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1.
BMC Plant Biol ; 19(1): 355, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31416418

RESUMO

BACKGROUND: To compensate for the lack of information about the molecular mechanism involved in Arundo donax L. response to salt stress, we de novo sequenced, assembled and analyzed the A. donax leaf transcriptome subjected to two levels of long-term salt stress (namely, S3 severe and S4 extreme). RESULTS: The picture that emerges from the identification of differentially expressed genes is consistent with a salt dose-dependent response. Hence, a deeper re-programming of the gene expression occurs in those plants grew at extreme salt level than in those subjected to severe salt stress, probably representing for them an "emergency" state. In particular, we analyzed clusters related to salt sensory and signaling, transcription factors, hormone regulation, Reactive Oxygen Species (ROS) scavenging, osmolyte biosynthesis and biomass production, all of them showing different regulation either versus untreated plants or between the two treatments. Importantly, the photosynthesis is strongly impaired in samples treated with both levels of salinity stress. However, in extreme salt conditions, a dramatic switch from C3 Calvin cycle to C4 photosynthesis is likely to occur, this probably being the more impressive finding of our work. CONCLUSIONS: Considered the distinct response to salt doses, genes either involved in severe or in extreme salt response could constitute useful markers of the physiological status of A. donax to deepen our understanding of its biology and productivity in salinized soil. Finally, many of the unigenes identified in the present study have the potential to be used for the development of A. donax varieties with improved productivity and stress tolerance, in particular the knock out of the GTL1 gene acting as negative regulator of water use efficiency has been proposed as good target for genome editing.


Assuntos
Folhas de Planta/fisiologia , Poaceae/fisiologia , RNA de Plantas/análise , Estresse Salino/genética , Transcriptoma/fisiologia , Folhas de Planta/efeitos dos fármacos , Poaceae/efeitos dos fármacos , Poaceae/genética , Estresse Salino/efeitos dos fármacos , Análise de Sequência de RNA , Transcriptoma/efeitos dos fármacos
2.
BMC Plant Biol ; 19(1): 365, 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31426739

RESUMO

BACKGROUND: Gossypium australe F. Mueller (2n = 2x = 26, G2 genome) possesses valuable characteristics. For example, the delayed gland morphogenesis trait causes cottonseed protein and oil to be edible while retaining resistance to biotic stress. However, the lack of gene sequences and their alternative splicing (AS) in G. australe remain unclear, hindering to explore species-specific biological morphogenesis. RESULTS: Here, we report the first sequencing of the full-length transcriptome of the Australian wild cotton species, G. australe, using Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq) from the pooled cDNA of ten tissues to identify transcript loci and splice isoforms. We reconstructed the G. australe full-length transcriptome and identified 25,246 genes, 86 pre-miRNAs and 1468 lncRNAs. Most genes (12,832, 50.83%) exhibited two or more isoforms, suggesting a high degree of transcriptome complexity in G. australe. A total of 31,448 AS events in five major types were found among the 9944 gene loci. Among these five major types, intron retention was the most frequent, accounting for 68.85% of AS events. 29,718 polyadenylation sites were detected from 14,536 genes, 7900 of which have alternative polyadenylation sites (APA). In addition, based on our AS events annotations, RNA-Seq short reads from germinating seeds showed that differential expression of these events occurred during seed germination. Ten AS events that were randomly selected were further confirmed by RT-PCR amplification in leaf and germinating seeds. CONCLUSIONS: The reconstructed gene sequences and their AS in G. australe would provide information for exploring beneficial characteristics in G. australe.


Assuntos
Processamento Alternativo/genética , Gossypium/genética , Isoformas de Proteínas/genética , Transcriptoma , Perfilação da Expressão Gênica , Genes de Plantas , Gossypium/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/análise , Isoformas de Proteínas/metabolismo , RNA Longo não Codificante/análise , RNA de Plantas/análise
3.
BMC Plant Biol ; 19(1): 362, 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31426743

RESUMO

BACKGROUND: The MYB transcription factor family is one of the largest transcriptional factor families in plants and plays a multifaceted role in plant growth and development. However, MYB transcription factors involved in pathogen resistance in apple remain poorly understood. RESULTS: We identified a new MYB family member from apple, and named it MdMYB30. MdMYB30 was localized to the nucleus, and was highly expressed in young apple leaves. Transcription of MdMYB30 was induced by abiotic stressors, such as polyethylene glycol and abscisic acid. Scanning electron microscopy and gas chromatograph-mass spectrometry analyses demonstrated that ectopically expressing MdMYB30 in Arabidopsis changed the wax content, the number of wax crystals, and the transcription of wax-related genes. MdMYB30 bound to the MdKCS1 promoter to activate its expression and regulate wax biosynthesis. MdMYB30 also contributed to plant surface properties and increased resistance to the bacterial strain Pst DC3000. Furthermore, a virus-based transformation in apple fruits and transgenic apple calli demonstrated that MdMYB30 increased resistance to Botryosphaeria dothidea. Our findings suggest that MdMYB30 plays a vital role in the accumulation of cuticular wax and enhances disease resistance in apple. CONCLUSIONS: MdMYB30 bound to the MdKCS1 gene promoter to activate its transcription and regulate cuticular wax content and composition, which influenced the surface properties and expression of pathogenesis-related genes to resistance against pathogens. MdMYB30 appears to be a crucial element in the formation of the plant cuticle and confers apple with a tolerance to pathogens.


Assuntos
Ascomicetos/fisiologia , Resistência à Doença , Malus/genética , Doenças das Plantas/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Ceras/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/fisiologia , Expressão Ectópica do Gene , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , Malus/metabolismo , Malus/microbiologia , Doenças das Plantas/microbiologia , Epiderme Vegetal/genética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , RNA de Plantas/análise , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
4.
BMC Plant Biol ; 19(1): 235, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159732

RESUMO

BACKGROUND: Elymus L. is the largest genus in the tribe Triticeae Dumort., encompassing approximately 150 polyploid perennial species widely distributed in the temperate regions of the world. It is considered to be an important gene pool for improving cereal crops. However, a shortage of molecular marker limits the efficiency and accuracy of genetic breeding for Elymus species. High-throughput transcriptome sequencing data is essential for gene discovery and molecular marker development. RESULTS: We obtained the transcriptome dataset of E. sibiricus, the type species of the genus Elymus, and identified a total of 8871 putative EST-SSRs from 6685 unigenes. Trinucleotides were the dominant repeat motif (4760, 53.66%), followed by dinucleotides (1993, 22.47%) and mononucleotides (1876, 21.15%). The most dominant trinucleotide repeat motif was CCG/CGG (1119, 23.5%). Sequencing of PCR products showed that the sequenced alleles from different Elymus species were homologous to the original SSR locus from which the primer was designed. Different types of tri-repeats as abundant SSR motifs were observed in repeat regions. Two hundred EST-SSR primer pairs were designed and selected to amplify ten DNA samples of Elymus species. Eighty-seven pairs of primer (43.5%) generated clear and reproducible bands with expected size, and showed good transferability across different Elymus species. Finally, thirty primer pairs successfully amplified ninety-five accessions of seventeen Elymus species, and detected significant amounts of polymorphism. In general, hexaploid Elymus species with genomes StStHHYY had a relatively higher level of genetic diversity (H = 0.219, I = 0.330, %P = 63.7), while tetraploid Elymus species with genomes StStYY had low level of genetic diversity (H = 0.182, I = 0.272, %P = 50.4) in the study. The cluster analysis showed that all ninety-five accessions were clustered into three major clusters. The accessions were grouped mainly according to their genomic components and origins. CONCLUSIONS: This study demonstrated that transcriptome sequencing is a fast and cost-effective approach to molecular marker development. These EST-SSR markers developed in this study are valuable tools for genetic diversity, evolutionary, and molecular breeding in E. sibiricus, and other Elymus species.


Assuntos
Elymus/classificação , Elymus/genética , Etiquetas de Sequências Expressas , Variação Genética , Repetições de Microssatélites , Marcadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia , Proteínas de Plantas/análise , RNA de Plantas/análise , Alinhamento de Sequência , Análise de Sequência de RNA
5.
BMC Plant Biol ; 19(1): 224, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31142280

RESUMO

BACKGROUND: Clubroot, caused by Plasmodiophora brassicae Woronin, is a very important disease of Brassica species. Management of clubroot relies heavily on genetic resistance. In a cross of Brassica nigra lines PI 219576 (highly resistant, R) × CR2748 (highly susceptible, S) to clubroot, all F1 plants were resistant to clubroot. There was a 1:1 ratio of R:S in the BC1 and 3R:1S in the F2, which indicated that a single dominant gene controlled clubroot resistance in PI 219576. This gene was designated Rcr6. Mapping of Rcr6 was performed using genome sequencing information from A-genome of B. rapa and B-genome of B. nigra though bulked segregant RNA sequencing (BSR-Seq) and further mapping with Kompetitive Allele Specific PCR (KASP) analysis. RESULTS: Reads of R and S bulks from BSR-Seq were initially aligned onto B. rapa (A-genome; B. nigra has the B-genome) where Rcr6 was associated with chromosome A08. KASP analysis showed that Rcr6 was flanked by SNP markers homologous to the region of 14.8-15.4 Mb of chromosome A08. There were 190 genes annotated in this region, with five genes (Bra010552, Bra010588, Bra010589, Bra010590 and Bra010663) identified as encoding the toll-interleukin-1 receptor / nucleotide-binding site / leucine-rich-repeat (TIR-NBS-LRR; TNL) class of proteins. The reads from BSR-Seq were then aligned into a draft B-genome of B. nigra, where Rcr6 was mapped on chromosome B3. KASP analysis indicated that Rcr6 was located on chromosome B3 in a 0.5 Mb region from 6.1-6.6 Mb. Only one TNL gene homologous to the B. rapa gene Bra010663 was identified in the target region. This gene is a likely candidate for Rcr6. Subsequent analysis of the Rcr6 equivalent region based on a published B. nigra genome was performed. This gene is located into chromosome B7 of the published B-genome, homologous to BniB015819. CONCLUSION: Rcr6 was the first gene identified and mapped in the B-genome of Brassica species. It resides in a genomic region homologous to chromosome A08 of A-genome. Based on this finding, it could possibly integrate into A08 of B. napus using marker assisted selection with SNP markers tightly linked to Rcr6 developed in this study.


Assuntos
Mostardeira/genética , Mostardeira/microbiologia , Doenças das Plantas/parasitologia , Proteínas de Plantas/genética , Plasmodioforídeos/fisiologia , Polimorfismo de Nucleotídeo Único , Mapeamento Cromossômico , Resistência à Doença/genética , Marcadores Genéticos , Proteínas de Plantas/metabolismo , RNA de Plantas/análise , Análise de Sequência de RNA
6.
Plant Mol Biol ; 99(4-5): 461-476, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30710225

RESUMO

KEY MESSAGE: ABA/GA4 ratio, stress resistance, carbon and nitrogen metabolism, and chromatin structure play important roles in vigour differences of seeds located at different maize ear positions. Seed vigour, which ensures rapid and uniform field emergence across diverse environments, differs at different maize ear positions. However, little is known regarding the associated mechanisms. In this study, we determined that seed vigour, stress resistance, and carbon and nitrogen metabolism were higher in seeds from middle and bottom section of the ear, while the ABA/GA4 ratio in the embryos was significantly lower. Compared with the seeds subjected to repeated pollination during silking, less variation in seed vigour and the ABA/GA4 ratio in the embryos was observed in seeds at different ear positions subjected to single pollination after complete silking. This indicated that single pollination can reduce, but not eliminate, the differences in seed vigour at different ear positions. RNA-seq analysis indicated that the seed vigour differences at the different locations of the maize ears of the single pollinated treatment were related to carbon and nitrogen metabolism. In contrast, the differences in seed vigour under repeated pollination were related to chromatin structure. The present study contributes to our understanding of the mechanisms underlying differences in seed vigour at different positions on the maize ear.


Assuntos
Adaptação Fisiológica , Reguladores de Crescimento de Planta/metabolismo , Sementes/metabolismo , Análise de Sequência de RNA , Estresse Fisiológico , Zea mays/metabolismo , Ácido Abscísico/genética , Ácido Abscísico/metabolismo , Carbono/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Giberelinas/genética , Giberelinas/metabolismo , Nitrogênio/metabolismo , Reguladores de Crescimento de Planta/genética , Polinização , RNA de Plantas/análise , Sementes/genética , Zea mays/genética
7.
Methods Mol Biol ; 1864: 397-410, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30415348

RESUMO

To develop a commercial trait product, a large number of transgenic events are often produced to obtain the event with desired level of expression. It is crucial to develop efficient and sensitive molecular characterization methods to advance events with stable transgene expression, free of vector backbone sequences and without major changes to the native genome caused by transgene insertion. Here, we discuss a variety of analytical tools, including quantitative PCR (qPCR), Southern blot analysis, and various sequencing technologies, which have been widely used to determine the insert copy number, presence/absence of vector backbone sequences, integrity of the T-DNA, and genomic location of the T-DNA insertion. Moreover, since the discovery of RNA interference in 1998 (Fire et al., Nature 391:806-811, 1998), RNAi has emerged as another powerful tool in in the development of a new transgenic trait for insect control. RNAi creates a double-stranded RNA duplex as the active molecule which forms a strong secondary structure, resulting in challenges for detection. In addition to molecular analysis at the DNA level, this chapter describes detection methods of the active molecules (i.e., double-stranded RNA) for RNAi-based traits.


Assuntos
Biotecnologia/métodos , Produtos Agrícolas/genética , DNA de Plantas/análise , Plantas Geneticamente Modificadas/genética , RNA de Plantas/análise , Biotecnologia/instrumentação , Southern Blotting , Comércio , DNA Bacteriano/genética , DNA de Plantas/genética , Genoma de Planta/genética , Reação em Cadeia da Polimerase , Locos de Características Quantitativas/genética , Interferência de RNA , RNA de Cadeia Dupla/análise , RNA de Cadeia Dupla/genética , RNA de Plantas/genética , Transformação Genética , Transgenes/genética
8.
Environ Sci Pollut Res Int ; 25(32): 32433-32446, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30232771

RESUMO

Cadmium (Cd) stress is one of the most serious threats to agriculture in the world. Oilseed rape (Brassica napus L.) is an important oil crop; however, Cd can easily accumulate in rapeseed and thus harm human health through the food chain. In the first experiment, our purpose was to measure the Cd accumulation in mature B. napus plants and its influences on fatty acid composition. The results showed that most Cd was accumulated in the root, and the seed fatty acid content was considerably different at different Cd toxicity levels. In the second experiment, 7-day-old B. napus seedlings stressed by Cd (1 mM) for 0 h (CK-0h), 24 h (T-24h), or 72 h (T-72h) were submitted to physiological and biological analyses, RNA-Seq and qRT-PCR. In total, 5469 and 6769 differentially expressed genes (DEGs) were identified in the comparisons of "CK-0h vs T-24h" and "CK-0h vs T-72h", respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that the photosynthetic and glutathione (GSH) pathways were significantly enriched in response to Cd stress. Key factors in the response to Cd stress included BnPCS1, BnGSTU12, BnGSTU5, and BnHMAs. The transcription factors BnWRKY11 (BnaA03g51590D), BnWRKY28 (BnaA03g43640D), BnWRKY33 (BnaA03g17820D), and BnWRKY75 (BnaA03g04160D) were upregulated after Cd exposure. The present study revealed that upregulation of the genes encoding GST and PCS under Cd stress promoted the formation of low-molecular weight complexes (PC-Cd), and upregulation of heavy metal ATPase genes induced PC-Cd transfer to vacuoles. These findings may provide the basis for the molecular mechanism of the response of B. napus to Cd.


Assuntos
Adaptação Fisiológica/genética , Brassica napus/genética , Cádmio/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Proteínas de Plantas/genética , Estresse Fisiológico , Adenosina Trifosfatases/genética , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Transporte Biológico , Brassica napus/efeitos dos fármacos , Brassica napus/metabolismo , Cádmio/farmacologia , Produtos Agrícolas/efeitos dos fármacos , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Ácidos Graxos/metabolismo , Glutationa/genética , Glutationa/metabolismo , Humanos , Metais Pesados/metabolismo , Metais Pesados/farmacologia , Fotossíntese , Desenvolvimento Vegetal , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , RNA de Plantas/análise , Plântula/metabolismo , Sementes/metabolismo , Análise de Sequência de RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima
9.
Am J Bot ; 105(3): 587-601, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29746718

RESUMO

PREMISE OF THE STUDY: The relative ease of high throughput sequencing is facilitating comprehensive phylogenomic and gene expression studies, even for nonmodel groups. To date, however, these two approaches have not been merged; while phylogenomic methods might use transcriptome sequences to resolve relationships, assessment of gene expression patterns in a phylogenetic context is less common. Here we analyzed both carbon assimilation and gene expression patterns of closely related species within the Agavoideae (Asparagaceae) to elucidate changes in gene expression across weak and strong phenotypes for Crassulacean acid metabolism (CAM). METHODS: Gene expression patterns were compared across four genera: Agave (CAM), which is paraphyletic with Polianthes (weak CAM) and Manfreda (CAM), and Beschorneria (weak CAM). RNA-sequencing was paired with measures of gas exchange and titratable acidity. Climate niche space was compared across the four lineages to examine abiotic factors and their correlation to CAM. KEY RESULTS: Expression of homologous genes showed both shared and variable patterns in weak and strong CAM species. Network analysis highlights that despite shared expression patterns, highly connected genes differ between weak and strong CAM, implicating shifts in regulatory gene function as key for the evolution of CAM. Variation in carbohydrate metabolism between weak and strong CAM supports the importance of sugar turnovers for CAM physiology. CONCLUSIONS: Integration of phylogenetics and RNA-sequencing provides a powerful tool to study the evolution of CAM photosynthesis across closely related but photosynthetically variable species. Our findings regarding shared or shifted gene expression and regulation of CAM via carbohydrate metabolism have important implications for efforts to engineer the CAM pathway into C3 food and biofuel crops.


Assuntos
Asparagaceae/genética , Evolução Biológica , Metabolismo dos Carboidratos/genética , Clima , Fenótipo , Fotossíntese/genética , Transcriptoma , Adaptação Biológica , Agave , Asparagaceae/metabolismo , Carbono/metabolismo , Expressão Gênica , Genes de Plantas , Genoma de Planta , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia , RNA de Plantas/análise , Análise de Sequência de RNA , Especificidade da Espécie , Açúcares/metabolismo
10.
PLoS One ; 13(4): e0195142, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29664904

RESUMO

The usual cultivation mode of the green microalga Chlamydomonas is liquid medium and light. However, the microalga can also be grown on agar plates and in darkness. Our aim is to analyze and compare gene expression of cells cultivated in these different conditions. For that purpose, RNA-seq data are obtained from Chlamydomonas samples of two different labs grown in four environmental conditions (agar@light, agar@dark, liquid@light, liquid@dark). The RNA seq data are analyzed by surprisal analysis, which allows the simultaneous meta-analysis of all the samples. First we identify a balance state, which defines a state where the expression levels are similar in all the samples irrespectively of their growth conditions, or lab origin. In addition our analysis identifies additional constraints needed to quantify the deviation with respect to the balance state. The first constraint differentiates the agar samples versus the liquid ones; the second constraint the dark samples versus the light ones. The two constraints are almost of equal importance. Pathways involved in stress responses are found in the agar phenotype while the liquid phenotype comprises ATP and NADH production pathways. Remodeling of membrane is suggested in the dark phenotype while photosynthetic pathways characterize the light phenotype. The same trends are also present when performing purely statistical analysis such as K-means clustering and differentially expressed genes.


Assuntos
Chlamydomonas/crescimento & desenvolvimento , Chlamydomonas/genética , Perfilação da Expressão Gênica , Transcriptoma , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Microalgas/genética , Microalgas/crescimento & desenvolvimento , RNA de Plantas/análise , Transdução de Sinais/genética
11.
J Phycol ; 54(4): 483-493, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29679512

RESUMO

Dunaliella, a commercially important chlorophyte, is globally distributed in saline habitats. Morphological species have not been definitively reconciled with phylogenetic analyses. Considerable genetic diversity continues to be discovered in new isolates, especially from soil and benthic habitats. Twenty-nine new isolates from Great Salt Lake, Utah, many from benthic or supralittoral habitats, were phylogenetically analyzed using ITS1+5.8S+ITS2 in comparison to a broad sampling of available sequences. A few new isolates align in one branch of a bifurcated monophyletic Dunaliella salina clade and several cluster within monophyletic D. viridis. Several others align with relatively few unnamed strains from other locations, comprising a diverse clade that may represent two or more new species. The overall Dunaliella clade is relatively robust, but the nearest outgroups are ambiguously placed with extremely long branches. About half of the isolates, all from benthic or supralittoral habitats, have been persistently sarcinoid in liquid media since isolation. This trait is spread across the Dunaliella phylogeny. The morphology of two sarcinoid strains was documented with light microscopy, revealing an extensive glycocalyx. Clumping behavior of unicellular and sarcinoid strains was unaffected by presence or absence of Mg2+ or Ca2+ , addition of lectin-inhibiting monosaccharides, or water-soluble factors from morphologically opposite strains. Results from this investigation have significantly expanded our current understanding of Dunaliella diversity, but it seems likely that much remains to be discovered with additional sampling.


Assuntos
Clorofíceas/classificação , Filogenia , Clorofíceas/genética , DNA Espaçador Ribossômico/análise , Lagos , RNA de Algas/análise , RNA de Plantas/análise , RNA Ribossômico 5,8S/análise , Utah
12.
Biosens Bioelectron ; 107: 34-39, 2018 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29427884

RESUMO

MicroRNAs play crucial role in regulating gene expression in organism, thus it is very necessary to exploit an efficient method for the sensitive and specific detection of microRNA. Herein, a signal-on electrochemiluminescence biosensor was fabricated for microRNA-319a detection based on two-stage isothermal strand-displacement polymerase reaction (ISDPR). In the presence of target microRNA, amounts of trigger DNA could be generated by the first ISDPR. Then, the trigger DNA and the primer hybridized simultaneously with the hairpin probe to open the stem of the probe, and then the ECL signal will be emitted. In the presence of phi29 DNA polymerase and dNTPs, the trigger DNA could be displaced to initiate a new cycle which was the second ISDPR. Due to the two-stage amplification, this method presented excellent detection sensitivity with a low detection limit of 0.14 fM. Moreover, the applicability of the developed method was demonstrated by detecting the change of microRNA-319a content in the leaves of rice seedlings after the rice seeds were incubated with chemical mutagen of ethyl methanesulfonate.


Assuntos
Técnicas Biossensoriais/métodos , Medições Luminescentes/métodos , MicroRNAs/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Oryza/genética , RNA de Plantas/análise , Técnicas Eletroquímicas/métodos , MicroRNAs/genética , RNA de Plantas/genética , Sementes/genética
13.
Sci Rep ; 7(1): 11052, 2017 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-28887495

RESUMO

To investigate changes in transcript and relative protein levels in response to salicylic acid regulation of the thermotolerance in U. prolifera, complementary transcriptome and proteome analyses were performed with U. prolifera grown at 35 °C (UpHT) and with the addition of SA at high temperature (UpSHT). At mRNA level,12,296 differentially expressed genes (DEGs) were obtained from the comparison of UpSHT with UpHT. iTRAQ-labeling proteome analysis showed that a total of 4,449 proteins were identified and reliably quantified. At mRNA level, the up-regulated genes involved in antioxidant activity were thioredoxin,peroxiredoxin,FeSOD, glutathione peroxidase, partion catalase and MnSOD. The down-regulated genes were ascorbate peroxidase, glutathione S-transferase, catalase and MnSOD. In addition, the DEGs involved in plant signal transduction pathway (such as auxin response factors, BRI1 and JAZ) were down-regulated. At protein level, the up-regulated proteins involved in carbon fixation and the down-regulated protein mainly were polyubiquitin, ascorbate peroxidase. The expression of Ca2+-binding protein, heat shock protein and photosynthesis-related proteins, EDS1 were also significantly regulated both at mRNA and protein level. The results indicated that SA alleviated the high-temperature stimulus through partion antioxidant related proteins up-regulated, JA signal pathway enchanced, Ca2+-binding proteins, photosynthesis-related proteins significantly changed, antioxidant enzyme activities increased and photosynthesis index changed.


Assuntos
Perfilação da Expressão Gênica , Temperatura Alta , Proteoma/análise , Ácido Salicílico/metabolismo , Estresse Fisiológico , Ulva/efeitos dos fármacos , Ulva/efeitos da radiação , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/análise , RNA Mensageiro/análise , RNA de Plantas/análise , Ulva/genética , Ulva/fisiologia
14.
Virol J ; 14(1): 129, 2017 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-28716126

RESUMO

BACKGROUND: Potato virus Y (PVY) is one of the most economically important pathogen of potato that is present as biologically distinct strains. The virus-derived small interfering RNAs (vsiRNAs) from potato cv. Russet Burbank individually infected with PVY-N, PVY-NTN and PVY-O strains were recently characterized. Plant defense RNA-silencing mechanisms deployed against viruses produce vsiRNAs to degrade homologous viral transcripts. Based on sequence complementarity, the vsiRNAs can potentially degrade host RNA transcripts raising the prospect of vsiRNAs as pathogenicity determinants in virus-host interactions. This study investigated the global effects of PVY vsiRNAs on the host potato transcriptome. METHODS: The strain-specific vsiRNAs of PVY, expressed in high copy number, were analyzed in silico for their proclivity to target potato coding and non-coding RNAs using psRobot and psRNATarget algorithms. Functional annotation of target coding transcripts was carried out to predict physiological effects of the vsiRNAs on the potato cv. Russet Burbank. The downregulation of selected target coding transcripts was further validated using qRT-PCR. RESULTS: The vsiRNAs derived from biologically distinct strains of PVY displayed diversity in terms of absolute number, copy number and hotspots for siRNAs on their respective genomes. The vsiRNAs populations were derived with a high frequency from 6 K1, P1 and Hc-Pro for PVY-N, P1, Hc-Pro and P3 for PVY-NTN, and P1, 3' UTR and NIa for PVY-O genomic regions. The number of vsiRNAs that displayed interaction with potato coding transcripts and number of putative coding target transcripts were comparable between PVY-N and PVY-O, and were relatively higher for PVY-NTN. The most abundant target non-coding RNA transcripts for the strain specific PVY-derived vsiRNAs were found to be MIR821, 28S rRNA,18S rRNA, snoR71, tRNA-Met and U5. Functional annotation and qRT-PCR validation suggested that the vsiRNAs target genes involved in plant hormone signaling, genetic information processing, plant-pathogen interactions, plant defense and stress response processes in potato. CONCLUSIONS: The findings suggested that the PVY-derived vsiRNAs could act as a pathogenicity determinant and as a counter-defense strategy to host RNA silencing in PVY-potato interactions. The broad range of host genes targeted by PVY vsiRNAs in infected potato suggests a diverse role for vsiRNAs that includes suppression of host stress responses and developmental processes. The interactome scenario is the first report on the interaction between one of the most important Potyvirus genome-derived siRNAs and the potato transcripts.


Assuntos
Interações Hospedeiro-Patógeno , Doenças das Plantas/virologia , Potyvirus/patogenicidade , RNA de Plantas/análise , RNA Interferente Pequeno/metabolismo , RNA Viral/metabolismo , Solanum tuberosum/virologia , Análise por Conglomerados , DNA Fúngico/química , DNA Fúngico/genética , DNA de Plantas/química , DNA de Plantas/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Perfilação da Expressão Gênica , Filogenia , Potyvirus/genética , RNA Ribossômico 18S/genética , RNA Ribossômico 28S/genética , RNA Viral/genética , Análise de Sequência de DNA
15.
FEBS Lett ; 591(15): 2261-2268, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28686301

RESUMO

miRPursuit is a pipeline developed for running end-to-end analyses of high-throughput small RNA (sRNA) sequence data in model and nonmodel plants, from raw data to identified and annotated conserved and novel sequences. It consists of a series of UNIX shell scripts, which connect open-source sRNA analysis software. The involved parameters can be combined with convenient workflow management by users without advanced computational skills. miRPursuit presents several advantages when compared to other tools, including the possibility of processing several sRNA libraries in parallel, thus easily allowing a comparison of the differences in sRNA read accumulation among sRNA libraries. We validate miRPursuit by using datasets from a model plant and discuss its performance with the analysis of sRNAs from non-model species.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/análise , Quercus/genética , RNA de Plantas/análise , Software , Automação , Bases de Dados Genéticas , Reprodutibilidade dos Testes
16.
Methods Mol Biol ; 1640: 1-21, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28608331

RESUMO

ARGONAUTEs (AGOs) are the effector proteins in eukaryotic small RNA (sRNA)-based gene silencing pathways controlling gene expression and transposon activity. In plants, AGOs regulate key biological processes such as development, response to stress, genome structure and integrity, and pathogen defense. Canonical functions of plant AGO-sRNA complexes include the endonucleolytic cleavage or translational inhibition of target RNAs and the methylation of target DNAs. Here, I provide a brief update on the major features, molecular functions, and biological roles of plant AGOs. A special focus is given to the more recent discoveries related to emerging molecular or biological functions of plant AGOs, as well as to the major unknowns in the plant AGO field.


Assuntos
Proteínas Argonauta/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Proteínas Argonauta/análise , Proteínas Argonauta/genética , Metilação de DNA , Interações Hospedeiro-Patógeno , Desenvolvimento Vegetal , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Fenômenos Fisiológicos Vegetais , Proteínas de Plantas/análise , Proteínas de Plantas/genética , Plantas/genética , Plantas/microbiologia , Mapas de Interação de Proteínas , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA de Plantas/análise , RNA de Plantas/genética , Estresse Fisiológico
17.
Methods Mol Biol ; 1640: 211-217, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28608345

RESUMO

Small noncoding RNAs are essential for gene expression at transcriptional and posttranscriptional levels. Northern blot is the most used method for small RNA detection in tissues. Here we present an improved protocol for the Northern blot-based small RNA detection from plant tissues by using biotin-labeled probes. MicroRNAs and small interfering RNAs derived from Arabidopsis and Oryza sativa, respectively, have been detected with this methodology. Results suggest that this method is sensitive and efficient enough to detect small RNAs from plant tissues by using as low as 5 µg of total RNA. Furthermore, biotin-labeled probes are safer and easier to store for long term than radiolabeled probes.


Assuntos
Arabidopsis/química , Northern Blotting/métodos , Hibridização de Ácido Nucleico/métodos , Oryza/química , RNA de Plantas/análise , Pequeno RNA não Traduzido/análise , Arabidopsis/genética , Biotina/química , Sondas de Oligonucleotídeos/química , Oryza/genética , RNA de Plantas/genética , Pequeno RNA não Traduzido/genética
18.
Mol Nutr Food Res ; 61(9)2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28319645

RESUMO

SCOPE: The uptake of dietary plant small RNAs (sRNAs) in consumers remains controversial, which is mainly due to low dietary content in combination with poor fractional absorption. MIR2911, among all the plant sRNAs including microRNAs, has been shown to be one of the most robustly absorbed sRNAs. Here we analyze the unusual abundance and unique genesis of MIR2911 during vegetable processing. METHODS AND RESULTS: Using qRT-PCR, the abundance of MIR2911 increased dramatically in macerated tissues while other microRNAs degraded. The accumulation of MIR2911 correlated with the degradation of the rRNAs, consistent with MIR2911 being derived from the 26S rRNA. Bioinformatic analysis predicts a microRNA-like precursor structure for MIR2911; however, no reciprocal increase in the putative star-strand was noted, and using an Arabidopsis mutation deficient in miRNA processing the accumulation of MIR2911 appeared Dicer independent. MIR2911 was incorporated into the mammalian RNA-induced silencing complex as demonstrated in HEK293T cells, where transfected synthetic MIR2911 modestly suppressed the activity of a cognate luciferase reporter. CONCLUSION: The genesis and amplification of MIR2911 post-harvest is atypical, as traditional plant bioactives are less plentiful as vegetables lose freshness. These findings offer an explanation to the disparity in serum detection between MIR2911 and canonical plant-based miRNAs.


Assuntos
MicroRNAs/fisiologia , RNA de Plantas/fisiologia , Disponibilidade Biológica , Brassica/genética , Manipulação de Alimentos , Células HEK293 , Humanos , MicroRNAs/análise , RNA de Plantas/análise
19.
Plant Cell Physiol ; 58(3): 574-586, 2017 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-28184867

RESUMO

ABA plays a critical role in regulating seed germination and stomatal movement in response to drought stress. Screening ABA-responsive genes led to the identification of a novel Arabidopsis gene encoding a protein which contained a conserved F-box-associated (FBA) domain, subsequently named ABA-responsive FBA domain-containing protein 1 (AFBA1). Expression of ProAFBA1:GUS revealed that this gene was mainly expressed in guard cells. Expression of AFBA1 increased following the application of exogenous ABA and exposure to salt (NaCl) and drought stresses. Seed germination of the loss-of-function mutant (afba1) was insensitive to ABA, salt or mannitol, whereas AFBA1-overexpressing (Ox) seeds were more sensitive to these stresses than the wild-type seeds. The afba1 plants showed decreased drought tolerance, increased water loss rate and ABA-insensitive stomatal movement compared with the wild-type. In contrast, AFBA1-Ox plants exhibited enhanced drought tolerance and a rapid ABA-induced stomatal closure response. The expression of genes encoding serine/threonine protein phosphatases that are known negative regulators of ABA signaling increased in afba1 plants but decreased in AFBA1-Ox plants. AFBA1 was also found to be localized in the nucleus and to interact with an R2R3-type transcription factor, MYB44, leading to the suggestion that it functions in the stabilization of MYB44. Based on these results, we suggest that AFBA1 functions as a novel positive regulator of ABA responses, regulating the expression of genes involved in ABA signal transduction in Arabidopsis through its interaction with positive regulators of ABA signaling including MYB44, and increasing their stability during ABA-mediated responses.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Adaptação Fisiológica/efeitos dos fármacos , Arabidopsis/efeitos dos fármacos , Secas , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Germinação , Manitol/metabolismo , Mutação , Estômatos de Plantas/efeitos dos fármacos , Estômatos de Plantas/fisiologia , Plantas Geneticamente Modificadas , RNA de Plantas/análise , RNA de Plantas/metabolismo , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Transdução de Sinais , Cloreto de Sódio/farmacologia , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
Gene ; 608: 66-72, 2017 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-28122266

RESUMO

MicroRNAs (miRNAs) are non-coding small RNAs which play an important regulatory role in various biological processes. Previous studies have reported that miRNAs are involved in fruit development in model plants. However, the miRNAs related to fruit development and quality in hot pepper (Capsicum annuum L.) remains unknown. In this study, small RNA populations from different fruit ripening stages and different varieties were compared using next-generation sequencing technology. Totally, 59 known miRNAs and 310 novel miRNAs were identified from four libraries using miRDeep2 software. For these novel miRNAs, 656 targets were predicted and 402 of them were annotated. GO analysis and KEGG pathways suggested that some of the predicted miRNAs targeted genes involved in starch sucrose metabolism and amino sugar as well as nucleotide sugar metabolism. Quantitative RT-PCR validated the contrasting expression patterns between several miRNAs and their target genes. These results will provide an important foundation for future studies on the regulation of miRNAs involved in fruit development and quality.


Assuntos
Capsicum/crescimento & desenvolvimento , Capsicum/genética , Frutas/crescimento & desenvolvimento , Frutas/genética , MicroRNAs/genética , Capsicum/química , Clonagem Molecular , Frutas/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/análise , RNA de Plantas/análise , RNA de Plantas/genética , Análise de Sequência de RNA , Transcriptoma
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