The polymorphisms of genes associated with the profile of fatty acids of sheep / Os polimorfismos de genes associados ao perfil de ácidos graxos de ovinos

The present study aimed to evaluate the occurrence of polymorphisms in Diacylglycerol acyltransferase (DGTA-1 and 2), Fatty acid synthase (FASN), Stearoyl-CoA desaturase (SCD) genes and the Thioesterase domain of FASN (TE-FASN) gene that may be related to the lipid profile. In the experiment, a total of 84 sheep from different genetic groups were used. For the evaluation of the polymorphism of the genes, PCR-Single Strand Conformation Polymorphism (SSCP) technique and subsequent sequencing were used. In DGAT-2 gene, four genotypes were identified with the presence of 6 polymorphisms, with two (c.229T> C; c.255T> C) that resulted into the exchange of phenylalanine by leucine. In FASN gene, two genotypes were identified. In TE-FASN gene, three genotypes and 17 polymorphisms were identified. DGAT-1 and SCD genes did not reveal the occurrence of polymorphism. There was difference in relation to C14: 0, C18: 0 fatty acids and Δ9-desaturase C18 for DGAT-2 gene and of C18: 2ω6t for TE-FASN. There were differences among the genetic groups for C10: 0, C12: 0, C17: 0, C18: 2ω6t, C18: 3ω3, C20: 2, total of ω3, ω3/ω6 and atherogenicity index. There is occurrence of polymorphism of DGAT-2 and TE-FASN genes and these should be further studied in sheep since they revealed influence of the genotypes on the fatty acid profile.(AU)

O presente estudo teve o objetivo de avaliar a ocorrência de polimorfismos nos genes Diacilglicerol aciltransferase (DGTA1 e 2), Ácido graxo sintase (FASN), Estearoil-CoA dessaturase (SCD) e o Domínio da tioesterase do gene FASN (TE-FASN), que possam estar relacionados ao perfil lipídico. No experimento, foram utilizados um total de 84 ovinos de diferentes grupos genéticos. Para avaliação do polimorfismo dos genes, foi utilizada a técnica de polimorfismo de conformação de cadeia simples (PCR-SSCP) e posterior sequenciamento. No gene DGAT-2, foram identificados quatro genótipos com a presença de seis polimorfismos, com dois (c.229T>C; c.255T>C) que resultaram na troca da fenilalanina por leucina. No gene FASN, foram identificados dois genótipos. No gene TE-FASN, foram identificados três genótipos e 17 polimorfismos. Os genes DGAT-1 e SCD não revelaram a ocorrência de polimorfismo. Houve diferença em relação aos ácidos graxos C14:0, C18:0 e ∆9-desaturaseC18 para o gene DGAT-2 e de C18:2ω6t para TE-FASN. Houve diferença entre os grupos genéticos para C10:0, C12:0, C17:0, C18:2ω6t, C18:3ω3, C20:2, total de ω3, ω3/ω6 e índice de aterogenicidade. Há ocorrência de polimorfismo dos genes DGAT-2 e TE-FASN, e estes devem ser mais estudados em ovinos, pois revelaram influência dos genótipos sobre o perfil de ácidos graxos.(AU)

Histodifferentiation of oil palm somatic embryo development at low auxin concentration.

Large-scale propagation of oil palm (Elaeis guineensis, Jacq.) is difficult due to its single apical meristem. Thus, obtaining plants is mainly through seed germination, and a long growing period is required before oil production is possible. An alternative to large-scale seedling production is indirect somatic embryogenesis. The aim of this study was to analyze the somatic embryogenesis process in oil palm (E. guineensis Jacq.) with amino acids and low concentrations of auxins. The Tenera hybrid was analyzed by cytochemical and ultrastructural methods and was used to regenerate oil palm plants. First, calli were induced in MS culture media supplemented with 2,4-D and picloram. Two types of calli were obtained, characterized by beige or translucent color. Beige calli had embryogenic characteristics, such as large nuclei with prominent nucleoli, and they were multiplied for 8 months in MM culture (half strength MS, 1 mg L-1 2,4-D, 2 mg L-1 2iP, 1 mg L-1 IBA, 250 mg L-1 citric acid, 10 mg L-1 cysteine, 100 mg L-1 inositol, 1 mg L-1 thiamine, 1 mg L-1 pyridoxine, 1 mg L-1 nicotinic acid, 1 mg L-1 glycine, 200 mg L-1 malt extract, and 100 mg L-1 casein hydrolysate). After multiplication, the MCB culture medium (half strength MS, supplemented with 0.25 mg L-1 NAA, 2 mg L-1 BAP, MM vitamins and 200 mg L-1 malt extract, and 100 mg L-1 casein hydrolysate) was the most efficient for embryo formation, showing meristematic centers with totipotent cells in histochemical analyses. The somatic embryos were developed and germinated in MG medium (half strength MS, 0.45 mg L-1 IAA, 0.25 mg L-1 BAP, and MM vitamins), transplanted into polyethylene tubes containing pine bark substrates, and acclimatized in a greenhouse, achieving a 97% survival rate. The use of picloram for callus induction and somatic embryogenesis is advantageous and multiplication in MM medium is an important step for increasing cell mass. The calli with light beige color and nodular structures have meristematic cells with dense cytoplasm and totipotential features that later give rise to protoderm, procambium, and ground meristem during the globular, cordiform, and torpedo embryogenesis phases. In MCB medium, the concentration of vitamins and amino acids are crucial for somatic embryogenesis.

Expression of genes related to the regulation of muscle protein turnover in Angus and Nellore bulls.

We aimed to evaluate the expression of genes related to the regulation of muscle protein turnover in the longissimus dorsi (LD) muscle of Angus and Nellore bulls and to estimate the within-breed correlations of gene expression and performance traits. Thirteen genes related to the IGF-1 and myostatin pathways were studied. Thirteen animals, with an initial average BW of 381.2 ± 11.8 kg, from each breed were used in a completely randomized 2 × 2 factorial design (2 breeds and 2 feeding levels). The diet consisted of corn silage and a corn-soybean meal concentrate in a roughage-to-concentrate ratio of 30:70. Cattle were fed ad libitum (with 9 animals from each breed) or feed restricted (a 55% restriction of total DMI of ad libitum-fed animals, calculated as percentage of metabolic BW, with 4 animals of each breed). The experimental period lasted for 82 d and it was preceded by a 28-d adaptation period. The performance traits evaluated were slaughter body weight, total ADG (from d 1 to 82 of the trial), initial ADG (from d 1 to 41 of the trial), final ADG (from d 42 to 82 of the trial), total DMI (from d 1 to 82 of the trial), initial DMI (from d 1 to 41 of the trial), final DMI (from d 42 to 82 of the trial), HCW, LD weight (LDW), and rib eye area (REA). After slaughter, samples were taken from the LD muscle between the 12th and 13th ribs for gene expression analysis by quantitative reverse transcription PCR. There was no difference ( > 0.05) in the expression of any of the genes studied between ad libitum-fed Angus and ad libitum-fed Nellore, whereas feed restriction increased the expression of (; < 0.001), (; = 0.05), and (; = 0.04) and decreased the expression of ( < 0.01). The REA was negatively correlated to (; = 0.01), (; = 0.02), and ( = 0.05). The HCW was negatively correlated to ( = 0.01) and ( = 0.01) and tended to be negatively correlated to ( = 0.07), whereas the LDW tended to be negatively correlated to ( = 0.08). The genes , , and seem to be important for muscle growth and may be worthy of further investigation as future strategies for increasing muscle in livestock.

New Insights on Coffea miRNAs: Features and Evolutionary Conservation.

Small RNAs influence the gene expression at the post-transcriptional level by guiding messenger RNA (mRNA) cleavage, translational repression, and chromatin modifications. In addition to model plants, the microRNAs (miRNAs) have been identified in different crop species. In this work, we developed a specific pipeline to search for coffee miRNA homologs on expressed sequence tags (ESTs) and genome survey sequences (GSS) databases. As a result, 36 microRNAs were identified and a total of 616 and 362 potential targets for Coffea arabica and Coffea canephora, respectively. The evolutionary analyses of these molecules were performed by comparing the primary and secondary structures of precursors and mature miRNAs with their orthologs. Moreover, using a stem-loop RT-PCR assay, we evaluated the accumulation of mature miRNAs in genomes with different ploidy levels, detecting an increase in the miRNAs accumulation according to the ploidy raising. Finally, a 5' RACE (Rapid Amplification of cDNA Ends) assay was performed to verify the regulation of auxin responsive factor 8 (ARF8) by MIR167 in coffee plants. The great variety of target genes indicates the functional plasticity of these molecules and reinforces the importance of understanding the RNAi-dependent regulatory mechanisms. Our results expand the study of miRNAs and their target genes in this crop, providing new challenges to understand the biology of these species.

Expression of genes involved in lipid metabolism in the muscle of beef cattle fed soybean or rumen-protected fat, with or without monensin supplementation.

Degree of unsaturation of fatty acids, which is influenced by lipid source and level of metabolism in the rumen, is a major determinant in how dietary lipids affect genes that regulate beef marbling. A total of 28 Red Norte bulls with an initial live weight of 361±32 kg (P>0.05) were used in a completely randomized experimental design to analyze the expression of genes that are involved in lipid metabolism in the longissimus dorsi (LD) when diets contained soybean grain or rumen-protected fat, with or without monensin. Treatments were arranged as a 2×2 factorial, with 4 treatments and 7 replicates per treatment. Half of the animals that received soybean or rumen-protected fat were supplemented with 230 mg head(-1) d(-1) of monensin. Gene expression was analyzed by reverse-transcription quantitative PCR (RT-qPCR). Expression of sterol regulatory element-binding protein-1c (SREBP-1c) in the LD muscle was not affected by lipid source or monensin (P>0.05). There was an interaction effect (P<0.05) between lipid source and monensin for peroxisome proliferator-activated receptor α (PPAR-α) and stearoyl-CoA desaturase (SCD) expression, where greater gene expression was found in animals fed soybean plus monensin and the lower gene expression was found in animals fed rumen-protected fat plus monensin. Expression of lipoprotein lipase (LPL) and fatty acid-binding protein 4 (FABP4) were greater (P<0.05) in the LD muscle of animals fed soybean. Monensin had no effect on LPL and FABP4 expression when soybean without monensin was fed, but when rumen-protected fat was fed, monensin increased LPL expression and decreased FABP4 expression (P<0.05). Linoleic and arachidonic acids had negative correlations (P<0.05) with the expression of PPAR-α, SCD, FABP4, and LPL genes. PPAR-α gene expression was not correlated with SREBP-1c but was positively correlated with SCD, FABP4, LPL, and glutathione peroxidase (GPX1) gene expression (P<0.001). Lipid sources and monensin interact and alter the expression of PPAR-α, SCD, acetyl CoA carboxylase α (ACACA), LPL, FABP4, and GPX1. These changes in gene expression were most associated with arachidonic and α-linolenic acids and the ability of lipid sources and monensin to increase these fatty acids in tissues.

Identification of expressed sequences in the coffee genome potentially associated with somatic embryogenesis.

Brazil possesses the most modern and productive coffee growing farms in the world, but technological development is desired to cope with the increasing world demand. One way to increase Brazilian coffee growing productivity is wide scale production of clones with superior genotypes, which can be obtained with in vitro propagation technique, or from tissue culture. These procedures can generate thousands of clones. However, the methodologies for in vitro cultivation are genotype-dependent, which leads to an almost empirical development of specific protocols for each species. Therefore, molecular markers linked to the biochemical events of somatic embryogenesis would greatly facilitate the development of such protocols. In this context, sequences potentially involved in embryogenesis processes in the coffee plant were identified in silico from libraries generated by the Brazilian Coffee Genome Project. Through these in silico analyses, we identified 15 EST-contigs related to the embryogenesis process. Among these, 5 EST-contigs (3605, 9850, 13686, 17240, and 17265) could readily be associated with plant embryogenesis. Sequence analysis of EST-contig 3605, 9850, and 17265 revealed similarity to a polygalacturonase, to a cysteine-proteinase, and to an allergenine, respectively. Results also show that EST-contig 17265 sequences presented similarity to an expansin. Finally, analysis of EST-contig 17240 revealed similarity to a protein of unknown function, but it grouped in the similarity dendrogram with the WUSCHEL transcription factor. The data suggest that these EST-contigs are related to the embryogenic process and have potential as molecular markers to increase methodological efficiency in obtaining coffee plant embryogenic materials.

Population diversity of Staphylococcus aureus isolated from bovine mastitis in Brazilian dairy herds.

Population diversity was evaluated in strains of Staphylococcus aureus isolated from bovine mastitis in Brazilian dairy herds by PCR-RFLP and sequencing of the 3'-terminal portion of the coagulase gene, and the susceptibility of strains to antimicrobials. The results showed great diversity in S. aureus population studied and the existence of predominant clones that account for most infections. No associations between the predominant types observed in the PCR-RFLP and the forms of presentation of the mastitis or to any of the different patterns of antimicrobial resistance were observed.

In silico characterization of putative members of the coffee (Coffea arabica) ethylene signaling pathway.

The plant hormone ethylene is involved in several developmental and physiological processes in plants, including senescence, fruit ripening and organ abscission, as well as in biotic and abiotic stress responses. Initiation of these processes involves complex regulation of both ethylene biosynthesis and the ability of cells to perceive the hormone and respond in an appropriate manner, a process which is regulated both spatially and temporally. Ethylene is a gaseous hormone whose sensitivity is a key factor to limiting its response in target cells. We made a search of the Coffee Expressed Sequence Tag (CAFEST) database for expressed sequence tags related to known elements of the ethylene signaling pathway. Sequences showing a reliable similarity were clusterized, annotated and analyzed for conserved domains. Multiple alignments comprising the sequences that we found and sequences of ethylene signaling elements from other species were made, and their phylogeny was assessed by phylogenetic trees constructed with the MEGA4 software. The expression profile was assessed by in silico Northern blot analysis performed using the Cluster and TreeView programs. The CAFEST database was found to have a large number of sequences related to previously described ethylene signaling pathway elements, allowing identification of putative members from almost every step of this pathway. The phylogenetic trees demonstrated high similarity between the sequences found in the CAFEST and those from other species, and the electronic Northern blot analysis detected their expression in various tissues, development stages and stress conditions.