Bioinformatics Analysis And Heterologous Expression Of Microcystin Transporter MlrD

Eutrophication of water body leads to frequent blooms of cyanobacteria,and the rupture of cyanobacteria cells will produce toxic secondary metabolites,which will endanger human health.Microorganisms play an important role in the degradation of microcystins(MCs),and MCs-degrading bacteria mostly follow the mlr gene cluster(mlr ABCD)mechanism.In the past studies,some achievements have been made on the structure,function and degradation mechanism of Mlr A,Mlr B and Mlr C,but there is little research on MlrD,so the molecular structure and specific catalytic process of MlrD are still unclear.In this paper,we studied the MlrD in Sphingopyxis sp.USTB-05,analyze and predict by bioinformatics means,and conduct homology analysis to construct the molecular structure of MlrD;The subcellular location of MlrD protein was determined by subcellular localization experiment.And the heterologous expression of MlrD in E.coli was studied,which laid the foundation for further research of MlrD in the later period.The main results are as follows:(1)Bioinformatics analysis of MlrD shows that MlrD is a kind of alkaline hydrophobin with good thermal stability,and its subcellular localization is predicted to be cell membrane,which belongs to membrane protein and there is no signal peptide.Homology analysis shows that MlrD belongs to PTR2 protein family and has PTR2 domain.Phylogenetic tree analysis shows that MlrD not only follows the vertical genetic law,but also follows the horizontal genetic law in the evolution process.The three-dimensional molecular structure of MlrD was simulated by homologous modeling.It was found that the structure was mainly composed of12 α-helices,and the conserved residues between the N-terminal and C-terminal domains formed a large reaction gap.(2)The two genes of mlr D and egfp were accurately linked by overlapping PCR amplification,and the fusion gene was successfully constructed into p ET-30a(+)vector containing His tag,and transferred into E.coli BL21(DE3).The recombinant strain USTB05-mlr D-egfp was successfully obtained,and the fusion expression of MlrD and EGFP in E.coli BL21(DE3)was realized.The observation results by laser confocal microscope showed that most of the recombinant bacteria USTB05-mlr D-egfp could observe fluorescence on the cell membrane after induced expression for 3 h,but there was no fluorescence in the cytoplasm,indicating that the expression site of MlrD protein was on the cell membrane.(3)The mlr D gene sequence obtained from Sphingopyxis sp.USTB-05 was amplified by PCR,and the recombinant plasmid p ET-30a(+)-mlr D was constructed by connecting the sequence with the plasmid p ET-30a(+).Finally,the recombinant plasmid was transferred into E coli BL21(DE3),and the recombinant strain USTB-05-mlr D was obtained.USTB-05-mlr D induced by IPTG has a visible protein band at 50 k Da.This experiment only preliminarily established the method of heterologous expression of MlrD,which laid a certain foundation for the follow-up study of heterologous overexpression of MlrD and optimization of systematic fermentation conditions.