Estabelecimento de protocolo para genotipagem do mutante Prosystemin overexpressor em tomateiro

Abstract

Plants, as sessile organisms, needs to use hormones to alter their development in the face of the environmental adversities. One of the main hormones produced to activate defense responses is jasmonic acid, which, in plants of the Solanaceae family, is produced through the action of sistemin, a polypeptide derived from a larger precursor called prosistemin. For this reason, transgenic tomato plants that carry the overexpressing prosystemin gene (Prosystemin overexpressor - Pex) present the constitutive production of defense structures. The identification by morphological pathways of plants carrying the Pex gene, however, is hampered by the little evident phenotype of these plants, which limits the application of external analyzes for screening individuals. Therefore, the objective of this work was to establish a protocol for Pex genotyping in Micro-Tom tomato plants, aiming at its use in the rapid detection of this gene in plants present in the laboratory. The first step was to establish a protocol for extracting genomic DNA from tomatoes and to develop primers for genotyping the Pex gene, to then establish a protocol for genotyping the gene in question via PCR. The effectiveness of the DNA extraction protocol with 3% CTAB was confirmed by an agarose gel analysis; among the 5 sets of primers developed, only 3 of them were able to differentiate the genotypes, the Fw3+Fw4+Rv2 primer being chosen for genotyping. Finally, the protocol for Pex gene genotyping via PCR was also effective in differentiating Pex plants from wild plants, thus performing the expected functions.