Cloning、Prokaryotic Expression And Subcellular Localization Of 16kDa Protein Of Lily Symptomless Virus

Lily symptomless virus is belonged to carlavirus. Lily symptomless virus (LSV) is one of the most common viruses infecting lily plants. The mode of transmission is aphid by unsustainable way or cotton aphid by sustainable way. The shape of lily symptomless virus is fibrous virus particles. The length of it is 640nm and the width of it is 17-18nm. The genome of lily symptomless virus is single-stranded RNA molecules, meanwhile, there is a PolyA tail in 3’terminal and a cap in 5’terminal. There are six open reading frames (ORF) and are coded four proteins RNA depend RNA polymerase (RdRp), three genetic box of protein(TGBp1～3), capsid protein (CP) and 16kDa protein of unkown function, respectively. This research is main centred on the 16kDa protein of unkown function of 3’terminal. The LSV-16kDa protein is a nucleotide binding protein. It is playing a important role in virus transcription and replication. It is a important role to study the basic physicochemical properties and structure characteristic of LSV-16kDa protein to illustrate the pathogenesis of LSV.It is cloned the LSV-16kDa gene by RT-PCR. The complete cDNA sequence of LSV-16kDa gene (ADO34176.1) is consisted of 423 nucleotides. To study the phylogenetic relationship of LSV-16kDa protein, compared with the known five different isolates, LSV-16kDa are the similarities around 97-99% and 89-99% between the DNA and the amino acids level. Phylogenetic analysis indicated that LSV-16kDa was closely related to the India-16kDa isolate, demonstrating it may be the same source of two species. Furthermore, we have study the same genus and belong to different species of LSV. We select the 12 species carlaviruses amino acid sequences similar with 16kDa protein amino acid sequence from GeneBank sequence database. Phylogenetic analysis indicated that 16kDa-DL was closely related to the Lily latent virus (LLV)-16kDa of the genus Carlavirus.The bioinformatics analysis of physicochemical property are aiming at LSV-16kDa protein of unknow function:the isoelectric point of 16kDa protein is 9.89, the molecular weight of 16kDa protein is 16194.8, negatively charged residues is 11, positively charged residues is 24, atomic number of 16kDa protein is 2288. LSV-16kDa is a strong hydrophilic protein. The major secondary structures of LSV-16kDa protein are two alpha helix and three extended strands. It is not a secretory protein and isn’t the structure of signal peptide. There is a protein superfamily Carla_C4. The conserved domain of Carla_C4 is located in 28～117 amino acids of 16kDa protein. The whole sequence of 91 amino acids, bit score:109.39, E-value:2.02e-31. It is a hydrophilic protein by hydrophobic analysis. The coils of which mainly occurs in the C-, N-termini and between the two helices. I-TASSER was predicted the tertiary structure of LSV-16kDa obtaining some conserved domains.We are used IPTG to induct expression and detect by SDS-PAGE. There is obvious specific bands in objective microbial, the size is 45kDa(contain the size of GST tag is 29kDa). It is illustrated that the expression of obvious of 16kDa protein. The best conditions of prokaryotic expression is 30℃, the concentration of IPTG is 0.1-0.5mmol/L, inducing 6h. The solubility form of GST-16kDa protein is more, the inclusion body is less. The molecular weight of GST-16kDa protein is 45kDa (GST tag is 29kDa). The concentration of protein is 0.65 mg/mL by Bradford. This result is the basis of antibody preparation, the analysis of protein function, the pathogensis mechanism of LSV. In addition, the protein is rich in cysteine, may be a pathogenic factor.We are used the bioinformatics software to analysis the subcellular localization of LSV-16kDa protein. It is used the method of instantaneous infection and fluorescence method to observe the fusion expression of 16kDa protein and GFP protein in onion epidermal cells. The result is mainly in nucleus and is evenly distributed in the nucleus.