Cloning,Expression And Localization For TGB Of Lily Symptomless Virus

Since scientists found the movement protein(MP) of plant virus, the structure and function of the different types of MP have been comparatively conducted the thorough research, but there was very few research about movement protein of Lily symptomless virus. The sequences of MP of LSV was cloned and provided a comparison with other genes. The evolutionary trees were built, using software of bio informatics to analyze gene. The constructed expression vectors were transformed into E. coli BL21 that was induced by IPTG for expressing the proteins of TGBpl. The movement protein localization was analysed by green fluorescent protein fusion technology. THis study resuLt was of important value to understand the structure and function of movement protein, and in the process of virus infection mechanism.The primers of MP of LSV were designed to amplified the target gene. The gene of MP was 1161 bp, containing three overlapping genes (TGB1,TGB2,TGB3), which was known as triple gene blocks(TGB). Bioinformatics analysis showed that gene encoded three movement proteins:TGBp1(228 aa), TGBp2(106aa), TGBp3(64 aa); Evolutionary tree analysis showed that LSV-TGB1 and TGB1 of Kalanchoe latent virus(AJ293571.1) shared a high similarity; LSV-TGB2 and TGB2 of lily latent virus(AJ131812.1) shared a high similarity; LSV-TGB3 and the TGB3 of mirabilis jalapa mottle virus shared a high similarity. The conservatism of the TGB1 was supreme and the conservatism of the TGB3 was lowest. TGBp3 was hydrophobic proteins. TGBp1 was muLtifunctional protein which had activity of helicase1 and nucleic acid hydrolysis enzyme, the amino acid sequence containing ATP-binding and RNA-binding, playing leading role in movement of virus.TGBp2 was conservative which was similar with TGBp2 of potato virus X. TGBp2 and TGBp3 were structural proteins, combining with the endoplasmic reticuLum to play supplementary role in virus movement. TGBpl hadn’t transmembrane domain. TGBp2 had two transmembrane domains and TGBp3 had one. Three proteins didn’t have curly spiral structure. The three-dimensional structure model of the proteins were built.TGB1 was inserted into pET-28a(+) vector for prokaryotic expression, and induced the expression of TGBpl with IPTG. The successfuL expression of TGBp1 with His label was demonstrated SDS-PAGE and Western-Blot. TGBpl purified from mycoprotein successfuLly by the principle of affinity chromatography.To study the three proteins in onion cells using GFP fusion gene technique,we connected the three target genes with eGFP, and then inserted into pTF101.1-35S. Agrobacterium strain of GV3101 was transformed with the vectors for onion epidermal cells infections. We founded that TGBpl-GFP mainly located in the cell walls and small amount located in the cytoplasm. TGBp2-GFP and TGBp3-GFP mainly located in cytoplasm and internal.