ABSTRACT
We constructed a His-tagged prokaryotic expression vector of WUSCHEL gene of Arabidopsis thaliana, pET-31b(+)-WUS-His(6). The induction condition of the fusion protein expression in Escherichia coli was optimized. After purified by affinity chromatography, the recombinant WUS protein was resolved by renaturation of gradient urea dialysis, then used as antigen to immune rabbit to prepare polyclonal antibody. The rabbit anti-WUS antibody titer and specificity were analyzed and confirmed by agarose immunodiffusion testing; the antiserum sensitivity was assayed by dot blot and Western blotting. The results showed that the A. thaliana WUS prokaryotic expression vector was successfully constructed, and the optimized protein expression induction condition in E. coli was 0.5 mmol/L IPTG (isopropy-beta-D-thiogalactoside) at 28 degrees C for 10 hours. The purity of the affinity purified protein was higher than 96%, and the prepared polyclonal antibody was with high specificity and sensitivity, it was able to detect protein antigen at ng level.
Subject(s)
Animals , Rabbits , Amino Acid Sequence , Antibodies , Metabolism , Arabidopsis , Genetics , Arabidopsis Proteins , Genetics , Chromatography, Affinity , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Genetics , Homeodomain Proteins , Genetics , Molecular Sequence Data , Plant Proteins , Genetics , Recombinant Fusion Proteins , Genetics , Allergy and ImmunologyABSTRACT
The natural isoflavones biosynthetic pathway is only limited in legumes plant. To study the isoflavone in bacteria by metabolic engineering requires transformation of multi-gene of the whole pathway into the host strain to resembling the expression and metabolism of the genes. The multi-gene transformation and expression strategy become necessary because of this. This article talks about the multi-gene transformation strategy using one or many vectors, taking the five genes of isoflavonoid biosynthetic pathway to E. coli. The recombinant bacteria carry five genes with two vectors, the whole Isoflavonoid biosynthetic pathway was constructed into E. coli. Fermented with L-tyrosine as substrate and IPTG as an inducer, the recombinant bacteria can produce a new isoflavone related metabolite showed on HPLC analysis profile.
Subject(s)
Amino Acid Sequence , Base Sequence , Biosynthetic Pathways , Genetics , Enzymes , Genetics , Escherichia coli , Genetics , Metabolism , Genetic Engineering , Methods , Isoflavones , Metabolism , Molecular Sequence Data , Oxygenases , Genetics , Glycine max , Chemistry , Transformation, BacterialABSTRACT
Hormones are the main materials which regulate the plant growth and the secondary metabolites formation of plant. The effects of hormones on cell biomass and metabolites content and the application progress of honrmones in plant cell suspension culture is reviewed. It covers the influence of exogenous hormones’ categories, concentration and proportion on cell biomass and metabolites content in plant cell suspension culture, the developmental course of the endogenous hormones determination, the changes of endogenous hormones content and its effects on the cell biomass and metabolites content, the relation of interaction between exogenous hormones and endogenous hormones, the study on the effect of new hormone varieties on the culture.