Análise in silico e expressão gênica de candidatos da família das aquaporinas em Coffea canephora submetidos ao estresse hídrico

Abstract

Aquaporins are proteins that belongs to a family of MIPs (Major Intrinsic Proteins) that are inserted into the plasmatic membrane forming channels that act directly in the transport of water and other essential solutes for the development and growth of plants. Genes of this family are the key to the study of the plants response of water stress. The objective of this work was to characterize in silico of the aquaporin family in Coffea Canephora and evaluate the relative expression of CcAQPs members in two contrasting of C. canephora genotypes submitted to water deficit, therefore, clone 14 is tolerant and clone 109A is susceptible to drought. The Coffee Genome Hub platform was used to search for the sequences of aquaporins in C. canephora. The sequences were analyzed regarding its structure, location, similarity, presence of domains and conserved motifs and phylogenetic relationship with other species. Were identified 33 aquaporins in C. canephora, subdivided into five families: PIPs, TIPs, NIPs, SIPs and XIPs. The size of the proteins varied between 118 to 327 amino acids. The molecular weight varied between 12364.41 to 35109.04 Mw and the isoelectric point varied between 5.12 to 9.66 pI. The aquaporins of C. canephora are located on the cell membrane and in the vacuole or simultaneously in both. The prediction of the aromatic / arginine selectivity filter (ar / R) Froger’s positions and specificity showed a noticeable difference in substrate specificity between subfamilies. The expression in silico varied between the members of each subfamily according to the tissue analyzed. In general, members of the PIP and TIP subfamilies showed higher levels of expression. The plants were subjected to four treatments: no stress (ψ - 1.34 MPa), moderate stress (ψ - 2.39 MPa), severe stress (ψ - 4.5 MPa) and re-irrigated treatment until the potential similar to the no stress treatment. The RNA was extracted and converted from the leaves to cDNA. Primers were designed to the members CcPIPs1;2, 2;3, 2;4 e CcTIPs1;2, 2;1. The quantification of the relative expression for each gene was performed by RTqPCR calculated by the ΔCt method using the UBIQ10 gene as a normalizer. The water deficit affected the relative expression of the CcPIPs and CcTIPs isoforms analyzed in C. canephora leaves. The relative expression of all isoforms decreased in the clone 14 while increased in the clone 109A when the plants were subjected to severe stress. This study was the first to characterize in silico genes of the aquaporin family and to analyze the relative expression of members of the subfamilies PIP and TIP in C. canephora.