| Lily Symptomless Virus(LSV)is a major virus endangering lily family plants,belonging to Carlavirus.This virus only infects lilium plants in a narrow host range.LSV is a single strand plus RNA virus,with a total length of 8394 bp,6 open reading frames,and 4 proteins encoded as RbRp,TGB1-3,CP and 16 kDa.Among them,LSV-16 kDa is a protein whose function is unknown.The literature speculated that it may be a gene of the silencing mechanism,which is related to RNA silencing mechanism.On the basis of previous studies,this paper analyzed the interaction between LSV-16 kDa protein and key proteins in RNA silencing process by methods of bioinformatics and molecular biology,and then clarified the function of LSV-16 kDa protein,laying a foundation for the totally cure of the pathogenic mechanism of LSV.The research results are as follows:By comparison with Blast sequence of LSV-16 kDa in Korea,Japan,Zhejiang province of China and other places.It was found that LSV-16 kDa was highly conservative,with a Caral4 family of nucleic acid binding functional domain,indicating that the function in the genome was very critical.Phosphorylation analysis showed the possibility of phosphorylation regulation,and Glycosylation site analysis showed that LSV-16 kDa had N-glycosylation and O-glycosylation sites.The N-terminal of LSV-16 kDa secondary structure was predicted to be an ?-helix coil ?-helix structure by multiple algorithms.Homologous modeling of the N-terminal domain of LSV-16 kDa is a two ?-helix connected by conserved random coil,which is consistent with the results calculated by multiple algorithms.Positively charged residues of the ?-helix-loop-?-helix structure are located on the two segments of the ?-helix,which can bind to nucleic acids through hydrogen bonds and electrostatic interactions.The leucine zipper structure on the ?-helix can further interact with siRNA through hook domain,suggesting that LSV-16 kDa has the basis functional of silencing suppressor.LSV-16 kDa was used as the material to mutate Positively charged residues at key sites and was named LSV-?16kDa.The purification label was added to LSV-16 kDa and LSV-?16kDa and the termination codon was removed,then the prokaryotic vector was transferred and the expression conditions were optimized to obtain soluble two proteins.Specific analysis by Western Blot showed that the expressed proteins were target proteins,and the target proteins were purified by nickel column chromatography.LSV-16 kDa was fused with green fluorescent protein to restructuring in PBI121 of eukaryotic dual-source expression vector then transformed into Agrobacterium GV3101.We recovered that LSV-16 kDa was located in nucleus by Laser Scanning Microscope,which is the key factor of silencing inhibitor.Physiological tests showed that the POD,SOD and PAL activity of the infected plants were rapidly up-regulated to resist LSV-16 kDa infection.Meanwhile,the expression of disease-course related genes HSP90 and PR1 were up-regulated,and the expression of HSP90 was up-regulated with the infection time.The expression of PR1 decreased with infection time.Studies on the key genes in the RNA silencing mechanism have found that both AGO and DCL were up-regulated,AGO2 was a faster response than AGO1 and AGO4,and DCL2 was a faster response than DCL1 and DCL3.It is speculated that AGO2 and DCL2 were the key pathways interacting with LSV-16 kDa in RNA silencing. |
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