Identificação de proteínas e validação de genes envolvidos na resistência de Brassica oleracea a Xanthomonas campestris pv. campestris

Abstract

Gene function determination, among at the confirmation of genes involvement in certain biological condition, is still a post-genomic era challenge, being that the proteomic analysis is a powerful technique for this purpose. Cellular organelles enrichment can also contribute for the less abundant proteins identification in the complex plant samples analysis. In the present study, young leaves of Brassica oleracea var. capitata (cabbage) of moderately resistant (Astrus Plus) and susceptible (Veloce) cultivars ware infiltrated with Xanthomonas campestris pv. campestris, (NaCl 0.85% and Xcc OD600 = 0.6 solution) and collected 24 h after infection. For the control condition, the both cultivars leaves were infiltrated with NaCl 0.85% solution. Proteins from leaf extract samples and chloroplasts were extracted with phenol and precipitated with ammonium acetate in methanol. The samples were analyzed by LC-MS/MS and the generated spectra were submitted to software’s Progenesis QI and PEAKS7® for protein identification and quantification. After the comparison of the resistant inoculated cultivar with the control (RI:RC) resulted in 2680 identified proteins, while 2686 proteins were found in the susceptible inoculated cultivar compared to the control (SI:SC). More than 300 differentially abundant proteins, in both cultivars, were identified. The chloroplast enrichment contributed with more than 600 proteins in RI:RC and more than 900 in SI:SC, not identified in leaf extract samples. We further analyzed 30 genes by RTq-PCR in both cultivars. In resistant cultivar, 9 genes had differential expression level statistically significant when compared with control (7 up-regulated and 2 down-regulates), while the susceptible cultivar showed 11 validated genes (10 up-regulated and 3 downregulated). Among the differentially expressed genes, with statistical significance, evaluated by RTq-PCR, three genes, potentially involved in plant defense were choose for functional validation by genic overexpression in model plant. The genes, BoCHB4, BoESP e BoRGP1, expressed in Arabidopsis, were validated and showed up-expression in transgenic plants in RTq-PCR analysis (578, 22,000 and 7.5-fold, respectively), when compared with non-transformed plants (wild type, WT). This work besides of to contribute for protein database enrichment and identified proteins of stress adaptation by the plant provide information of potential genes candidates in genetic enhancement programs.