Publications by authors named "Roberta Lane Oliveira Silva"

7 Publications

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Evaluation of quality and gene expression of goat embryos produced in vivo and in vitro after cryopreservation.

Cryobiology 2021 May 5. Epub 2021 May 5.

Laboratory of Reproductive Biotechniques, Department of Veterinary Medicine, Federal Rural University of Pernambuco, Brazil. Electronic address:

In the present study, we aimed to identify morphological and molecular changes of in vivo and in vitro-produced goat embryos submitted to cryopreservation. In vivo embryos were recovered by transcervical technique from superovulated goats, whereas in vitro produced embryos were produced from ovaries collected at a slaughterhouse. Embryos were frozen by two-steps slow freezing method, which is defined as freezing to -32 °C followed by transfer to liquid nitrogen. Morphological evaluation of embryos was carried out by assessing blastocoel re-expansion rate and the total number of blastomeres. The expression profile of candidate genes related to thermal and oxidative stress, apoptosis, epigenetic, and implantation control was measured using RT-qPCR based SYBR Green system. In silico analyses were performed to identify conserved genes in goat species and protein-protein interaction networks were created. In vivo-produced embryos showed greater blastocoel re-expansion and more blastomere cells (P < 0.05). The expression level of CTP2 and HSP90 genes from in vitro cryopreserved embryos was higher than their in vivo counterparts. Unlikely, no significant difference was observed in the transcription level of SOD gene between groups. The high similarity of CPT2 and HSP90 proteins to their orthologs among mammals indicates that they share conserved functions. In summary, cryopreservation negatively affects the morphology and viability of goat embryos produced in vitro and changes the CPT2 and HSP90 gene expression likely in response to the in vitro production process.
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http://dx.doi.org/10.1016/j.cryobiol.2021.04.008DOI Listing
May 2021

Cassava (Manihot esculenta) defensins: Prospection, structural analysis and tissue-specific expression under biotic/abiotic stresses.

Biochimie 2021 Jul 28;186:1-12. Epub 2021 Mar 28.

Departamento de Genética, Centro de Biociências, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, 1235, CEP 50.670-423, Recife, PE, Brazil. Electronic address:

Defensins are a prominent family of antimicrobial peptides. They play sophisticated roles in the defense against pathogens in all living organisms, but few works address their expression under different conditions and plant tissues. The present work prospected defensins of Manihot esculenta Crantz, popularly known as cassava. Five defensin candidates (MeDefs) were retrieved from the genome sequences and characterized. Considering chromosome distribution, only MeDef1 and 2 occupy adjacent positions in the same chromosome arm. All 3D structures had antiparallel ß-sheets, an α-helix, and amphipathic residues distributed throughout the peptides with a predominance of cationic surface charge. MeDefs expression was validated by RT-qPCR, including two stress types (biotic: fungus Macrophomina pseudophaseolina, and abiotic: mechanical injury) and a combination of both stresses (fungus+injury) in three different tissues (root, stem, and leaf). For this purpose, ten reference genes (RGs) were tested, and three were chosen to characterize MeDef expression. MeDef3 was up-regulated at roots in all stress situations tested. MeDef1 and MeDef5 were induced in leaves under biotic and abiotic stresses, but not in both stress types simultaneously. Only MeDef2 was down-regulated in the stem tissue also with biotic/abiotic combined stresses. These results indicate that although defensins are known to be responsive to pathogen infection, they may act as preformed defense or, still, have tissue or stress specificities. Aspects of their structure, stability and evolution are also discussed.
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http://dx.doi.org/10.1016/j.biochi.2021.03.012DOI Listing
July 2021

Plant Antimicrobial Peptides: State of the Art, In Silico Prediction and Perspectives in the Omics Era.

Bioinform Biol Insights 2020 2;14:1177932220952739. Epub 2020 Sep 2.

Departamento de Genética, Universidade Federal de Pernambuco, Recife, Brazil.

Even before the perception or interaction with pathogens, plants rely on constitutively guardian molecules, often specific to tissue or stage, with further expression after contact with the pathogen. These guardians include small molecules as antimicrobial peptides (AMPs), generally cysteine-rich, functioning to prevent pathogen establishment. Some of these AMPs are shared among eukaryotes (eg, defensins and cyclotides), others are plant specific (eg, snakins), while some are specific to certain plant families (such as heveins). When compared with other organisms, plants tend to present a higher amount of AMP isoforms due to gene duplications or polyploidy, an occurrence possibly also associated with the sessile habit of plants, which prevents them from evading biotic and environmental stresses. Therefore, plants arise as a rich resource for new AMPs. As these molecules are difficult to retrieve from databases using simple sequence alignments, a description of their characteristics and in silico (bioinformatics) approaches used to retrieve them is provided, considering resources and databases available. The possibilities and applications based on tools versus database approaches are considerable and have been so far underestimated.
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http://dx.doi.org/10.1177/1177932220952739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476358PMC
September 2020

Housekeeping genes for RT-qPCR in ovine preimplantation embryos.

Zygote 2020 Jul 30:1-8. Epub 2020 Jul 30.

Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Brazil.

Housekeeping genes (HKG) are paramount for accurate gene expression analysis during preimplantation development. Markedly, quantitative reverse transcription polymerase chain reaction (RT-qPCR) in ovine embryos currently lacks HKGs. Therefore, we tested 11 HKGs for RT-qPCR normalization during ovine parthenogenetic preimplantation development. Seven HKGs reached the qPCR efficiency threshold (97.20-105.96%), with correlation coefficients ranging from -0.922 to -0.998 and slopes from -3.22 to -3.59. GeNorm ranked glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and TATA-binding protein (TBP) as the best HKG pair, while H3 histone, family 3A (H3F3A) was the third HKG. Relative gene expression was measured for zinc finger protein X-linked (ZFX) and developmental pluripotency-associated 3 (DPPA3) transcripts during ovine parthenogenetic preimplantation development. ZFX did not show any transcript abundance fluctuation among oocytes, cleavage-stage embryos, and morulae. DPPA3 transcript abundance was also similar among all developmental stages, therefore suggesting that it may not display a maternal gene expression profile. In silico analysis of ovine DPPA3 mRNA and protein showed high conservation to bovine orthologues. However, DPPA3 orthologues differed in regulatory motifs. In conclusion, GAPDH, TBP and H3F3A are stable HKGs in ovine parthenogenetic embryos and allow accurate RT-qPCR-based gene expression analysis.
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http://dx.doi.org/10.1017/S0967199420000295DOI Listing
July 2020

Plant Thaumatin-like Proteins: Function, Evolution and Biotechnological Applications.

Curr Protein Pept Sci 2020 ;21(1):36-51

Departamento de Genetica, Centro de Biociencias, Universidade Federal de Pernambuco (UFPE), Recife, Pernambuco, Brazil.

Thaumatin-like proteins (TLPs) are a highly complex protein family associated with host defense and developmental processes in plants, animals, and fungi. They are highly diverse in angiosperms, for which they are classified as the PR-5 (Pathogenesis-Related-5) protein family. In plants, TLPs have a variety of properties associated with their structural diversity. They are mostly associated with responses to biotic stresses, in addition to some predicted activities under drought and osmotic stresses. The present review covers aspects related to the structure, evolution, gene expression, and biotechnological potential of TLPs. The efficiency of the discovery of new TLPs is below its potential, considering the availability of omics data. Furthermore, we present an exemplary bioinformatics annotation procedure that was applied to cowpea (Vigna unguiculata) transcriptome, including libraries of two tissues (root and leaf), and two stress types (biotic/abiotic) generated using different sequencing approaches. Even without using genomic sequences, the pipeline uncovered 56 TLP candidates in both tissues and stresses. Interestingly, abiotic stress (root dehydration) was associated with a high number of modulated TLP isoforms. The nomenclature used so far for TLPs was also evaluated, considering TLP structure and possible functions identified to date. It is clear that plant TLPs are promising candidates for breeding purposes and for plant transformation aiming a better performance under biotic and abiotic stresses. The development of new therapeutic drugs against human fungal pathogens also deserves attention. Despite that, applications derived from TLP molecules are still below their potential, as it is evident in our review.
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http://dx.doi.org/10.2174/1389203720666190318164905DOI Listing
January 2021

Genotype-dependent regulation of drought-responsive genes in tolerant and sensitive sugarcane cultivars.

Gene 2017 Oct 30;633:17-27. Epub 2017 Aug 30.

Federal University of Pernambuco (UFPE), Bioscience Center, Department of Genetics, 50670-420 Recife, PE, Brazil. Electronic address:

Drought is the most damaging among the major abiotic stresses. Transcriptomic studies allow a global overview of expressed genes, providing the basis for molecular markers development. Here, the HT-SuperSAGE technique allowed the evaluation of four drought-tolerant cultivars and four-sensitive cultivars, after 24h of irrigation suppression. We identified 9831 induced unitags from roots of the tolerant cultivars with different regulations by the -sensitive cultivars after the applied stress. These unitags allowed a proposal of 15 genes, whose expressed profiles were validated by RT-qPCR, evaluating each cultivar independently. These genes covered broad metabolic processes: ethylene stress attenuation (ACCD); root growth (β-EXP8); protein degradation [ubiquitination pathway (E2, 20SPβ4); plant proteases (AP, C13)]; oxidative detoxification (TRX); fatty acid synthesis (ACC); amino acid transport (AAT), and carbohydrate metabolism [glycolysis (PFK, TPI, FBA); TCA cycle (LDP, MDH); pentose phosphate pathway (TKT)]. The expressed profiles showed a genotype-dependent regulation of the target genes. Two drought-tolerant cultivars (SP83-2847; CTC6) presented each one, nine of the induced genes. Among the -sensitive cultivars, CTC13 induced only one, while SP90-1636 induced two genes. These genes should help breeders to identify accessions managing drought stress tolerance responses, showing better ethylene stress attenuation, energy allocation, amino acid transport, and protein homeostasis.
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http://dx.doi.org/10.1016/j.gene.2017.08.022DOI Listing
October 2017

Resistance (R) Genes: Applications and Prospects for Plant Biotechnology and Breeding.

Curr Protein Pept Sci 2017 ;18(4):323-334

Federal University of Pernambuco, Center of Biological Sciences, Department of Genetics, Av. Prof. Moraes Rego, 1235, CEP 50670-420, Recife, PE, Brazil.

The discovery of novel plant resistance (R) genes (including their homologs and analogs) opened interesting possibilities for controlling plant diseases caused by several pathogens. However, due to environmental pressure and high selection operated by pathogens, several crop plants have lost specificity, broad-spectrum or durability of resistance. On the other hand, the advances in plant genome sequencing and biotechnological approaches, combined with the increasing knowledge on Rgenes have provided new insights on their applications for plant genetic breeding, allowing the identification and implementation of novel and efficient strategies that enhance or optimize their use for efficiently controlling plant diseases. The present review focuses on main perspectives of application of R-genes and its co-players for the acquisition of resistance to pathogens in cultivated plants, with emphasis on biotechnological inferences, including transgenesis, cisgenesis, directed mutagenesis and gene editing, with examples of success and challenges to be faced.
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http://dx.doi.org/10.2174/1389203717666160724195248DOI Listing
November 2017