| The BTR1 protein is a protein that binds to tomato mosaic virus(ToMV)genomic RNA in Arabidopsis thaliana and inhibits the amplification of this virus.BTR1 consists of 334 amino acid residues harbored three heterogeneous nuclear ribonucleoprotein K-homology RNA binding domain(KH domain),which sequence-specifically bind to single-stranded nucleic acids.The BTR1 gene produce two splice variant mRNAs encoding the different protein products BTR1 L and BTR1 S respectively,mainly expressing the BTR1 S protein in Arabidopsis thaliana.The mRNA of BTR1 s can be expressed in the tissues of leaves,stems and fruit of Arabidopsis thaliana.The genomic RNA of ToMV is 6384 nts in length and harbors 5'UTR(untranslated region)of 71 nts and 3'UTR of 202 nts.BTR1 S preferentially and directly binds to the 5'UTR of ToMV genomic RNA.The knockout and overexpression of BTR1 resulted in an increase and decrease in ToMV multiplication in Arabidopsis thaliana,but in protoplasts,this effect is difficult to detect.And this effect is specific.So far,the study of BTR1 protein is very rare,the crystal structure and functional mechanism of the protein has not been reported,and the underlying mechanism of BTR1 protein on the negative effects to ToMV diffusion is unknown.In this study,we constructed prokaryotic expression vector of Arabidopsis thaliana BTR1 gene,and then induced prokaryotic expression,and got BTR1 protein by chromatographic purification.The protein homogeneity,aggregation state and binding activity with single-stranded nucleic acid were analyzed.This study provide basic data for further study of the structure and function of BTR1 protein.The main results are as follows:1.We synthesised codon-optimized sequence of Arabidopsis thaliana BTR1 gene and constructed a recombinant plasmid pET-15b(BE-)SUMO-BTR1,then transformed the plasmid into E.coli 2566 induced expression at 18?,finally got BTR1 protein with purity greater than 95%,its molecular weight is about 34 kD.2.The homogeneity and aggregation status of BTR1 protein were analyzed by gel filtration chromatography and dynamic light scattering technique(DLS).The results showed that BTR1 protein has a good homogeneity,and the aggregation state may be dimer,and theprotein is a single state in the solution.This is conducive to the crystallization experiments of this protein.3.The binding activity of BTR1 protein with single-stranded nucleic acid was analyzed by electrophoretic mobility shift assay(EMSA).The results showed that BTR1 can bind to single-stranded DNA and RNA,and can not bind to double-stranded DNA and G4.4.The binding activity of BTR1 protein with single-stranded nucleic acid was further determined by Fluorescence Anisotropy.The results showed that BTR1 can bind to single-stranded DNA and RNA,this results are consistent with the results measured by EMSA.For the same type of ssDNA and ssRNA substrate,binding activity of BTR1 tends to increase with the increase of the chain.BTR1 has a stronger binding activity for ssDNA which the sequence is random than ssDNA which the sequence contains a repeating base.The binding activity of BTR1 with ssRNA is stronger than that with ssDNA.5.Candida albicans Pif1(Capif1)can act as a helicase after impacting telomerase,performing a second round extension,and found that Capif1 has a 3'-5 'exonuclease activity.We transformed the recombinant plasmids into E.coli BL21(DE3)competent cell induced expression at 18 ?,and purified the protein by Ni-NTA nucleophilic column,SP cation column and Superdex 200.We got Capif1 Protein with purity greater than 95%,and its molecular weight is about 79 kD.In this study,we successfully constructed,expressed and purified BTR1 protein with high purity,and clarified its homogeneity and aggregation state,and determined the activity binding with single-stranded nucleic acid.This study laid the foundation for the crystal preparation and structural analysis of BTR1.Purified Capif1 protein with purity greater than95% was used in a series of experiments on its activity. |
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