Abstract
Key message
A novel Brassica oleracea lectin with two tandem MATH domains in its structure has been characterized. The expression of the lectin is regulated by light and by stimuli related to plant defence response. The coding sequence of the protein has been cloned and a fully functional recombinant protein has been obtained.
Abstract
BOL (Brassica oleracea Lectin) is a novel lectin isolated from Brassica oleracea ssp. Botrytis, composed of a single subunit, non-glycosylated peptide that contains two conserved MATH domains, not previously found in any other characterized lectin. The complete sequence encoding BOL was obtained by PCR using degenerated primers designed according to the N-terminal amino acid sequence and the sequences of various tryptic peptides obtained by the digestion of the purified native protein. The entire cDNA consists of 1053 bp, which encodes a 301-amino acid protein. In silico analysis of the Bol promoter sequence showed several regulatory motifs that could be involved in responses to various stresses, as well as various light regulatory elements. Determination of Bol expression in plant tissues showed higher transcript levels in the leaves than in the stems and the roots. The expression of Bol was regulated by light and by signalling compounds related to stress responses. Specifically, Bol expression was higher in light than in darkness, and was induced by methyl jasmonate, while it decreased in tissues treated with salicylic acid. The full-length BOL coding sequence was cloned, expressed in bacteria, and activity assays were performed to ascertain that the recombinant protein was correctly folded and was able to recognize and bind carbohydrates as the native lectin did. Three-dimensional structure prediction showed that this lectin had a dome-like structure with a closely related topology to l-lectins but with two characteristic MATH domains. Isolation, cloning and recombinant expression of BOL may be helpful to understand its physiological function in the plant and opens the possibility of subsequent investigations about the structural features of this novel lectin.
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Acknowledgements
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 474715/2013-2), by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG APQ-01463-17) and by Ministerio de Economia y Competitividad, Spain (AGL2015-69554-P); CEM Duarte was recipient of a PhD fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, and an ERASMUS+ CeiA3 exchange Grant from the European Union program.
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Duarte, C.E.M., Alamillo, J.M., Koehler, A.D. et al. Molecular and biochemical analyses of a novel lectin with MATH domains from Brassica oleracea. Acta Physiol Plant 42, 79 (2020). https://doi.org/10.1007/s11738-020-03070-8
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DOI: https://doi.org/10.1007/s11738-020-03070-8