.At the same time,can also be used for detailed study with p126 interaction,in the process of virus infection play an important role in the host protein.3.The identification of CytPM has a constant effect on TMV p126 protein expression and RNA synthesis.Northern blotting was used to detect the amount of TMV RNA accumulation and found that there was almost no accumulation of TMV RNA positive strand and negative strand in BY-2 tobacco protoplasts at the diluted 3200× CytPM treatment,indicating that the TMV RNA and the expression of p126 protein in BY-2 cells was detected by Western blot.The results showed that the BY-2 cells co-inoculated by TMV particles before treated with 3200x CytPM,did not detect the p126 and CP of TMV virus.After co-culture of treated BY-2 cells with TMV virus,Western blot analysis showed that after co-culture with TMV for 3h,The p126 protein fragment was detected.With the prolongation of culture time,the higher concentration of p126 protein was detected,and it was suggested that the CytPM could inhibit the synthesis of p126 protein and RNA sythesis of TMV virus.This study provides an important reference value for the understanding of the molecular mechanism of TMV virus.