Expressão do gene RcDREB1 de forma constitutiva em plantas de tabaco (Nicotiana tabacum L.)

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Botânica, 2015.A água é de importância fundamental para a vida, pois constitui a matriz onde ocorre a maioria dos processos bioquímicos que são essenciais. As estruturas e funções das proteínas dependem da presença de água. As plantas naturalmente têm a capacidade de suportar ambientes desfavoráveis e essa capacidade de resistir ao ambiente é determinada geneticamente. Atualmente, mecanismos de proteção em nível molecular têm sido estudados, como os fatores de transcrição DREB (Dehydration-Responsive Element- Binding) que foram identificados e caracterizados inicialmente em Arabidopsis thaliana. Fatores de transcrição (FT) iniciam uma cascata gênica conferindo tolerância à desidratação, salinidade ou altas e baixas temperaturas, ou seja, estresses abióticos. Em trabalho anterior o promotor do gene RcDREB1 dirigiu a expressão do gene gus que foi detectada apenas em grãos de pólen de plantas transgênicas de tabaco no momento da antese. Este trabalho visa obter plantas transgênicas de tabaco expressando o gene RcDREB1 constitutivamente para testar a hipótese de que essas plantas poderão ser tolerantes ao estresse hídrico. Para alcançar este objetivo, foi inserido o gene RcDREB1 no vetor pCAMBIA3300 onde o promotor constitutivo 35SCaMV dirigirá sua expressão. As plantas resistentes ao agente seletivo glufosinato de amônio foram analisadas para a presença do transgene por PCR e para a detecção da proteína fosfinotricina acetiltransferase (PAT). Essas análises confirmaram que 11 plantas eram transgênicas. Cinco linhagens de plantas transformadas foram testadas para tolerância a estresse hídrico onde a rega das plantas foi removida por nove dias. Quatro das linhagens utilizadas no teste anterior tiveram a viabilidade dos pólens avaliada por exposição a alta temperatura (37ºC). Os resultados mostraram que a linhagem T6 apresentou maior tolerância ao estresse hídrico assim como um ganho de biomassa durante o período do estresse. E o pólen da linhagem T11 apresentou viabilidade estatisticamente maior que a linhagem WT. O com os resultados obtidos gene RcDREB1 se mostrou um gene candidato a programas de melhoramento de cultivares comerciais.Water is fundamental for life, as it is the matrix, in which occurs most essential biochemical processes. The structures and functions of proteins depend on the presence of water. Plants have a natural ability to withstand harsh environments and ability to withstand this environment is genetically determined. Currently, protection mechanisms at the molecular level have been studied, such as DREB transcription factors (Dehydration- Responsive Element-Binding) that were identified and initially characterized in Arabidopsis thaliana. Transcription factors (TF) initiate a gene cascade conferring tolerance to dehydration, salinity or high and low temperatures, i.e. abiotic stresses. In our previous work, the promoter of the RcDREB1 gene, controlling the expression of the gus gene, have shown expression only in pollen grains of transgenic tobacco plants at the time of anthesis. This work aims to obtain transgenic tobacco plants constitutively expressing the RcDREB1gene to test the hypothesis that these plants may be tolerant to water stress. To accomplish this, the RcDREB1 gene was inserted into the pCAMBIA3300 vector under control of the constitutive promoter 35SCaMV. Plants resistant to the selective agent glufosinate ammonium were analyzed for the presence of the transgene by PCR and for the detection of protein phosphinothricin acetyltransferase (PAT). These analyzes confirmed that 11 plants were transgenic. Five transformed plants lines were tested for tolerance to drought stress by removing watering for nine days. Four of the lines were evaluated for the viability of pollen, exposed to high temperature (37 ° C). Results showed that the line T6 was more tolerant to water stress as well as a gain of biomass during the stress period. In addition, the pollen viability showed to be statistically higher in the line T11 when compared to the WT plants. These results demonstrated that RcDREB1 gene is a candidate for the improvement of commercial crops

Introdução do gene RcDREB1 de mamona em tabaco e soja visando tolerância ao estresse hídrico

Tese (doutorado)—Universidade de Brasília, Programa em Rede Multi-Institucional do Pró-Centro-Oeste de Pós-Graduação em Biotecnologia e Biodiversidade, 2020.As plantas possuem grande vulnerabilidade às condições ambientais sofrendo múltiplos tipos de estresses como frio, falta de água, salinidade e excesso de luminosidade. Ao longo da sua evolução, surgiram estratégias de adaptação a ambientes desfavoráveis como genes de proteção e fatores de transcrição. Dentre os genes relacionados à tolerância a estresses, foi observado que os genes DREB possuem o grande envolvimento com respostas a estresse abiótico. O promotor do gene RcDREB1 de mamona (Ricinus communis L.) foi estudado por Cipriano et al. (2013) que analisaram a expressão do gene gus com o promotor do gene RcDREB1 em plantas transgênicas de tabaco. A expressão da proteína GUS foi observada somente em grãos de pólen durante a antese. Ao expressar o gene RcDREB1 de forma constitutiva em tabaco foi observado um maior desenvolvimento das plantas transgênicas durante um período de estresse hídrico controlado. Como complemento a esses estudos este trabalho avaliou a variação interna de prolina durante o período de estresse hídrico e a resposta das plantas a ABA, NaCl e manitol. Os resultados obtidos indicam que o gene RcDREB1 tem potencial para ser usado nos programas de melhoramento de cultivares de grande importância econômica como soja. Além disso, no presente trabalho o gene RcDREB1 foi utilizado para gerar plantas transgênicas de soja com o intuito de obter plantas tolerantes a estresse hídrico, contudo somente uma linhagem de soja transgênica foi obtida e esta linhagem não mostrou alta tolerância a estresse hídrico quando submetida aos testes de seca.CAPESPlants are highly vulnerable to environmental conditions suffering different types of stresses such as cold, lack of water, salinity and excess of light. Throughout evolution, strategies for adapting to unfavorable environments have emerged, such as protective genes and transcription factors. Among the genes related to stress tolerance, it was observed that the DREB genes are highly involved with responses to abiotic stress. The promoter of the castor bean RcDREB1 gene (Ricinus communis L.) was studied by Cipriano et al. (2013) who analyzed the expression of the gus gene under the domain of the RcDREB1 gene promoter in transgenic tobacco plants. GUS protein expression was observed only in pollen grains during anthesis. When expressing the RcDREB1 gene constitutively in tobacco, a greater development of transgenic plants was observed during a period of controlled water stress. As a complement to these studies, this work evaluated the internal variation of proline during the period of water stress and the response of plants to ABA, NaCl and mannitol. The results obtained showed that the RcDREB1 gene shows potential to be used in breeding programs to generate tolerant cultivars of economic importance such as soybeans. In addition, in the present work the RcDREB1 gene was used to generate transgenic soybean plants in order to obtain water stress tolerant plants, however only one transgenic soybean strain was obtained and this strain did not show high tolerance to water stress when submitted to drought tests

Geoeconomic variations in epidemiology, ventilation management, and outcomes in invasively ventilated intensive care unit patients without acute respiratory distress syndrome: a pooled analysis of four observational studies

Background: Geoeconomic variations in epidemiology, the practice of ventilation, and outcome in invasively ventilated intensive care unit (ICU) patients without acute respiratory distress syndrome (ARDS) remain unexplored. In this analysis we aim to address these gaps using individual patient data of four large observational studies. Methods: In this pooled analysis we harmonised individual patient data from the ERICC, LUNG SAFE, PRoVENT, and PRoVENT-iMiC prospective observational studies, which were conducted from June, 2011, to December, 2018, in 534 ICUs in 54 countries. We used the 2016 World Bank classification to define two geoeconomic regions: middle-income countries (MICs) and high-income countries (HICs). ARDS was defined according to the Berlin criteria. Descriptive statistics were used to compare patients in MICs versus HICs. The primary outcome was the use of low tidal volume ventilation (LTVV) for the first 3 days of mechanical ventilation. Secondary outcomes were key ventilation parameters (tidal volume size, positive end-expiratory pressure, fraction of inspired oxygen, peak pressure, plateau pressure, driving pressure, and respiratory rate), patient characteristics, the risk for and actual development of acute respiratory distress syndrome after the first day of ventilation, duration of ventilation, ICU length of stay, and ICU mortality. Findings: Of the 7608 patients included in the original studies, this analysis included 3852 patients without ARDS, of whom 2345 were from MICs and 1507 were from HICs. Patients in MICs were younger, shorter and with a slightly lower body-mass index, more often had diabetes and active cancer, but less often chronic obstructive pulmonary disease and heart failure than patients from HICs. Sequential organ failure assessment scores were similar in MICs and HICs. Use of LTVV in MICs and HICs was comparable (42·4% vs 44·2%; absolute difference -1·69 [-9·58 to 6·11] p=0·67; data available in 3174 [82%] of 3852 patients). The median applied positive end expiratory pressure was lower in MICs than in HICs (5 [IQR 5-8] vs 6 [5-8] cm H2O; p=0·0011). ICU mortality was higher in MICs than in HICs (30·5% vs 19·9%; p=0·0004; adjusted effect 16·41% [95% CI 9·52-23·52]; p<0·0001) and was inversely associated with gross domestic product (adjusted odds ratio for a US$10 000 increase per capita 0·80 [95% CI 0·75-0·86]; p<0·0001). Interpretation: Despite similar disease severity and ventilation management, ICU mortality in patients without ARDS is higher in MICs than in HICs, with a strong association with country-level economic status

Strategies and performance of the CMS silicon tracker alignment during LHC Run 2

The strategies for and the performance of the CMS silicon tracking system alignment during the 2015–2018 data-taking period of the LHC are described. The alignment procedures during and after data taking are explained. Alignment scenarios are also derived for use in the simulation of the detector response. Systematic effects, related to intrinsic symmetries of the alignment task or to external constraints, are discussed and illustrated for different scenarios