Biodegradação de ácido 2,4 - Diclorofenoxiacético por burkholderia sp

Abstract

Bacteria of the genus Burkholderia have the capacity to degrade various toxic and recalcitrant compounds. Based on the foregoing, this study used real-time PCR (qRT-PCR) and two-dimensional electrophoresis (2DE) to investigate the gene expression profiles tfdB related to biodegradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and differentially expressed proteins qualitative and qualitative, respectively in the presence of 2,4-D Burkholderia sp. SMF07. In order to make the extraction of RNA and protein total bacterial growth was carried out in two TY culture media (Tryptone-Yeast Medium), a control and the other being supplemented with 1 mg / mL 2,4-D, both were incubated at 28 ° C. RNA extraction and total proteins was done after addition of the pollutant in time of 15 minutes, 1, 2, 3 and 5 hours and during the log phase bacterial growth, respectively. The quality of total RNA was measured by the integrity of the fragments of ribosomal RNA (rRNA) checked by electrophoresis on 1.2% agarose gel and the ratio between the absorbances at 260 nm and 280 nm. For quantitative and qualitative analysis of proteins, the method of Bradford and SDS-PAGE, respectively. The synthesis of complementary DNA (cDNA) was determined from the total RNA using the enzyme reverse transcriptase (Superscript III, Invitrogen) using random primers and 6-mer (Invitrogen). Relative expression of the gene was performed using tfdB qRT Power-PCR using SYBR® Green Master Mix (Applied Biosystems) and using the 16S rRNA gene as housekeeping. The synthesized cDNA was used as template DNA. Comparative analysis of qRT-PCR was performed by 2-ΔΔCT method. The total proteins extracted from the two-dimensional map was determined for each reference condition. The adjustment of the two-dimensional images of the gels, the detection of protein spots and evaluate data to determine quantitative and qualitative variations, molecular mass (MM) and isoelectric point (pI) of the spots was done by the program ImageMaster®. Proteins differentially expressed quantitatively and qualitatively in the presence of 2,4-D were identified using the values of pI and MM of the spot against a database of proteins available in UniProt ExPASy server. According to this work, the gene expression tfdB significantly increased 200% over the control after one hour of induction of the abovementioned herbicides. The mean number of spots of replication of protein was 836 2DE gel (control) and 803 (treatment). The greater abundance of proteins was observed in the 2DE gels in pH range 5-6 with MM between 20 and 40 quilodalton (kDa). It was verified in 2DE gels differentially expressed proteins quantitatively and qualitatively to the stress condition in response to bacterial 2,4-D compared to control. Most of these identified proteins belong to molecular function: hydrolase or transferase, expressed different quantitative and qualitative. For these reasons, the strain of Burkholderia sp. SMF07 introduced tfdB also the gene expression level was increased and it was possible to detect differentially expressed proteins qualitatively and quantitatively to grow in medium containing 2,4-D, indicating the importance of these bacteria on the biodegradation of the pollutant. In accordance with these data, we conclude that strain Burkholderia sp. SMF07 tfdB introduced gene, which demonstrated high expression in a medium containing 2,4-D. Adicionadamente, were differentially expressed proteins in medium supplemented above, indicating the importance of these bacteria as a biotechnological tool for the biodegradation potential of this pollutant