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1.
BMC Biotechnol ; 19(1): 71, 2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31684940

RESUMO

BACKGROUND: The CRISPR-Cas9 system is a powerful and versatile tool for crop genome editing. However, achieving highly efficient and specific editing in polyploid species can be a challenge. The efficiency and specificity of the CRISPR-Cas9 system depends critically on the gRNA used. Here, we assessed the activities and specificities of seven gRNAs targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in hexaploid wheat protoplasts. EPSPS is the biological target of the widely used herbicide glyphosate. RESULTS: The seven gRNAs differed substantially in their on-target activities, with mean indel frequencies ranging from 0% to approximately 20%. There was no obvious correlation between experimentally determined and in silico predicted on-target gRNA activity. The presence of a single mismatch within the seed region of the guide sequence greatly reduced but did not abolish gRNA activity, whereas the presence of an additional mismatch, or the absence of a PAM, all but abolished gRNA activity. Large insertions (≥20 bp) of DNA vector-derived sequence were detected at frequencies up to 8.5% of total indels. One of the gRNAs exhibited several properties that make it potentially suitable for the development of non-transgenic glyphosate resistant wheat. CONCLUSIONS: We have established a rapid and reliable method for gRNA validation in hexaploid wheat protoplasts. The method can be used to identify gRNAs that have favourable properties. Our approach is particularly suited to polyploid species, but should be applicable to any plant species amenable to protoplast transformation.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Genoma de Planta/genética , RNA Guia/genética , Triticum/genética , Protoplastos/metabolismo
2.
Plant Biotechnol J ; 17(10): 1905-1913, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30839150

RESUMO

The development and adoption of hybrid seed technology have led to dramatic increases in agricultural productivity. However, it has been a challenge to develop a commercially viable platform for the production of hybrid wheat (Triticum aestivum) seed due to wheat's strong inbreeding habit. Recently, a novel platform for commercial hybrid seed production was described. This hybridization platform utilizes nuclear male sterility to force outcrossing and has been applied to maize and rice. With the recent molecular identification of the wheat male fertility gene Ms1, it is now possible to extend the use of this novel hybridization platform to wheat. In this report, we used the CRISPR/Cas9 system to generate heritable, targeted mutations in Ms1. The introduction of biallelic frameshift mutations into Ms1 resulted in complete male sterility in wheat cultivars Fielder and Gladius, and several of the selected male-sterile lines were potentially non-transgenic. Our study demonstrates the utility of the CRISPR/Cas9 system for the rapid generation of male sterility in commercial wheat cultivars. This represents an important step towards capturing heterosis to improve wheat yields, through the production and use of hybrid seed on an industrial scale.


Assuntos
Sistemas CRISPR-Cas , Infertilidade das Plantas , Sementes , Triticum/genética , Mutação da Fase de Leitura , Técnicas de Inativação de Genes , Genes de Plantas , Poliploidia
3.
Rice (N Y) ; 11(1): 2, 2018 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-29313187

RESUMO

BACKGROUND: Flooding during seasonal monsoons affects millions of hectares of rice-cultivated areas across Asia. Submerged rice plants die within a week due to lack of oxygen, light and excessive elongation growth to escape the water. Submergence tolerance was first reported in an aus-type rice landrace, FR13A, and the ethylene-responsive transcription factor (TF) gene SUB1A-1 was identified as the major tolerance gene. Intolerant rice varieties generally lack the SUB1A gene but some intermediate tolerant varieties, such as IR64, carry the allelic variant SUB1A-2. Differential effects of the two alleles have so far not been addressed. As a first step, we have therefore quantified and compared the expression of nearly 2500 rice TF genes between IR64 and its derived tolerant near isogenic line IR64-Sub1, which carries the SUB1A-1 allele. Gene expression was studied in internodes, where the main difference in expression between the two alleles was previously shown. RESULTS: Nineteen and twenty-six TF genes were identified that responded to submergence in IR64 and IR64-Sub1, respectively. Only one gene was found to be submergence-responsive in both, suggesting different regulatory pathways under submergence in the two genotypes. These differentially expressed genes (DEGs) mainly included MYB, NAC, TIFY and Zn-finger TFs, and most genes were downregulated upon submergence. In IR64, but not in IR64-Sub1, SUB1B and SUB1C, which are also present in the Sub1 locus, were identified as submergence responsive. Four TFs were not submergence responsive but exhibited constitutive, genotype-specific differential expression. Most of the identified submergence responsive DEGs are associated with regulatory hormonal pathways, i.e. gibberellins (GA), abscisic acid (ABA), and jasmonic acid (JA), apart from ethylene. An in-silico promoter analysis of the two genotypes revealed the presence of allele-specific single nucleotide polymorphisms, giving rise to ABRE, DRE/CRT, CARE and Site II cis-elements, which can partly explain the observed differential TF gene expression. CONCLUSION: This study identified new gene targets with the potential to further enhance submergence tolerance in rice and provides insights into novel aspects of SUB1A-mediated tolerance.

4.
J Rheumatol ; 44(11): 1612-1618, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28864644

RESUMO

OBJECTIVE: Patients with myositis-associated interstitial lung disease (MA-ILD) are often refractory to conventional treatment, and predicting their response to therapy is challenging. Recent case reports and small series suggest that tacrolimus may be useful in refractory cases. METHODS: A retrospective cohort study of patients with MA-ILD comparing clinical characteristics between those who responded to or failed conventional treatment. In those who failed conventional treatment and received adjunctive tacrolimus, response to tacrolimus was measured by the improvement in myositis, ILD, and change in the dose of glucocorticoids. RESULTS: Thirty-one of 54 patients (57%) responded to conventional treatment based on the predefined variables of improvement in myositis and/or ILD. Patients with polymyositis (PM)-ILD were more likely to respond to conventional treatment than those with dermatomyositis (DM)-ILD (67% vs 35%, p = 0.013). Twenty-three patients failed conventional treatment, 18 of whom subsequently received adjunctive tacrolimus. Ninety-four percent had improvements in ILD and 72% showed improvement in both myositis and ILD. The mean doses of prednisone decreased from baseline by 65% at 3-6 months (p = 0.002) and 81% at 1 year (p < 0.001). CONCLUSION: Patients with PM-ILD were more likely to respond to conventional treatment than patients with DM-ILD, but clinical characteristics and serology did not otherwise predict response to therapy. A majority of patients with MA-ILD refractory to conventional therapy improved while receiving tacrolimus and were able to decrease their dose of both glucocorticoids and other disease-modifying antirheumatic drugs.


Assuntos
Anti-Inflamatórios/uso terapêutico , Imunossupressores/uso terapêutico , Doenças Pulmonares Intersticiais/tratamento farmacológico , Miosite/complicações , Prednisona/uso terapêutico , Tacrolimo/uso terapêutico , Adulto , Relação Dose-Resposta a Droga , Feminino , Humanos , Doenças Pulmonares Intersticiais/etiologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Falha de Tratamento , Estados Unidos
5.
PLoS One ; 12(1): e0169686, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28103252

RESUMO

Comparative genomics have facilitated the mining of biological information from a genome sequence, through the detection of similarities and differences with genomes of closely or more distantly related species. By using such comparative approaches, knowledge can be transferred from the model to non-model organisms and insights can be gained in the structural and evolutionary patterns of specific genes. In the absence of sequenced genomes for allergenic grasses, this study was aimed at understanding the structure, organisation and expression profiles of grass pollen allergens using the genomic data from Brachypodium distachyon as it is phylogenetically related to the allergenic grasses. Combining genomic data with the anther RNA-Seq dataset revealed 24 pollen allergen genes belonging to eight allergen groups mapping on the five chromosomes in B. distachyon. High levels of anther-specific expression profiles were observed for the 24 identified putative allergen-encoding genes in Brachypodium. The genomic evidence suggests that gene encoding the group 5 allergen, the most potent trigger of hay fever and allergic asthma originated as a pollen specific orphan gene in a common grass ancestor of Brachypodium and Triticiae clades. Gene structure analysis showed that the putative allergen-encoding genes in Brachypodium either lack or contain reduced number of introns. Promoter analysis of the identified Brachypodium genes revealed the presence of specific cis-regulatory sequences likely responsible for high anther/pollen-specific expression. With the identification of putative allergen-encoding genes in Brachypodium, this study has also described some important plant gene families (e.g. expansin superfamily, EF-Hand family, profilins etc) for the first time in the model plant Brachypodium. Altogether, the present study provides new insights into structural characterization and evolution of pollen allergens and will further serve as a base for their functional characterization in related grass species.


Assuntos
Alérgenos/genética , Brachypodium/genética , Brachypodium/imunologia , Poaceae/genética , Poaceae/imunologia , Pólen/genética , Pólen/imunologia , Alérgenos/química , Alérgenos/classificação , Cromossomos de Plantas/genética , Sequência Conservada , Evolução Molecular , Genoma de Planta , Humanos , Lolium/genética , Lolium/imunologia , Modelos Genéticos , Modelos Imunológicos , Modelos Moleculares , Phleum/genética , Phleum/imunologia , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Pólen/química , Domínios Proteicos , Rinite Alérgica Sazonal/etiologia , Rinite Alérgica Sazonal/imunologia
6.
Anal Sci ; 32(12): 1321-1325, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27941262

RESUMO

An online preconcentration system based on solid-phase extraction was developed for speciation and determinations of Cr in industrial water samples using flame atomic absorption spectrometry. A minicolumn packed with Amberlite XAD-16 loaded with salicylic acid was used to preconcentrate chromium species. All flow and chemical parameters that influence sorption were studied. The preconcentration factor and detection limit for the preconcentration time of 120 s were 79 and 0.10 µg L-1, respectively. The concentration of Cr(VI) species was calculated by the difference of the total Cr and Cr(III). The relative standard deviation (RSD) (five replicate of measurements) for 50 and 100 µg L-1 Cr(III) solution was 1.2 and 1.0% respectively. The accuracy of the method was verified by analyzing a standard reference material (NIST SRM 1640a, trace elements in natural water). The proposed preconcentration method was successfully applied to the determination of Cr species in industrial water samples with satisfactory results.

7.
J Clin Rheumatol ; 22(6): 320-3, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27556240

RESUMO

Atypical hemolytic uremic syndrome is characterized by the presence of thrombocytopenia, microangiopathic hemolytic anemia, and end-organ injury. In this report, we describe two patients with systemic lupus erythematosus who presented with findings compatible with atypical hemolytic uremic syndrome, complicated by acute kidney injury that was refractory to conventional therapies. Both patients exhibited a response to eculizumab, a monoclonal antibody to complement protein C5, with stabilization of their platelet count. On 1-year follow-up from their initial presentation, their hematologic disease remained in remission without recurrence.


Assuntos
Lesão Renal Aguda , Anticorpos Monoclonais Humanizados/administração & dosagem , Síndrome Hemolítico-Urêmica Atípica , Lúpus Eritematoso Sistêmico/complicações , Microangiopatias Trombóticas , Lesão Renal Aguda/diagnóstico , Lesão Renal Aguda/etiologia , Lesão Renal Aguda/terapia , Adulto , Síndrome Hemolítico-Urêmica Atípica/diagnóstico , Síndrome Hemolítico-Urêmica Atípica/etiologia , Síndrome Hemolítico-Urêmica Atípica/fisiopatologia , Síndrome Hemolítico-Urêmica Atípica/terapia , Feminino , Humanos , Fatores Imunológicos/administração & dosagem , Masculino , Contagem de Plaquetas/métodos , Microangiopatias Trombóticas/diagnóstico , Microangiopatias Trombóticas/etiologia , Microangiopatias Trombóticas/terapia , Resultado do Tratamento
8.
Indian J Endocrinol Metab ; 20(3): 354-8, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27186553

RESUMO

OBJECTIVES: To compare clinical characteristics, treatment, and utilization of evidence-based medicines at discharge from hospital in acute coronary syndrome (ACS) patients with or without diabetes at a tertiary care cardiac center in India. METHODS: We performed an observational study in consecutive patients discharged following management of ACS. We obtained demographic details, comorbid conditions, and cardiovascular risk factors, physical and biochemical parameters, and management. Descriptive statistics are reported. RESULTS: We enrolled 100 patients (diabetics = 28) with mean age of 59.0 ± 10.8 years (diabetics 59.3 ± 11.6, nondiabetics 58.9 ± 8.5). Forty-nine patients had ST-elevation myocardial infarction (STEMI) (diabetics = 14, 28.7%) while 51 had nonSTEMI/unstable angina (diabetics = 14, 27.4%) (P = nonsignificant). Among diabetics versus nondiabetics there was greater prevalence (%) of hypertension (78.6% vs. 44.4%), obesity (25.0% vs. 8.3%), abdominal obesity (85.7% vs. 69.4%) and sedentary activity (89.2% vs. 77.8%), and lower prevalence of smoking/tobacco use (10.7% vs. 25.0%) (P < 0.05). In STEMI patients 28 (57.1%) were thrombolysed (diabetes 17.8% vs. 31.9%), percutaneous coronary interventions (PCI) was in 67.8% diabetics versus 84.7% nondiabetics and coronary bypass surgery in 21.4% versus 8.3%. At discharge, in diabetics versus nondiabetics, there was similar use of angiotensin converting enzyme inhibitors (67.9% vs. 69.4%) and statins (100.0% vs. 98.6%) while use of dual antiplatelet therapy (85.7% vs. 95.8%) and beta-blockers (64.3% vs. 73.6%) was lower (P < 0.05). CONCLUSIONS: Diabetic patients with ACS have greater prevalence of cardiometabolic risk factors (obesity, abdominal obesity, and hypertension) as compared to nondiabetic patients. Less diabetic patients undergo PCIs and receive lesser dual anti-platelet therapy and beta-blockers.

9.
Curr Med Chem ; 23(15): 1549-70, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27048335

RESUMO

The isoprenoid stress-associated phytohormone abscisic acid (ABA) has recently been recognized to possess multifaceted biological functions in mammals and to exert potent curative effects in a number of clinically relevant human diseases. Studies with human specimens have unequivocally shown that ABA retains its stress-related functional attributes, previously identified in plants, which contribute to enhanced inflammatory defense mechanisms in mammals. Besides, studies performed in animal models revealed prominent anti-inflammatory properties of ABA as indicated by a marked reduction of immune cell infiltrates at the sites of inflammation. Thus, ABA treatment ultimately leads to the profound improvement of both non-communicable and communicable diseases which are associated with an overall alleviated course of inflammation. In addition to its action on the mammalian immune system, ABA was also shown to exert diverse physiological functions on non-immune components. One of the most remarkable features of ABA is to stimulate and expand mesenchymal stem cells, which may open a new avenue for its potential use in the field of regenerative medicine. Furthermore, ABA has been reported to play an important role in the maintenance of glycemic control. In this review, we summarize current understanding of the significance of ABA in the mammalian system, its prophylactic and therapeutic effects in various disease settings and the future directions for the development of ABA as novel drug candidate for the improved treatment of inflammatory and infectious human diseases.


Assuntos
Ácido Abscísico/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Doenças Transmissíveis/tratamento farmacológico , Inflamação/tratamento farmacológico , Ácido Abscísico/química , Animais , Anti-Inflamatórios não Esteroides/química , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos
10.
Anal Sci ; 31(12): 1303-8, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26656822

RESUMO

A speciation method for Cr(III) and Cr(VI) by on-line solid-phase extraction using a flow-injection system with FAAS has been proposed. The Cr(III) was selectively retained at pH 5 on the AXAD16-XO chelating resin and was eluted using 0.5 mol L(-1) HNO3. The preconcentration factor and the detection limit (3σ) of 73 and 0.11 µg L(-1), respectively, were obtained for a 120-s preconcentration time. The precision (%RSD) at the 100 µg L(-1) level was 1.08%. The linearity for Cr(III) measurements was up to 600 µg L(-1). The total chromium was determined by reducing Cr(VI) to Cr(III) using hydroxylamine hydrochloride, and the Cr(VI) concentration was obtained by subtraction from the total chromium concentration. The effect of interfering ions was examined towards the selectivity of the proposed system for Cr(III). Spike recovery studies in industrial water samples were carried out using standard Cr(III) and Cr(VI) solutions traceable to NIST. The proposed method was validated using SRM 1643e (Trace Elements in Water) supplied by NIST, USA.

11.
Viruses ; 7(10): 5274-304, 2015 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-26473911

RESUMO

Influenza A virus (IAV) infection represents a global threat causing seasonal outbreaks and pandemics. Additionally, secondary bacterial infections, caused mainly by Streptococcus pneumoniae, are one of the main complications and responsible for the enhanced morbidity and mortality associated with IAV infections. In spite of the significant advances in our knowledge of IAV infections, holistic comprehension of the interplay between IAV and the host immune response (IR) remains largely fragmented. During the last decade, mathematical modeling has been instrumental to explain and quantify IAV dynamics. In this paper, we review not only the state of the art of mathematical models of IAV infection but also the methodologies exploited for parameter estimation. We focus on the adaptive IR control of IAV infection and the possible mechanisms that could promote a secondary bacterial coinfection. To exemplify IAV dynamics and identifiability issues, a mathematical model to explain the interactions between adaptive IR and IAV infection is considered. Furthermore, in this paper we propose a roadmap for future influenza research. The development of a mathematical modeling framework with a secondary bacterial coinfection, immunosenescence, host genetic factors and responsiveness to vaccination will be pivotal to advance IAV infection understanding and treatment optimization.


Assuntos
Coinfecção/microbiologia , Coinfecção/virologia , Influenza Humana/complicações , Influenza Humana/virologia , Modelos Teóricos , Orthomyxoviridae/crescimento & desenvolvimento , Pneumonia Pneumocócica/microbiologia , Imunidade Adaptativa , Animais , Coinfecção/patologia , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Humanos , Influenza Humana/patologia , Orthomyxoviridae/imunologia , Pneumonia Pneumocócica/patologia
12.
PLoS One ; 9(5): e97497, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24841988

RESUMO

The liverwort Marchantia polymorpha is a member of the most basal lineage of land plants (embryophytes) and likely retains many ancestral morphological, physiological and molecular characteristics. Despite its phylogenetic importance and the availability of previous EST studies, M. polymorpha's lack of economic importance limits accessible genomic resources for this species. We employed Illumina RNA-Seq technology to sequence the gametophyte transcriptome of M. polymorpha. cDNA libraries from 6 different male and female developmental tissues were sequenced to delineate a global view of the M. polymorpha transcriptome. Approximately 80 million short reads were obtained and assembled into a non-redundant set of 46,533 transcripts (> =  200 bp) from 46,070 loci. The average length and the N50 length of the transcripts were 757 bp and 471 bp, respectively. Sequence comparison of assembled transcripts with non-redundant proteins from embryophytes resulted in the annotation of 43% of the transcripts. The transcripts were also compared with M. polymorpha expressed sequence tags (ESTs), and approximately 69.5% of the transcripts appeared to be novel. Twenty-one percent of the transcripts were assigned GO terms to improve annotation. In addition, 6,112 simple sequence repeats (SSRs) were identified as potential molecular markers, which may be useful in studies of genetic diversity. A comparative genomics approach revealed that a substantial proportion of the genes (35.5%) expressed in M. polymorpha were conserved across phylogenetically related species, such as Selaginella and Physcomitrella, and identified 580 genes that are potentially unique to liverworts. Our study presents an extensive amount of novel sequence information for M. polymorpha. This information will serve as a valuable genomics resource for further molecular, developmental and comparative evolutionary studies, as well as for the isolation and characterization of functional genes that are involved in sex differentiation and sexual reproduction in this liverwort.


Assuntos
Células Germinativas Vegetais/metabolismo , Marchantia/genética , Transcriptoma/genética , Etiquetas de Sequências Expressas , Biblioteca Gênica , Repetições de Microssatélites/genética
13.
BMC Genomics ; 14: 915, 2013 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-24365221

RESUMO

BACKGROUND: Transcription factors (TFs) are vital elements that regulate transcription and the spatio-temporal expression of genes, thereby ensuring the accurate development and functioning of an organism. The identification of TF-encoding genes in a liverwort, Marchantia polymorpha, offers insights into TF organization in the members of the most basal lineages of land plants (embryophytes). Therefore, a comparison of Marchantia TF genes with other land plants (monocots, dicots, bryophytes) and algae (chlorophytes, rhodophytes) provides the most comprehensive view of the rates of expansion or contraction of TF genes in plant evolution. RESULTS: In this study, we report the identification of TF-encoding transcripts in M. polymorpha for the first time, as evidenced by deep RNA sequencing data. In total, 3,471 putative TF encoding transcripts, distributed in 80 families, were identified, representing 7.4% of the generated Marchantia gametophytic transcriptome dataset. Overall, TF basic functions and distribution across families appear to be conserved when compared to other plant species. However, it is of interest to observe the genesis of novel sequences in 24 TF families and the apparent termination of 2 TF families with the emergence of Marchantia. Out of 24 TF families, 6 are known to be associated with plant reproductive development processes. We also examined the expression pattern of these TF-encoding transcripts in six male and female developmental stages in vegetative and reproductive gametophytic tissues of Marchantia. CONCLUSIONS: The analysis highlighted the importance of Marchantia, a model plant system, in an evolutionary context. The dataset generated here provides a scientific resource for TF gene discovery and other comparative evolutionary studies of land plants.


Assuntos
Evolução Molecular , Marchantia/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Transcriptoma , Perfilação da Expressão Gênica , Família Multigênica , Análise de Sequência de RNA
14.
Funct Integr Genomics ; 12(2): 229-48, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22466020

RESUMO

Carefully analyzed expression profiles can serve as a valuable reference for deciphering gene functions. We exploited the potential of whole genome microarrays to measure the spatial and temporal expression profiles of rice genes in 19 stages of vegetative and reproductive development. We could verify expression of 22,980 genes in at least one of the tissues. Differential expression analysis with respect to five vegetative tissues and preceding stages of development revealed reproductive stage-preferential/-specific genes. By using subtractive logic, we identified 354 and 456 genes expressing specifically during panicle and seed development, respectively. The metabolic/hormonal pathways and transcription factor families playing key role in reproductive development were elucidated after overlaying the expression data on the public databases and manually curated list of transcription factors, respectively. During floral meristem differentiation (P1) and male meiosis (P3), the genes involved in jasmonic acid and phenylpropanoid biosynthesis were significantly upregulated. P6 stage of panicle, containing mature gametophytes, exhibited enrichment of transcripts involved in homogalacturonon degradation. Genes regulating auxin biosynthesis were induced during early seed development. We validated the stage-specificity of regulatory regions of three panicle-specific genes, OsAGO3, OsSub42, and RTS, and an early seed-specific gene, XYH, in transgenic rice. The data generated here provides a snapshot of the underlying complexity of the gene networks regulating rice reproductive development.


Assuntos
Genes de Plantas , Inflorescência/genética , Oryza/genética , Sementes/genética , Transcriptoma , Vias Biossintéticas/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Inflorescência/crescimento & desenvolvimento , Inflorescência/metabolismo , Família Multigênica , Análise de Sequência com Séries de Oligonucleotídeos , Oryza/crescimento & desenvolvimento , Oryza/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Reprodução/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Transcrição Genética
15.
BMC Genomics ; 13: 126, 2012 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-22462805

RESUMO

BACKGROUND: Picrorhiza kurrooa Royle ex Benth. is an endangered plant species of medicinal importance. The medicinal property is attributed to monoterpenoids picroside I and II, which are modulated by temperature. The transcriptome information of this species is limited with the availability of few hundreds of expressed sequence tags (ESTs) in the public databases. In order to gain insight into temperature mediated molecular changes, high throughput de novo transcriptome sequencing and analyses were carried out at 15 °C and 25 °C, the temperatures known to modulate picrosides content. RESULTS: Using paired-end (PE) Illumina sequencing technology, a total of 20,593,412 and 44,229,272 PE reads were obtained after quality filtering for 15 °C and 25 °C, respectively. Available (e.g., De-Bruijn/Eulerian graph) and in-house developed bioinformatics tools were used for assembly and annotation of transcriptome. A total of 74,336 assembled transcript sequences were obtained, with an average coverage of 76.6 and average length of 439.5. Guanine-cytosine (GC) content was observed to be 44.6%, while the transcriptome exhibited abundance of trinucleotide simple sequence repeat (SSR; 45.63%) markers.Large scale expression profiling through "read per exon kilobase per million (RPKM)", showed changes in several biological processes and metabolic pathways including cytochrome P450s (CYPs), UDP-glycosyltransferases (UGTs) and those associated with picrosides biosynthesis. RPKM data were validated by reverse transcriptase-polymerase chain reaction using a set of 19 genes, wherein 11 genes behaved in accordance with the two expression methods. CONCLUSIONS: Study generated transcriptome of P. kurrooa at two different temperatures. Large scale expression profiling through RPKM showed major transcriptome changes in response to temperature reflecting alterations in major biological processes and metabolic pathways, and provided insight of GC content and SSR markers. Analysis also identified putative CYPs and UGTs that could help in discovering the hitherto unknown genes associated with picrosides biosynthesis.


Assuntos
Perfilação da Expressão Gênica/métodos , Picrorhiza/genética , Análise de Sequência de RNA/métodos , Temperatura , Composição de Bases , Cinamatos/metabolismo , Análise por Conglomerados , Biologia Computacional , Sistema Enzimático do Citocromo P-450/genética , Éxons/genética , Etiquetas de Sequências Expressas/metabolismo , Glicosiltransferases/genética , Glucosídeos Iridoides/metabolismo , Repetições de Microssatélites/genética , Anotação de Sequência Molecular , Picrorhiza/enzimologia , Picrorhiza/metabolismo , RNA Mensageiro/genética , RNA de Plantas/genética , Reprodutibilidade dos Testes , Homologia de Sequência do Ácido Nucleico
16.
BMC Res Notes ; 4: 319, 2011 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-21892935

RESUMO

BACKGROUND: The male germ line in flowering plants is initiated within developing pollen grains via asymmetric division. The smaller cell then becomes totally encased within a much larger vegetative cell, forming a unique "cell within a cell structure". The generative cell subsequently divides to give rise to two non-motile diminutive sperm cells, which take part in double fertilization and lead to the seed set. Sperm cells are difficult to investigate because of their presence within the confines of the larger vegetative cell. However, recently developed techniques for the isolation of rice sperm cells and the fully annotated rice genome sequence have allowed for the characterization of the transcriptional repertoire of sperm cells. Microarray gene expression data has identified a subset of rice genes that show unique or highly preferential expression in sperm cells. This information has led to the identification of cis-regulatory elements (CREs), which are conserved in sperm-expressed genes and are putatively associated with the control of cell-specific expression. FINDINGS: We aimed to identify the CREs associated with rice sperm cell-specific gene expression data using in silico prediction tools. We analyzed 1-kb upstream regions of the top 40 sperm cell co-expressed genes for over-represented conserved and novel motifs. Analysis of upstream regions with the SIGNALSCAN program with the PLACE database, MEME and the Mclip tool helped to find combinatorial sets of known transcriptional factor-binding sites along with two novel motifs putatively associated with the co-expression of sperm cell-specific genes. CONCLUSIONS: Our data shows the occurrence of novel motifs, which are putative CREs and are likely targets of transcriptional factors regulating sperm cell gene expression. These motifs can be used to design the experimental verification of regulatory elements and the identification of transcriptional factors that regulate sperm cell-specific gene expression.

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