Desenho de primers para estudo de expressão gênica, via PCR em tempo real, em mamona (Ricinus communis L.)

Abstract

The species Ricinus communis L., known as castor bean has a high industrial interest because of the oil extracted from its seeds. Diseases such as gray mold, macrophomine rot, botryodiplodia rot fusarium wilt, among others, are diseases that affect the crop, interfering with its productivity. Thus, the objective of this work was to design primer pairs for castor bean resistance genes aiming at the study of gene expression for pathogen resistance via real time PCR. Oligonucleotides (primers) were designed using castor bean ESTs (expressed sequence markers) involved in pathogen resistance metabolic pathways. The sequences were retrieved through the NCBI database. Primer pairs were designed using Primer3 software and their quality measured by NetPrime software. For standardization of PCR reactions, the following criteria were used in primer selection: size ranging from 20 to 24 bp (base pairs); percentage of GC (Guanine and Cytosine) in the range of 40 to 60%; and meltin temperature between 60 and 63oC. Thus, it was possible to design 100 primer pairs that flank the ESTs regions for castor disease resistance. Presenting an average size of 22 nucleotides, flanking regions to be amplified, via PCR, with an average size of 199 bp. Also, they had Tm averages of 60.1° C and GC concentration of 47.1%. The average specificity was 94.3%. In addition, 8 primer pairs were synthesized for identification of the RPM1 gene, 5 for RPS2, 6 for the TMV N gene, 36 for identification of genes encoding leucine repeating rich proteins, 2 for RPS5 and 3 for the RPP8. The primer pairs developed in this work are of great contribution to differential gene expression studies, related to plant-pathogen interaction in castor bean.