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23 Congress of the International Union for Biochemistry and Molecular Biology
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44 Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology
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Foz do Iguaçu, PR, Brazil, August 24 to 28 , 2015
CLONING AND SEQUENCING OF A NEW PROTEIN TRAF-LIKE ISOLATED
FROM BRASSICA OLERACEA VAR. BOTHRYTIS WITH LECTINIC ACTIVITY
Duarte, C.E.M.1; Koehler, A.D.2; Abranches, M.V.1,3; Silva, P.F.1; Oliveira, L.L.1
1
Departamento de Biologia Geral, Centro de Ciências Biológicas e da Saúde,
Universidade Federal de Viçosa, Minas Gerais, Brazil; 2Departamento de Biologia
Vegetal, Instituto de Biotecnologia Aplicada à Agropecuária, Universidade Federal
de Viçosa, Minas Gerais, Brazil; 3Departamento de Nutrição e Saúde, Instituto de
Ciências Biológicas e da Saúde, Universidade Federal de Viçosa, Minas Gerais,
Brazil.
Brassica oleracea is a very morphologically diverse species including common
cabbage, broccoli, kale, kohlrabi, Brussels sprouts, and cauliflower. This species
has become established as an important human food crop plant, used because of
its large food reserves, which are stored over the winter in its leaves. Yet, there
have been no reports on lectins from Brassica. Lectins are a group of
carbohydrate-binding proteins found in virus, bacteria, fungi, plants, and animals,
which are involved in various biological process, such as host defense, cell–cell
interaction, folding of glycoproteins, and other functions. Our research group
recently identified a new protein with lectinic activity isolated from Brassica
oleracea var. Botrytis, called BOL. In order to better characterize this protein its
cDNA and deduced amino acid sequences were determined and analyzed. The
protein was purified in fetuin affinity column and eluted with 1M NaCl. Degenerate
primers 5'-garacncgngcntty-3' were designed from the N-terminal sequence
ETRAFREERPSSKIVTIAG obtained by Edman degradation. Then, B. oleracea
var. Botrytis seeds were germinated in Murashige and Skoog medium. Total RNA
was extracted from seedlings using Trizol reagent and cDNA was synthesized by
M-MLV Reverse Transcriptase with oligodT16N as primer. Subsequent PCR
fragments were sequenced. The deduced polypeptide with 297 amino acids
consisting of two in tandem arrayed MATH-domain. The predict amino acid
sequence of BOL showed no significant sequence identities to other known
lectins. On the other hand, BOL showed 99% sequence identity to a predicted
protein of the Brassica rapa (XM_009113448.1) suggesting that BOL belong to a
TRAF-like family in plants. Native BOL possess carbohydrate-binding activity and
hence can be considered as lectin. This is the first report to describe the
characterization and cDNA cloning of a Brassica lectin. The next step in this study
involve the expression of recombinant BOL for further characterization of their
biological functions.
Acknowledgements: The authors would like to thank the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Fundação de
Amparo à Pesquisa do estado de Minas Gerais (FAPEMIG) for financial support.
Keywords: carbohydrate-binding proteins; cauliflower; MATH-domain.
Brazilian Society for Biochemistry and
Molecular Biology (SBBq)