Extraction And Mass Spectrometry Analysis Of Viral Particle Of Tomato Spotted Wilt Virus

Tomato spotted wilt virus(TSWV)is the representative that belongs to the genus of Tospovirus,which belongs to the family of Bunyaviridae.Due to the host of the virus is widespread and the mainly spreading vector western flower thrips distribution range is wide,caused serious economic losses worldwide.So it is very urgent and meaningful for the study of Tomato Spotted Wilt Virus.Through inoculation TSWV in Nicotiana benthamiana and Nicotiana rustica,and collecting the incidence system leaves,TSWV virus particles were extracted by sucrose gradient centrifugation.Virus particles were gained with high purity through SDS-PAGE verification,further analysis of mass spectrometry analysis showed that the extracted virus particles containing viral proteins such as replication protein RdRp and glycoprotein Gn Gc,non structural protein NSs,nucleocapsid protein N,in addition to plant protein hsp70,Sar1 etc.Because the TSWV protein N can be combined with RdRp to form Ribonucleoprotein(RNP),which is responsible for the replication and transcription of the virus.However,the RdRp protein is too large to be expressed completely.In this study,the fragment was cloned into the vector,and then the interaction between N and the protein was tested by prokaryotic expression of different seperated fragments.It was found that the RdRp fragments of prokaryotic expression were interacting with N,and the results showed that the RdRp2380-2878aa fragment with His tag had a weak interaction with GST-N,and the negative control could exclude the interaction between GST protein and RdRp.The mass spectrometric analysis of TSWV virus particles found that the extraction contained protein Sar1 which belongs to N.benthamiana.The protein is involved in the formation of endoplasmic reticulum COP ? clothing,which is very valuable for the study of replication and transfer mechanism of virus particle in plants.So the preparation of polyclonal antibody and transgenic N.benthamiana plants to the next step of research are very necessary.The polyclonal antibody of Sar1 against 1:4000 was obtained by immunizing mice.The results showed that the polyclonal antibody could specifically detect Sar1 protein by Western Blot.The Sar1 gene with YFP labeled was transformed into N.benthamiana by Agrobacterium mediated transformation.The Sar1 gene was successfully transferred into N.benthamiana tested by laser confocal microscopy and Western Blot.Tobacco mosaic virus is a single stranded RNA virus that can infect tobacco and other Solanaceae plants,so that these infected leaves appear to be mottled fouling,hence the name of the virus.CRISPR-Cas9 is the most efficient genome editing system,which has been successfully applied in plants,bacteria,yeast,fish and mammalian cells.This paper constructed the CRISPR/Cas9 vector infection attack TMV genome to inhibit TMV,and through agrobacterium co infiltration N.benthamiana.Fluorescent spots were observed to verify the number and scope,and found two attack sites effectively,inhibit TMV infection,the other two had no obvious effect,preliminary inference attack site is not correct.