Ital.J.anIm.ScI. vol. 6, (Suppl. 2), 287-290, 2007 287 vIII World Buffalo congreSS Partial genetic characterization of Stearoyl Coa-Desaturase´s structural region in Bubalus bubalis A.L.F. Lima, A.R. Otaviano, M.M.M. Laureano, P.M. Galeazzi, G. M.F. de Camargo, R.B. Thomazine, H. Tonhati São Paulo State University - F.C.A.V / Unesp / Jaboticabal, Brasil Corresponding author: H. Tonhati. Via de Acesso - Paulo Donato Castellane s.nº, F.C.A.V / Unesp / Jabotica- bal, Estado de São Paulo, Brasil, CEP – 14884-900, Tel. +55 16 3209-2678 - Email: tonhati@fcav.unesp.br ABSTRACT: Conjugated Linoleic Acids (CLAs) comprise a family of positional and geo- metric isomers of linoleic acid. The main form of CLA, cis-9, trans-11-C18:2 show positive effects in cancer prevention and treatment. The major dietary sources of these fatty acids are derived from ruminant animals, in particular dairy products. In these animals, the endogenous synthesis mainly occurs in mammary gland by the action of enzyme Stearoyl CoA Desaturase (SCD). Different levels of expression and activity of SCD in mammary gland can explain partially the variation of CLA levels in fat milk. Considering a great fat concentration in bubaline milk and the benefit of a high and positive correlation between fat milk and CLA production, this study was carried on with the intention of sequencing and characterizing part of the gene that codifies SCD in buffaloes. Genomic DNA was ex- tracted from blood samples of lactating bubaline which begins to the breed Murrah. After the (acho que nao precisa desse the) extractions, PCR (Polymerase Chain Reaction) reac- tions were made by using primers Z43D1 and E143F1. The fragments obtained in PCR were cloned into “T” vectors and transformed in competent cells DH10B line. After this, three samples of each fragment were sequenced from 5’ and 3’ extremities using a BigDye kit in an automatic sequencer. Sequences were edited in a consensus of each fragment and were submitted to BLAST-n / NCBI for similarity comparisions among other species. The sequence obtained with Z43D1 primers shows 938 bp enclosing exons 1 and 2 and intron 1. The primers E143F1 show 70 bp corresponding to exon 3 of bubaline SCD gene. Simi- larities were obtained between 85% and 97% among bubaline sequences and sequences of SCD gene described in human, mouse, rat, swine, bovine, caprine and ovine species. This study has permitted the identification and partial characterization of SCD codifing region in Bubalus bubalis specie. Key words: Milk, CLA, PCR, Genomic DNA sequence. INTRODUCTION - Conjugated linoleic acids comprise a family of positional and geo- metric isomers of a octadecadienoic acid with two double-bonds conjugated by a single bound (Ha et al. 1990). The isomer cis-9, trans-11/C18:2 CLA isomer shows positive effects in the prevention and treatment of many types of cancer, such as mammary gland (Park et al., 2000, Miller et al., 2001), prostate (Palombo et al. 2002) and colon (Miller et al., 2001). The main source of this isomer available to human nutrition are dairy products from ruminants (Parodi et al., 1997). In these animals, the edogenous synthesis occours in mammary gland by the action of a ∆9 enzyme named Stearoyl Coa Desaturase (SCD) (Ke- ating et al. 2006). Considering that different levels of expression and activity of SCD in mammary gland can, partially, explain the variation of CLA levels in fat milk, this study was carried on with the intention of sequencing and characterizing part of the structural region of SCD in buffaloes. MATERIAL AND METHODS - Genomic DNA extractions - A sample of hair’s tails was collected from 5 Murrah breed lactating buffaloes. The genomic DNA was extracted using PCL (phenol-chloroform-isoamilic alcohol/25:24:1) method. After extractions, 2µL of each DNA sample were transfered in a agarose gel (0,8%) stained with Ethidium Bromide to electrophoresis and visualized under UV light in a Gel Logic system (Kodak). PCR reac- tions – Considering the existence of high similarity for SCD gene among ruminants spe- cies, the primers used in this study were designed from Capra hircus SCD complete cDNA sequence described by Bernard et al. (2001). The primers used were: Z4 (forward)> 5´- CAG-CAC-AGC-AGG-TCG-GGT-C –3`; D13 (reverse)> 5´- CCA-TGA-GGA-TGA-TGT- TTC-TCC –3´; and E14 (forward)> 5´- GTC-CAT-GCG-CTG-TGG-AGT-CA –3´; F13 (reverse)> 5´- GTT-GGC-GAT-GAT-GAG-GAA-GAC –3´. PCR were made using 100 ng de DNA, 0.5µM from each primer, 1X PCR buffer [10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl2], 50 mM KCl, 100 µM de dNTPs e 0.5 U of Platinum Taq DNA polymerase, Invitrogen® in a final volume of 25 µl. The amplifying cycles follow the steps: 95 °C for 5 min., 30 cycles (95 °C, 30 seconds; 59 °C, 60 seconds; 72 °C, 60 seconds) and 72 °C, 5 min. After amplifications, 5µL of each PCR sample were transfered in a agarose gel (1,5%) stained with Ethidium Bromide (5ug/ml) to electrophoresis and visualized under UV light in a Gel Logic system (Kodak). Cloning reactions - The PCR products obtained were ligated into plasmids using the kit pGEM®-T-Easy Vector Systems (Promega). After ligation re- actions, the recombinant vectors were inserted by transformation reactions into E. coli DH10B competent cells. After this step, colonies of competent cells were cultivated and the plasmid DNA containing the specific vectors with PCR fragments was identified by white color and extracted as described by Sambrook & Russel (2001). Sequencing reac- tions and edition of sequences: Cloned fragments of SCD gene were analyzed from 5’ and 3’extremes using a BigDye Terminator Cycle Sequence Ready ABI Prism Version 3 kit in an automatic sequencer ABI PRISM 3700 DNA Analyzer. The sequences obtained (3 samples per fragment) were edited in a consensus sequence and analyzed using softwares CodonCode Aligner and CLC Free WorkBench 3 to estimate the exon-intron limits using Staden weight matrix method (WMM). After this analisys the sequences obtained were submitted to BLASTnucleotide-to-nucleotide (NCBI) for verify similarity among buffaloes and other ruminant species. RESULTS AND CONCLUSION - The genomic DNA extractions were effective, allow- ing good results in PCR amplifications. The fragments amplified by primers Z43D1 prim- ers show 938 bp enclosing exons 1 and 2 and intron 1. The primers E143F1 show 70 bp corresponding to exon 3 of bubaline SCD gene. Figure 1 shows the estimative of the struc- tural region evaluated in this study using CLC Free WorkBench 3 software. The FASTA Ital.J.anIm.ScI. vol. 6, (Suppl. 2), 287-290, 2007288 vIII World Buffalo congreSS sequence of nucleotides obtained was described in Figure 2. The analisys in BLASTn shows similarity between 85% and 97% among these sequences of bubaline´s SCD gene and the sequences of SCD gene described in human, mouse, rat, swine, bovine, caprine and ovine species. This study has allowed the identification and partial characterization of SCD codif- ing region in Bubalus bubalis specie and the sequences obtained were included in GenBank (Acession Number: EF122150). Table 1. FASTA sequence of the partial Stearoyl Coa-Desaturasé s structural region in Bubalus bubalis; black boxes are indicating the splicing signal estimated by WMM; black points (.) are indicating the region of intron II (not sequenced). Figure 2. Partial structure of SCD Gene in Bubalus bubalis; arrows are indicating the number of base pairs in each exon and intron, respectively. ACKNOWLEDGMENTS - FAPESP ( Fundação de Amparo à Pesquisa do Estado de São Paulo); CNPq ( Conselho Nacional de Desenvolvimento Científico e Tecnológico). Ital.J.anIm.ScI. vol. 6, (Suppl. 2), 287-290, 2007 289 vIII World Buffalo congreSS REFERENCES - Bernard, L., Leroux, C., Hayes, H., Gautier, M., Chilliard, Y., Martin, P. 2001 Characterization of the caprine stearoyl-CoA desaturase gene and its mRNA showing na unusually long 3´-UTR sequence arising from a single exon., Gene 281 53–61. Ha, Y.C., Storkson, J.M., Pariza, M.W., 1990. Inhibition benzo[a]pyrene-induced mouse forestomach neoplasia by conjugated dienoic derivatives of linoleic acid. Cancer Res. 50, 1097–1101. Keating AF, Kennelly JJ, Zhao FQ. Characterization and regulation of the bovine stearoyl- CoA desaturase gene promoter.Biochem Biophys Res Commun. 2006 May 26;344(1):233-40. 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