Prokaryotic Expression, Antiserum Preparation And Detection In Chloroplasts Of LMoV CP

Lily mottle virus (LMoV) is belonged to Potyvirus, which is one of the most common viruses infecting lily. LMoV can cause mottled or striped symptom after infecting lily. Moreover, LMoV can reduce the production and quality of lily bulbs. At present, the pathogenesis of LMoV is not clear, the research of proteins function which encoded by LMoV is still blank, which seriously hinders the prevention and treatment of lily virus diseases. In this paper, a serological method for detection of LMoV was established, and the effect of LMoV on lily chloroplasts was preliminarily proved, which lays a foundation for elucidating the pathogenesis of LMoV.According to the sequence of LMoV gene (HM222521.1) reported on GenBank, we designed the primers and amplified the coat gene of LMoV from the total RNA of lily leaves by RT-PCR and obstained about 822 bp DNA fragment. The fragment was cloned to the cloning vector pMDTM18-T. The recombinant plasmid pMDTM18-CP) was transformed into Escherichia coli DH5α, and then was sequenced after identified by PCR and restriction enzymes. The results showed that we had successfully constructed recombinant plasmid pMDTM18-CP.We amplified the coat gene of LMoV from the recombinant plasmid pMDTM18-CP by PCR, then the gene fragment was cloned to the expression vector pET-28a(+). The recombinant plasmid pET28a-CP was transformed into Escherichia coli BL21(DE3) and was induced by IPTG. The expression products were analyzed by SDS-PAGE. The results showed that approximately a 34 kDa recombinant protein with His tag was expressed. The expression yield of recombinant protein was increased by optimizing induction temperature and time. The result showed that the optimal expression condition of recombinant protein was 30℃,4 h.We purified the recombinant protein by Ni-IDA-Sefinose under denaturing conditions and obtained a single recombinant protein band with higher expression level. Then we used the recombinant protein as an antigen to prepare antiserum for the virus. The preparation of the antiserum was tested by ID-ELISA and Western blot, the results showed that the titer of the antiserum was 1:52000 with high specificity. Seven lily samples were randomly selected to be detected by ID-ELISA and RT-PCR, the results of these two tests were consistent, which proved that the antiserum can be used for LMoV detection on Lily.Electron microscopic photography of lily leaves infected by LMoV showed that chloroplasts were swollen and deformed, and most of stroma lamellae were messy, starch grains in chloroplast were also swollen. The result indicated that LMoV could destroy the structure of chloroplasts. The detection of immunogold-particles showed that colloidal gold particles were mainly localized in the thylakoid membrane. SDS-PAGE and Western blot proved that LMoV CP was present in the chloroplast, which was consistent with the observed results of the immuno-gold labeling. The system of LMoV CP imported into chloroplasts in vitro, and the effects of the incubation time and the concentration of LMoV CP on import efficiency were studied. The results showed that LMoV CP was able to import into the isloated chloroplasts in 1 minute when the concentration of LMoV CP was 30 μg/μL.