title: Structural and functional characterisation of a class I endochitinase of the carnivorous sundew (Drosera rotundifolia L.).
abstract: Chitinase gene from the carnivorous plant, Drosera rotundifolia , was cloned and functionally characterised. Plant chitinases are believed to play an important role in the developmental and physiological processes and in responses to biotic and abiotic stress. In addition, there is growing evidence that carnivorous plants can use them to digest insect prey. In this study, a full-length genomic clone consisting of the 1665-bp chitinase gene (gDrChit) and adjacent promoter region of the 698 bp in length were isolated from Drosera rotundifolia L. using degenerate PCR and a genome-walking approach. The corresponding coding sequence of chitinase gene (DrChit) was obtained following RNA isolation from the leaves of aseptically grown in vitro plants, cDNA synthesis with a gene-specific primer and PCR amplification. The open reading frame of cDNA clone consisted of 978 nucleotides and encoded 325 amino acid residues. Sequence analysis indicated that DrChit belongs to the class I group of plant chitinases. Phylogenetic analysis within the Caryophyllales class I chitinases demonstrated a significant evolutionary relatedness of DrChit with clade Ib, which contains the extracellular orthologues that play a role in carnivory. Comparative expression analysis revealed that the DrChit is expressed predominantly in tentacles and is up-regulated by treatment with inducers that mimick insect prey. Enzymatic activity of rDrChit protein expressed in Escherichia coli was confirmed and purified protein exhibited a long oligomer-specific endochitinase activity on glycol-chitin and FITC-chitin. The isolation and expression profile of a chitinase gene from D. rotundifolia has not been reported so far. The obtained results support the role of specific chitinases in digestive processes in carnivorous plant species.
authors.affiliation: Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, P.O. Box 39A, 950 07, Nitra, Slovak Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Jopcik
authors.affiliation: Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, P.O. Box 39A, 950 07, Nitra, Slovak Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Moravcikova
authors.affiliation: Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, P.O. Box 39A, 950 07, Nitra, Slovak Republic.
authors.forename: Ildiko
authors.initials: I
authors.lastname: Matusikova
authors.affiliation: NAFC Research Institute for Animal Production, Nitra, Hlohovska 2, 951 41, Lužianky, Slovak Republic.|Department of Botany and Genetics, Faculty of Natural Sciences, Constantine the Philosopher University, Nábrežie mládeže 91, 949 74, Nitra, Slovak Republic.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Bauer
authors.affiliation: Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, P.O. Box 39A, 950 07, Nitra, Slovak Republic.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Rajninec
authors.affiliation: Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, P.O. Box 39A, 950 07, Nitra, Slovak Republic. jana.libantova@savba.sk.
authors.forename: Jana
authors.initials: J
authors.lastname: Libantova
mesh_terms: D000818:Animals; D002688:Chitinases / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D029787:Drosera / Q000201:enzymology* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D007313:Insecta; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism*; D011235:Predatory Behavior; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D002688:Chitinases
keywords: Caryophyllales; Chitinase; Digestive process; Expression profile; Pathogenesis-related proteins; Recombinant protein