ABSTRACT
Saline stress is one of the primary factors limiting increased rice productivity in the southern region of Brazil. Farming can be affected by salinity that is due to both the origin of the soils as well as the irrigation water. Lipid transfer proteins (LTPs) have many physiological functions, including in the response to saline stress. Therefore, the objective of this study was to quantify the relative expression of 11 genetic isoforms that encode LTP1-type proteins in rice genotypes tolerant and sensitive to saline stress in the vegetative period. When the plants reached development stage V4, alternating irrigation was started with nutritive solution and water containing 150 mM NaCl. The LTP7 gene showed an increase in expression by 13.81-fold after 96 h of stress exposure in the saline-tolerant group, whereas the LTP10 gene expression level was increased by 71.10-fold after 96 h in the saline-sensitive group. The LTP26, LTP23, and LTP18 genes showed increased expression in both genotypes; however, the expression levels and response times were different. Thus, LTP7 and LTP10 showed the highest response to salinity. The LTP18, LTP23, and LTP26 genes were negatively correlated with the response to salinity.
Subject(s)
Antigens, Plant/biosynthesis , Carrier Proteins/biosynthesis , Oryza/genetics , Plant Proteins/biosynthesis , Salinity , Seedlings/genetics , Antigens, Plant/genetics , Brazil , Carrier Proteins/genetics , Gene Expression Regulation, Plant , Genotype , Multigene Family/genetics , Oryza/growth & development , Plant Proteins/genetics , Seedlings/drug effects , Sodium Chloride/toxicity , Stress, PhysiologicalABSTRACT
To obtain accurate and reliable results for the expression of genes of interest using quantitative real-time polymerase chain reaction (RT-qPCR) techniques, it is necessary to normalize the data by comparing them to constitutive genes that exhibit uniform expression levels under experimental conditions. In this study, the stability of expression was evaluated for the following ten candidate reference genes in rice leaves (Oryza sativa L.) from the BRS Bojuru and BRS Ligeirinho genotypes that were subjected to salt stress (150 mM): actin 11 (ACT11), beta-tubulin (ß-TUB), eukaryote elongation factor 1-α (Eef-1), eukaryotic initiation factor 4-α (eIF-4-α), E2 ubiquitin-conjugating enzyme (UBC-E2), ubiquitin 5 (UBQ5), ubiquitin 10 (UBQ10), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TIP41-like, and cyclophilin. The stability of expression for the aforementioned genes was then compared to that of three LTP genes using UBQ10, Eef-1, and eIF-4-α as references. After analyzing the expression levels using analysis of variance tests, the results indicated that UBQ10 was the most stable in all treatments (M = 0.404 and SV = 0.327). Furthermore, the eIF-4-α, TIP41-like, and cyclophilin genes exhibited the highest total coefficient of variation (CV = 269, 169.2, 179.2, respectively), which signifies that they exhibited the least stable expression. The expression levels of each candidate gene (LTP7, LTP10, and LTP13) were in contrast to the reference genes. Therefore, we concluded that UBQ10 is the best reference gene for RT-qPCR reactions under the experimental conditions. The expression analysis of LTP7, LTP10, and LTP13 confirmed the importance of validating reference genes to achieve accurate RT-qPCR results.
Subject(s)
Gene Expression Regulation, Plant , Genes, Plant/genetics , Oryza/genetics , Plant Leaves/genetics , Real-Time Polymerase Chain Reaction/standards , Seedlings/genetics , Real-Time Polymerase Chain Reaction/methods , Reference Standards , Reproducibility of Results , Sodium Chloride/pharmacology , Stress, Physiological/drug effects , Stress, Physiological/geneticsABSTRACT
Maytenus ilicifolia Mart. ex Reis., popularmente conhecida como espinheira-santa, é espécie autóctone pertencente à família Celastraceae, usada para tratamento de úlceras gástricas e gastrites. Devido à importância medicinal, houve aumento no extrativismo das populações naturais, tornando-a uma espécie prioritária para a conservação, a fim de evitar a erosão genética. Buscou-se com este trabalho analisar a diversidade genética de 20 acessos de M. ilicifolia coletados em diferentes localidades no Rio Grande do Sul. Utilizando marcadores moleculares do tipo AFLP, foram testadas oito combinações de primers, que geraram 455 bandas eletroforéticas, 100 por cento polimórficas. As combinações de primers E-ACC/M-CAA, E-ACG/M-CTA, E-ACG/M-CTC apresentaram o maior número de bandas eletroforéticas, 71 cada, totalizando 46,80 por cento do polimorfismo total. Os valores de similaridade genética calculada pelo coeficiente simple matching foram utilizados para gerar o dendrograma de similaridade pelo método UPGMA. Foi obtido alto coeficiente de correlação cofenética (r=0,94), demonstrando elevada representatividade dos dados de similaridade genética no dendrograma. Pela AMOVA verificou-se que 89,33 por cento da diversidade total ocorreram entre indivíduos dentro das populações. A caracterização molecular de acessos de Maytenus ilicifolia por meio de AFLP foi eficiente para identificar diversidade genética. Através da análise de similaridade genética o banco de germoplasma poderia ser composto com os acessos que apresentaram menor similaridade e maior número de alelos, permitindo com que estes fornecessem ampla cobertura do genoma que compõem Maytenus ilicifolia. Os acessos que ficaram agrupados em mesmo cluster e com número reduzido de alelos podem ser descartados deste banco. A diversidade genética intrapopulacional identificada por esse marcador foi muito maior do que aquela entre populações.
Maytenus ilicifolia Mart. ex Reis., popularly known as "espinheira-santa", is an autochthonous species belonging to the Celastraceae family. This species is used to treat ulcers and gastritis. Due to its medicinal importance, the exploitation of natural populations has increased, making the conservation of this species essential to prevent genetic erosion. The aim of this work was to analyze the genetic diversity of 20 M. ilicifolia accessions collected in different localities of Rio Grande do Sul State. Using AFLP-type molecular markers, eight primer combinations were tested, producing 455 electrophoretic profiles, with 100 percent polymorphism. The primer combinations E-ACC/M-CAA, E-ACG/M-CTA and E-ACG/M-CTC presented the largest number of electrophoretic profiles, 71 each, totaling 46.80 percent of the total polymorphism. The values of genetic similarity estimated by Simple Matching Coefficient were used to produce the dendrogram of similarity by the UPGMA method. A high cophenetic correlation coefficient (r = 0.94) was obtained, demonstrating high representativeness of the data of genetic similarity in the dendrogram. Using AMOVA, 89.33 percent of the total diversity were observed among individuals from the same population. The molecular characterization of Maytenus ilicifolia accessions by AFLP allowed the identification of genetic diversity. The genetic similarity analysis indicated that the germplasm bank could be composed of the accessions presenting the lowest similarity and the largest numbers of alleles, providing a comprehensive coverage of Maytenus ilicifolia genome. The accessions that were grouped into one same cluster and with a reduced number of alleles could be disposed of this bank. The genetic diversity identified by this marker within populations was much greater than that between populations.
