Genetic expression by real time PCR of bgl, bglA, CspA, GyrB and 16S DNA genes

HÉCTOR A. CRISTÓBAL; RAMIRO POMA; VERÓNICA RAJAL; CARLOS M ABATE

Villa Carlos Paz, Cordoba

Congreso; Sociedad Argentina de Bioloía Molecular y Bioquímica (SAIB); 2008

SAIB

Shewanella sp. G5 is a psychrotolerant bacteria, Gram negative and ¥â-glucosidases (BGs) producers. This marine bacteria was isolate from the intestinal content of Munida subrrugosa in the Beagle Channel, Tierra del Fuego (Argentina); and grown between 4 and 37 ºC using cellobiose as carbon source. BGs are a heterogeneous group of enzymes with a broad substrate specificity range over different B-glucosides. The genes (bglA and bgl) that encoding of two ¥âGs, were characterized molecularly in previous studies. The primers design, RNA extraction, synthesis of cDNA and real time PCR assay using SYBRGreen were performed. The parameters assays to each condition were: temperature (10 and 30 ºC), carbon source (cellobiose and glucose) and culture media. bglA, bgl, CspA, gyrB and ADNr 16S were quantified and expressed as relative expression genetic from of each assays using 2-¥Ä¥ÄCt method. Positives results were obtained for all genes during the optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-¥Ä¥ÄCt method were carried out with the housekeeping gene (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 26,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose.