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Molecular cloning and expression profiling of a chalcone synthase gene from hairy root cultures of Scutellaria viscidula Bunge.

ABSTRACT: A cDNA encoding chalcone synthase (CHS), the key enzyme in flavonoid biosynthesis, was isolated from hairy root cultures of Scutellaria viscidula Bunge by rapid amplification of cDNA ends (RACE). The full-length cDNA of S. viscidula CHS, designated as Svchs (GenBank accession no. EU386767), was 1649 bp with a 1170 bp open reading frame (ORF) that corresponded to a deduced protein of 390 amino acid residues, a calculated molecular mass of 42.56 kDa and a theoretical isoelectric point (pI) of 5.79. Multiple sequence alignments showed that SvCHS shared high homology with CHS from other plants. Functional analysis in silico indicated that SvCHS was a hydrophilic protein most likely associated with intermediate metabolism. The active sites of the malonyl-CoA binding motif, coumaroyl pocket and cyclization pocket in CHS of Medicago sativa were also found in SvCHS. Molecular modeling indicated that the secondary structure of SvCHS contained mainly ?-helixes and random coils. Phylogenetic analysis showed that SvCHS was most closely related to CHS from Scutellaria baicalensis. In agreement with its function as an elicitor-responsive gene, the expression of Svchs was induced and coordinated by methyl jasmonate. To our knowledge, this is the first report to describe the isolation and expression of a gene from S. viscidula.

SUBMITTER: Lei W

PROVIDER: S-EPMC3036846 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Molecular cloning and expression profiling of a chalcone synthase gene from hairy root cultures of Scutellaria viscidula Bunge.

Lei Wei W Tang Shao-Hu SH Luo Ke-Ming KM Sun Min M

Genetics and molecular biology 20100401 2

A cDNA encoding chalcone synthase (CHS), the key enzyme in flavonoid biosynthesis, was isolated from hairy root cultures of Scutellaria viscidula Bunge by rapid amplification of cDNA ends (RACE). The full-length cDNA of S. viscidula CHS, designated as Svchs (GenBank accession no. EU386767), was 1649 bp with a 1170 bp open reading frame (ORF) that corresponded to a deduced protein of 390 amino acid residues, a calculated molecular mass of 42.56 kDa and a theoretical isoelectric point (pI) of 5.79 ...[more]

PMID: 21637484

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Project description:BACKGROUND: Hairy root cultures produced via Agrobacterium rhizogenes-mediated transformation have emerged as practical biological models to elucidate the biosynthesis of specialized metabolites. To effectively understand the expression patterns of the genes involved in the metabolic pathways of these compounds, reference genes need to be systematically validated under specific experimental conditions as established by the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. In the present report we describe the first validation of reference genes for RT-qPCR in hairy root cultures of peanut which produce stilbenoids upon elicitor treatments. RESULTS: A total of 21 candidate reference genes were evaluated. Nineteen genes were selected based on previous qPCR studies in plants and two were from the T-DNAs transferred from A. rhizogenes. Nucleotide sequences of peanut candidate genes were obtained using their homologous sequences in Arabidopsis. To identify the suitable primers, calibration curves were obtained for each candidate reference gene. After data analysis, 12 candidate genes meeting standard efficiency criteria were selected. The expression stability of these genes was analyzed using geNorm and NormFinder algorithms and a ranking was established based on expression stability of the genes. Candidate reference gene expression was shown to have less variation in methyl jasmonate (MeJA) treated root cultures than those treated with sodium acetate (NaOAc). CONCLUSIONS: This work constitutes the first effort to validate reference genes for RT-qPCR in hairy roots. While these genes were selected under conditions of NaOAc and MeJA treatment, we anticipate these genes to provide good targets for reference genes for hairy roots under a variety of stress conditions. The lead reference genes were a gene encoding for a TATA box binding protein (TBP2) and a gene encoding a ribosomal protein (RPL8C). A commonly used reference gene GAPDH showed low stability of expression suggesting that its use may lead to inaccurate gene expression profiles when used for data normalization in stress-stimulated hairy roots. Likewise the A. rhizogenes transgene rolC showed less expression stability than GAPDH. This study proposes that a minimum of two reference genes should be used for a normalization procedure in gene expression profiling using elicited hairy roots.

Project description:Human papillomavirus (HPV) tumor disease is a critical public health problem worldwide, especially in the developing countries. The recognized pathogenic function of E5, E6, and E7 oncoproteins offers the opportunity to devise therapeutic vaccines based on engineered recombinant proteins. The potential of plants to manufacture engineered compounds for pharmaceutical purposes, from small to complex protein molecules, allows the expression of HPV antigens and, possibly, the regulation of immune functions to develop very specific therapies as a reinforcement to available nonspecific therapies and preventive vaccination also in developed countries. Among plant-based expression formats, hairy root cultures are a robust platform combining the benefits of eukaryotic plant-based bioreactors, with those typical of cell cultures. In this work, to devise an experimental therapeutic vaccine against HPV, hairy root cultures were used to express a harmless form of the HPV type 16 E7 protein (E7*) fused to SAPKQ, a noncytotoxic form of the saporin protein from Saponaria officinalis, that we had shown to improve E7-specific cell-mediated responses as a fusion E7*-SAPKQ DNA vaccine. Hairy root clones expressing the E7*-SAPKQ candidate vaccine were obtained upon infection of leaf explants of Solanum lycopersicum using a recombinant plant expression vector. Yield was approximately 35.5 μg/g of fresh weight. Mouse immunization with vaccine-containing crude extracts was performed together with immunological and biological tests to investigate immune responses and anticancer activity, respectively. Animals were primed with either E7*-SAPKQ DNA-based vaccine or E7*-SAPKQ root extract-based vaccine and boosted with the same (homologous schedule) or with the other vaccine preparation (heterologous schedule) in the context of TC-1 experimental mouse model of HPV-associated tumor. All the formulations exhibited an immunological response associated to anticancer activity. In particular, DNA as prime and hairy root extract as boost demonstrated the highest efficacy. This work, based on the development of low-cost technologies, highlights the suitability of hairy root cultures as possible biofactories of therapeutic HPV vaccines and underlines the importance of the synergic combination of treatment modalities for future developments in this field.

Dataset Information

Molecular cloning and expression profiling of a chalcone synthase gene from hairy root cultures of Scutellaria viscidula Bunge.

Publications

Molecular cloning and expression profiling of a chalcone synthase gene from hairy root cultures of Scutellaria viscidula Bunge.

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