ABSTRACT
In order to identify a promoter system for high-level expression of transgenes in hairy roots, we characterized the chimeric super-promoter fused to the translational enhancer from tobacco etch virus (TEV). Transgenic tobacco plants and hairy roots were generated with the super-promoter:TEV sequence and a modified green fluorescence protein (mGFP5) as a reporter gene. To exploit the utility of hairy root cultures as a secretion-based expression system, the signal peptide of patatin was fused to mGFP5 to direct its secretion into the culture medium. Levels of mGFP5 RNA were on average sixfold higher in hairy roots than leaves. Likewise, GFP protein levels per gram of fresh weight were at least tenfold higher in hairy roots than leaves. Furthermore, more than 10% of the recombinant protein produced in the hairy root culture system was found in the medium. Immunoblotting with anti-GFP antibodies showed two products of 27.1 and 29.9 kDa in all leaf and hairy root tissue extracts, whereas a single 27.1-kDa product was detected in the medium. Inducibility of the promoter was studied with mature leaves and 14-day (midlog phase) hairy roots. A twofold increase in mRNA levels was found immediately after wounding in both mature leaves and hairy roots, with a corresponding increase in mGFP5 protein after 24 h. Our studies demonstrate the utility of the super-promoter:TEV system for high-level expression of recombinant proteins in hairy root bioreactors.
Subject(s)
Bioreactors , Gene Expression Regulation, Plant/physiology , Nicotiana/metabolism , Plant Roots/metabolism , Promoter Regions, Genetic/genetics , Recombinant Proteins/biosynthesis , Amino Acid Sequence , Base Sequence , Blotting, Northern , Blotting, Southern , Blotting, Western , Carboxylic Ester Hydrolases/genetics , Carboxylic Ester Hydrolases/metabolism , DNA Primers/genetics , Enhancer Elements, Genetic/genetics , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation, Plant/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Molecular Sequence Data , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/genetics , Polymerase Chain Reaction , Potyvirus/genetics , Recombinant Proteins/metabolism , Nicotiana/genetics , Transgenes/geneticsABSTRACT
Nicotine biosynthesis in Nicotiana tabacum is under genetic control by the A and B loci. Plants containing semi-dominant mutations at both the A and B loci (i.e. aabb genotype) have lower nicotine levels, reduced nicotine biosynthetic enzyme activities, and reduced mRNA levels of the corresponding biosynthetic genes. The A and B loci therefore appear to be coordinate regulators of several nicotine biosynthetic genes and define a group of co-regulated genes called the A-B regulon. To investigate the composition of genes in the A-B regulon, a fluorescent differential display (FDD) screen was used to randomly sample the transcriptomes of wild type and mutant aabb roots. This resulted in the isolation of 64 FDD clones, representing 49 unique genes or gene families. Four genes associated with nicotine biosynthesis were identified, whereas most of the other FDD clones were homologous with an assortment of stress response genes. Thirty-three genes or gene families showed reproducible aabb genotype effects, representing seven distinct mRNA expression patterns in response media treatments that increase the mRNA levels of known alkaloid biosynthetic genes. Thus, the A and B loci regulate the mRNA levels of some target genes differently than others. Eleven genes or gene families showed only treatment-specific effects, representing four mRNA accumulation patterns. These results indicate the A-B regulon is complex network of differentially expressed stress response genes, only a small subset of which are involved in nicotine biosynthesis.
Subject(s)
Gene Expression Regulation, Plant/genetics , Genes, Regulator/genetics , Nicotiana/genetics , Nicotine/biosynthesis , Acetates/pharmacology , Alkaloids/biosynthesis , Base Sequence , Cyclopentanes/pharmacology , Gene Expression Regulation, Plant/drug effects , Genes, Plant/genetics , Genotype , Molecular Sequence Data , Mutation , Oxylipins , Plant Growth Regulators/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Sequence Homology, Nucleic Acid , Nicotiana/drug effects , Nicotiana/metabolismABSTRACT
Ricin B (RTB), the lectin subunit of ricin, shows promise as an effective mucosal adjuvant and carrier for use in humans. In order to obtain a recombinant plant source of RTB that is devoid of the toxic ricin A subunit, we expressed RTB in Nicotiana tabacum. RTB was engineered with an N-terminal hexahistidine tag (His-RTB), which may affect protein stability. Lactose-affinity purification of His-RTB from leaves yielded three major glycosylated products of 32, 33.5 and 35 kDa. Their identity as RTB was verified by mass spectrometry and immunoblotting with anti-ricin antibodies. Functionality of His-RTB was confirmed by binding to asialofetuin, lactose and galactose.
