Clonagem molecular de genes de Vigna Unguiculata em vetores de entrada e expressão

Abstract

The cowpea, Vigna unguiculata (L.) Walp, has been the focus of studies at the Plant Cell Signaling Laboratory (LabSin) due to its significance as a legume providing a valuable source of proteins. Additionally, its high annual production in Brazil makes it crucial for the country's economy. However, various abiotic and biotic stresses adversely affect its productivity. In response to these challenges, plants have developed physiological and molecular adaptations to protect themselves against stress. Previous studies with V. unguiculata have revealed that the homologous protein to peroxisomal glycolate oxidase (GOX1-like), apoptosis and chromatin condensation inducer (ACINUS), and the enzyme catalase had their genes differentially expressed in response to stresses, indicating their importance in plant defense. The SAP domain present in ACINUS has been shown to be necessary for apoptosis events and is consequently involved in the defense process. Therefore, the investigation of these genes is of great relevance. In the initial stage, competent cells were prepared from the strains DH10B, TOP10 f’, and DB3.1. The DB 3.1 strain underwent thermal shock transformation with the Gateway vector pDEST17 for its amplification. Additionally, the cloning protocols for the genes VuGOX1-like, in the Gateway expression vector pDEST17, VuSAP, and VuCATC-like, in the entry vector pGEM-T easy, were executed. The competency result for the DH10B, TOP10f’, and DB3.1 strains was 2.47 x 105 cfu/μg, 7.9 x 104 cfu/μg, and 4.61 x 105 cfu/μg, respectively. The amplification of the pDEST17 vector was completed and confirmed with restriction enzyme reactions. The molecular cloning of the VuGOX1-like gene in pDEST17 was not successful, emphasizing the crucial need to obtain cells with higher competency. On the other hand, our results indicate that the cloning of VuSAP in the entry vector pGEM-T easy was completed in practically all extractions, but confirmation through restriction enzyme reactions is needed. In the samples of VuCATC-like, positive results were not obtained, requiring a better evaluation of possible bottlenecks in the process. The cloned genes can be transferred to expression vectors for the study of the gene product.