Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 57.975
Filtrar
Mais filtros








Intervalo de ano de publicação
1.
BMC Plant Biol ; 22(1): 144, 2022 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-35337273

RESUMO

Sophora davidii is an important plant resource in the karst region of Southwest China, but S. davidii plant-height mutants are rarely reported. Therefore, we performed phenotypic, anatomic structural, transcriptomic and metabolomic analyses to study the mechanisms responsible for S. davidii plant-height mutants. Phenotypic and anatomical observations showed that compared to the wild type, the dwarf mutant displayed a significant decrease in plant height, while the tall mutant displayed a significant increase in plant height. The dwarf mutant cells were smaller and more densely arranged, while those of the wild type and the tall mutant were larger and loosely arranged. Transcriptomic analysis revealed that differentially expressed genes (DEGs) involved in cell wall biosynthesis, expansion, phytohormone biosynthesis, signal transduction pathways, flavonoid biosynthesis and phenylpropanoid biosynthesis were significantly enriched in the S. davidii plant-height mutants. Metabolomic analysis revealed 57 significantly differential metabolites screened from both the dwarf and tall mutants. A total of 8 significantly different flavonoid compounds were annotated to LIPID MAPS, and three metabolites (chlorogenic acid, kaempferol and scopoletin) were involved in phenylpropanoid biosynthesis and flavonoid biosynthesis. These results shed light on the molecular mechanisms of plant height in S. davidii mutants and provide insight for further molecular breeding programs.


Assuntos
Sophora , Transcriptoma , Perfilação da Expressão Gênica , Metabolômica , Reguladores de Crescimento de Plantas/metabolismo , Sophora/genética , Sophora/metabolismo
2.
Gigascience ; 112022 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-35333302

RESUMO

BACKGROUND: Cassava (Manihot esculenta) is an important clonally propagated food crop in tropical and subtropical regions worldwide. Genetic gain by molecular breeding has been limited, partially because cassava is a highly heterozygous crop with a repetitive and difficult-to-assemble genome. FINDINGS: Here we demonstrate that Pacific Biosciences high-fidelity (HiFi) sequencing reads, in combination with the assembler hifiasm, produced genome assemblies at near complete haplotype resolution with higher continuity and accuracy compared to conventional long sequencing reads. We present 2 chromosome-scale haploid genomes phased with Hi-C technology for the diploid African cassava variety TME204. With consensus accuracy >QV46, contig N50 >18 Mb, BUSCO completeness of 99%, and 35k phased gene loci, it is the most accurate, continuous, complete, and haplotype-resolved cassava genome assembly so far. Ab initio gene prediction with RNA-seq data and Iso-Seq transcripts identified abundant novel gene loci, with enriched functionality related to chromatin organization, meristem development, and cell responses. During tissue development, differentially expressed transcripts of different haplotype origins were enriched for different functionality. In each tissue, 20-30% of transcripts showed allele-specific expression (ASE) differences. ASE bias was often tissue specific and inconsistent across different tissues. Direction-shifting was observed in <2% of the ASE transcripts. Despite high gene synteny, the HiFi genome assembly revealed extensive chromosome rearrangements and abundant intra-genomic and inter-genomic divergent sequences, with large structural variations mostly related to LTR retrotransposons. We use the reference-quality assemblies to build a cassava pan-genome and demonstrate its importance in representing the genetic diversity of cassava for downstream reference-guided omics analysis and breeding. CONCLUSIONS: The phased and annotated chromosome pairs allow a systematic view of the heterozygous diploid genome organization in cassava with improved accuracy, completeness, and haplotype resolution. They will be a valuable resource for cassava breeding and research. Our study may also provide insights into developing cost-effective and efficient strategies for resolving complex genomes with high resolution, accuracy, and continuity.


Assuntos
Manihot , Alelos , Cromossomos , Diploide , Haplótipos , Manihot/genética , Melhoramento Vegetal , Análise de Sequência de DNA , Transcriptoma
3.
Methods Mol Biol ; 2418: 405-424, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35119677

RESUMO

With the ability to obtain several millions of reads per sample, high-throughput RNA sequencing (RNA-Seq) enables investigation of any transcriptome at a fine resolution. Not just the messenger RNA (mRNA), but a wide variety of different RNA populations (e.g., total RNA, microRNA, long ncRNA, pre-mRNA) can also be investigated using RNA-Seq. While facilitating accurate quantification of gene expression, RNA-Seq offers the opportunity to estimate abundance of isoforms and find novel transcripts and allele-specific transcripts. In this chapter, we describe a protocol to construct an RNA-Seq library for sequencing on Illumina NGS platforms and a computational pipeline to perform RNA-Seq data analysis. The protocols described in this chapter can be applied to the analysis of differential gene expression in control versus 17ß-estradiol treatment of in vivo or in vitro systems.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Análise de Dados , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA-Seq , Transcriptoma
4.
PLoS One ; 17(5): e0268295, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35536827

RESUMO

The red color in radish taproots is an important quality index and is mainly affected by anthocyanins. However, the metabolite components and gene expression underlying dark red taproot color formation in radish remain elusive. In this study, the metabolites and gene expression patterns affecting anthocyanin biosynthesis were monitored in the dark red taproots. Comparative analysis of anthocyanin metabolites between dark red taproots and white taproots indicated that pelargonin and pelargonidin 3-O-beta-D-glucoside were the most promising dark red pigments responsible for the coloration of the taproots. Transcriptomic analysis of gene expression between dark red taproots and white taproots revealed that most of genes involved in the anthocyanin biosynthesis pathway were up-regulated in dark red taproots. In particular, RsCHS and RsDFR were the two most up-regulated genes in the dark red taproots. Moreover, the higher coexpression of two R2R3-Myb transcription factors, RsMYB1 and RsMYB2, may contribute to dark red color formation. Our work documents metabolomic and transcriptomic changes related to the dark red color formation in taproots radish and provides valuable data for anthocyanin-rich radish breeding.


Assuntos
Raphanus , Antocianinas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Melhoramento Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raphanus/genética , Raphanus/metabolismo , Transcriptoma
5.
Sci Rep ; 12(1): 7671, 2022 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-35538164

RESUMO

Since global temperature is expected to rise by 2 °C in 2050 heat stress may become the most severe environmental factor. In the study, we illustrate the application of mixed linear models for the analysis of whole transcriptome expression in livers and adrenal tissues of Sprague-Dawley rats obtained by a heat stress experiment. By applying those models, we considered four sources of variation in transcript expression, comprising transcripts (1), genes (2), Gene Ontology terms (3), and Reactome pathways (4) and focussed on accounting for the similarity within each source, which was expressed as a covariance matrix. Models based on transcripts or genes levels explained a larger proportion of log2 fold change than models fitting the functional components of Gene Ontology terms or Reactome pathways. In the liver, among the most significant genes were PNKD and TRIP12. In the adrenal tissue, one transcript of the SUCO gene was expressed more strongly in the control group than in the heat-stress group. PLEC had two transcripts, which were significantly overexpressed in the heat-stress group. PER3 was significant only on gene level. Moving to the functional scale, five Gene Ontologies and one Reactome pathway were significant in the liver. They can be grouped into ontologies related to DNA repair, histone ubiquitination, the regulation of embryonic development and cytoplasmic translation. Linear mixed models are valuable tools for the analysis of high-throughput biological data. Their main advantages are the possibility to incorporate information on covariance between observations and circumventing the problem of multiple testing.


Assuntos
Perfilação da Expressão Gênica , Transtornos de Estresse por Calor , Animais , Biodiversidade , Resposta ao Choque Térmico/genética , Modelos Lineares , Ratos , Ratos Sprague-Dawley , Temperatura , Transcriptoma
6.
BMC Genomics ; 23(1): 358, 2022 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-35538402

RESUMO

BACKGROUND AND AIM: Yak estrus is a seasonal phenomenon, probably involving epigenetic regulation of synthesis and secretion of sex hormones as well as growth and development of follicles. N6-methyladenosine (m6A) is the most common internal modification of the eukaryotic mRNA. However, there are no detailed reports on the m6A transcriptome map of yak ovary. Therefore, this study aimed to collected the yak ovarian tissues at three different states of anestrus (YO-A), estrus (YO-F), and pregnancy (YO-P), and obtained the full transcriptome m6A map in yak by MeRIP-seq. RESULTS: The HE staining revealed that the number of growing follicles and mature follicles in the ovary during the estrus period was relatively higher than those in the anestrus period and the pregnancy period. The RT-qPCR showed that the expression of METTL3, METTL14, FTO, YTHDC1 were significantly different across different periods in the ovaries, which suggests that m6A may play a regulatory role in ovarian activity. Next, we identified 20,174, 19,747 and 13,523 m6A peaks in the three ovarian samples of YO-A, YO-F and YO-P using the methylated RNA immunoprecipitation sequencing (MeRIP-seq). The m6A peaks are highly enriched in the coding sequence (CDS) region and 3'untranslated region (3'UTR) as well as the conserved sequence of "RRACH." The GO, KEGG and GSEA analysis revealed the involvement of m6A in many physiological activities of the yak's ovary during reproductive cycle. The association analysis found that some genes such as BNC1, HOMER1, BMP15, BMP6, GPX3, and WNT11 were related to ovarian functions. CONCLUSIONS: The comparison of the distribution patterns of methylation peaks in the ovarian tissues across different periods further explored the m6A markers related to the regulation of ovarian ovulation and follicular development in the yak ovary. This comprehensive map provides a solid foundation for revealing the potential function of the mRNA m6A modification in the yak ovary.


Assuntos
Ovário , Transcriptoma , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Animais , Bovinos , Epigênese Genética , Feminino , Ovário/metabolismo , Gravidez , RNA Mensageiro/genética
7.
BMC Plant Biol ; 22(1): 237, 2022 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-35538406

RESUMO

BACKGROUND: Quinoa (Chenopodium quinoa), a dicotyledonous species native to Andean region, is an emerging crop worldwide nowadays due to its high nutritional value and resistance to extreme abiotic stresses. Although it is well known that seed germination is an important and multiple physiological process, the network regulation of quinoa seed germination is largely unknown. RESULTS: Here, we performed transcriptomic study in five stages during transition from quinoa dry seed to seedling. Together with the GC-MS based metabolome analysis, we found that seed metabolism is reprogrammed with significant alteration of multiple phytohormones (especially abscisic acid) and other nutrients during the elongation of radicels. Cell-wall remodeling is another main active process happening in the early period of quinoa seed germination. Photosynthesis was fully activated at the final stage, promoting the biosynthesis of amino acids and protein to allow seedling growth. The multi-omics analysis revealed global changes in metabolic pathways and phenotype during quinoa seed germination. CONCLUSION: The transcriptomic and metabolomic landscape depicted here pave ways for further gene function elucidation and quinoa development in the future.


Assuntos
Chenopodium quinoa , Chenopodium quinoa/fisiologia , Germinação/genética , Plântula/genética , Plântula/metabolismo , Sementes , Transcriptoma
8.
BMC Genomics ; 23(1): 357, 2022 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-35538420

RESUMO

BACKGROUND: The DoG (Delay of Germination1) family plays a key regulatory role in seed dormancy and germination. However, to date, there is no complete genomic overview of the DoG gene family of any economically valuable crop, including moso bamboo (Phyllostachys edulis), and no studies have been conducted to characterize its expression profile. To identify the DoG gene members of moso bamboo (PeDoG) and to investigate their family structural features and tissue expression profile characteristics, a study was conducted. Based on the whole genome and differential transcriptome data, in this investigation, we have scrutinized the physicochemical properties, gene structure, cis-acting elements, phylogenetic relationships, conserved structural (CS) domains, CS motifs and expression patterns of the PeDoG1 family of moso bamboo. RESULTS: The DoG family genes of moso bamboo were found distributed across 16 chromosomal scaffolds with 24 members. All members were found to carry DoG1 structural domains, while 23 members additionally possessed basic leucine zipper (bZIP) structural domains. We could divide the PeDoG genes into three subfamilies based on phylogenetic relationships. Covariance analysis revealed that tandem duplication was the main driver of amplification of the PeDoG genes. The upstream promoter of these genes containing several cis-acting elements indicates a plausible role in abiotic stress and hormone induction. Gene expression pattern according to transcriptome data revealed participation of the PeDoG genes in tissue and organ development. Analysis using Short Time-series Expression Miner (STEM) tool revealed that the PeDoG gene family is also associated with rapid early shoot growth. Gene ontology (GO) and KEGG analyses showed a dual role of the PeDoG genes. We found that PeDoGs has a possible role as bZIP transcription factors by regulating Polar like1 (PL1) gene expression, and thereby playing a disease response role in moso bamboo. Quantitative gene expression of the PeDoG genes revealed that they were abundantly expressed in roots and leaves, and could be induced in response to gibberellin (GA). CONCLUSION: In this study, we found that the PeDoG genes are involved in a wide range of activities such as growth and development, stress response and transcription. This forms the first report of PeDoG genes and their potential roles in moso bamboo.


Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Poaceae/genética , Poaceae/metabolismo , Transcriptoma
9.
Genome Biol ; 23(1): 113, 2022 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-35538548

RESUMO

BACKGROUND: Colorectal cancer (CRC) consensus molecular subtypes (CMS) have different immunological, stromal cell, and clinicopathological characteristics. Single-cell characterization of CMS subtype tumor microenvironments is required to elucidate mechanisms of tumor and stroma cell contributions to pathogenesis which may advance subtype-specific therapeutic development. We interrogate racially diverse human CRC samples and analyze multiple independent external cohorts for a total of 487,829 single cells enabling high-resolution depiction of the cellular diversity and heterogeneity within the tumor and microenvironmental cells. RESULTS: Tumor cells recapitulate individual CMS subgroups yet exhibit significant intratumoral CMS heterogeneity. Both CMS1 microsatellite instability (MSI-H) CRCs and microsatellite stable (MSS) CRC demonstrate similar pathway activations at the tumor epithelial level. However, CD8+ cytotoxic T cell phenotype infiltration in MSI-H CRCs may explain why these tumors respond to immune checkpoint inhibitors. Cellular transcriptomic profiles in CRC exist in a tumor immune stromal continuum in contrast to discrete subtypes proposed by studies utilizing bulk transcriptomics. We note a dichotomy in tumor microenvironments across CMS subgroups exists by which patients with high cancer-associated fibroblasts (CAFs) and C1Q+TAM content exhibit poor outcomes, providing a higher level of personalization and precision than would distinct subtypes. Additionally, we discover CAF subtypes known to be associated with immunotherapy resistance. CONCLUSIONS: Distinct CAFs and C1Q+ TAMs are sufficient to explain CMS predictive ability and a simpler signature based on these cellular phenotypes could stratify CRC patient prognosis with greater precision. Therapeutically targeting specific CAF subtypes and C1Q + TAMs may promote immunotherapy responses in CRC patients.


Assuntos
Neoplasias Colorretais , Complemento C1q , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Complemento C1q/genética , Complemento C1q/uso terapêutico , Humanos , Instabilidade de Microssatélites , Transcriptoma , Microambiente Tumoral/genética
10.
Elife ; 112022 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-35543322

RESUMO

Human myelin disorders are commonly studied in mouse models. Since both clades evolutionarily diverged approximately 85 million years ago, it is critical to know to what extent the myelin protein composition has remained similar. Here, we use quantitative proteomics to analyze myelin purified from human white matter and find that the relative abundance of the structural myelin proteins PLP, MBP, CNP, and SEPTIN8 correlates well with that in C57Bl/6N mice. Conversely, multiple other proteins were identified exclusively or predominantly in human or mouse myelin. This is exemplified by peripheral myelin protein 2 (PMP2), which was specific to human central nervous system myelin, while tetraspanin-2 (TSPAN2) and connexin-29 (CX29/GJC3) were confined to mouse myelin. Assessing published scRNA-seq-datasets, human and mouse oligodendrocytes display well-correlating transcriptome profiles but divergent expression of distinct genes, including Pmp2, Tspan2, and Gjc3. A searchable web interface is accessible via www.mpinat.mpg.de/myelin. Species-dependent diversity of oligodendroglial mRNA expression and myelin protein composition can be informative when translating from mouse models to humans.


Like the electrical wires in our homes, the processes of nerve cells ­ the axons, thin extensions that project from the cell bodies ­ need to be insulated to work effectively. This insulation takes the form of layers of a membrane called myelin, which is made of proteins and fats and produced by specialized cells called oligodendrocytes in the brain and the spinal cord. If this layer of insulation becomes damaged, the electrical impulses travelling along the nerves slow down, affecting the ability to walk, speak, see or think. This is the cause of several illnesses, including multiple sclerosis and a group of rare genetic diseases known as leukodystrophies. A lot of the research into myelin, oligodendrocytes and the diseases caused by myelin damage uses mice as an experimental model for humans. Using mice for this type of research is appropriate because of the ethical and technical limitations of experiments on humans. This approach can be highly effective because mice and humans share a large proportion of their genes. However, there are many obvious physical differences between the two species, making it important to determine whether the results of experiments performed in mice are applicable to humans. To do this, it is necessary to understand how myelin differs between these two species at the molecular level. Gargareta, Reuschenbach, Siems, Sun et al. approached this problem by studying the proteins found in myelin isolated from the brains of people who had passed away and donated their organs for scientific research. They used a technique called mass spectrometry, which identifies molecules based on their weight, to produce a list of proteins in human myelin that could then be compared to existing data from mouse myelin. This analysis showed that myelin is very similar in both species, but some proteins only appear in humans or in mice. Gargareta, Reuschenbach, Siems, Sun et al. then compared which genes are turned on in the oligodendrocytes making the myelin. The results of this comparison reflected most of the differences and similarities seen in the myelin proteins. Despite the similarities identified by Gargareta, Reuschenbach, Siems, Sun et al., it became evident that there are unexpected differences between the myelin of humans and mice that will need to be considered when applying results from mice research to humans. To enable this endeavor, Gargareta, Reuschenbach, Siems, Sun et al. have created a searchable web interface of the proteins in myelin and the genes expressed in oligodendrocytes in the two species.


Assuntos
Bainha de Mielina , Proteoma , Animais , Conexinas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Proteína Proteolipídica de Mielina , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Oligodendroglia/metabolismo , Proteoma/metabolismo , Tetraspaninas/genética , Tetraspaninas/metabolismo , Transcriptoma
11.
Front Immunol ; 13: 840811, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35515000

RESUMO

Background: The tumor microenvironment (TME) plays a pivotal role in cancer progression in papillary thyroid carcinoma (PTC), yet the composition and the phenotype of cells within the TME in bilateral PTC are poorly understood. Methods: We performed unbiased transcriptome-wide single-cell RNA sequencing (scRNA-seq) analysis on 29,561 cells from 3 pairs of bilateral PTC and 1 non-tumor thyroid sample. The results of the analysis were validated by a large-scale bulk transcriptomic dataset deposited in The Cancer Genome Atlas (TCGA) database. Results: Our integrative analysis of thyroid follicular cells revealed 42 signaling pathways enriched in malignant follicular cells, including cytokine-cytokine receptor interaction, PI3K/Akt signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, and tumor necrosis factor (TNF) signaling pathway. A 6-gene signature (CXCL3, CXCL1, IL1A, CCL5, TNFRSF12A, and IL18) in the cytokine-cytokine receptor interaction pathway was constructed to predict the prognosis of patients with PTC, with high risk scores being associated with decreased overall survival [hazard ratio (HR) = 3.863, 95% CI = 2.233-6.682, p < 0.001]. Gene set variation analysis (GSVA) indicated that the pathways enriched in bilateral PTC were significantly different, indicating great heterogeneity in bilateral PTC, even with the same BRAF V600E mutation. Comprehensive analysis of T cells revealed that the proportion of CD8+ tissue-resident memory T cells expressing IFNG decreased in tumor samples with advanced N stage. Within the myeloid compartment, the ratio of suppressive M2-like to pro-inflammatory M1-like macrophages increased with advanced disease stage, which was confirmed in the bulk dataset using transcriptomic profiles. In addition, we also identified numerous biologically critical interactions among myeloid cells, T cells, and follicular cells, which were related to T-cell recruitment, M2-like macrophage polarization, malignant follicular cell progression, and T-cell inhibitory signaling. Conclusion: Our integrative analyses revealed great inter-tumor heterogeneity within the TME in bilateral PTC, which will offer assistance for precise diagnosis and treatment.


Assuntos
Neoplasias da Glândula Tireoide , Transcriptoma , Citocinas/genética , Perfilação da Expressão Gênica , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Citocinas/genética , Análise de Célula Única , Câncer Papilífero da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Microambiente Tumoral/genética
12.
Front Cell Infect Microbiol ; 12: 853064, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35521218

RESUMO

Salmonella enterica serovars Enteritidis (S. Enteritidis) can survive extreme food processing environments including bactericidal sodium hypochlorite (NaClO) treatments generally recognized as safe. In order to reveal the molecular regulatory mechanisms underlying the phenotypes, the overall regulation of genes at the transcription level in S. Enteritidis after NaClO stimulation were investigated by RNA-sequencing. We identified 1399 differentially expressed genes (DEG) of S. Enteritidis strain CVCC 1806 following treatment in liquid culture with 100 mg/L NaClO for 20 min (915 upregulated and 484 downregulated). NaClO stress affects the transcription of genes related to a range of important biomolecular processes such as membrane damage, membrane transport function, energy metabolism, oxidative stress, DNA repair, and other important processes in Salmonella enterica. First, NaClO affects the structural stability of cell membranes, which induces the expression of a range of outer and inner membrane proteins. This may lead to changes in cell membrane permeability, accelerating the frequency of DNA conversion and contributing to the production of drug-resistant bacteria. In addition, the expression of exocytosis pump genes (emrB, yceE, ydhE, and ydhC) was able to expel NaClO from the cell, thereby increasing bacterial tolerance to NaClO. Secondly, downregulation of genes related to the Kdp-ATPase transporter system (kdpABC) and the amino acid transporter system (aroP, brnQ and livF) may to some extent reduce active transport by bacterial cells, thereby reducing their own metabolism and the entry of disinfectants. Downregulation of genes related to the tricarboxylic acid (TCA) cycle may drive bacterial cells into a viable but non-culturable (VBNC) state, resisting NaClO attack by reducing energy metabolism. In addition, significant upregulation of genes related to oxidative stress could mitigate damage caused by disinfectants by eliminating alkyl hydroperoxides, while upregulation of genes related to DNA repair could repair damage to bacterial cells caused by oxidative stress. Therefore, this study indicated that S. Enteritidis has genomic mechanisms to adapt to NaClO stress.


Assuntos
Desinfetantes , Salmonella enterica , Desinfetantes/metabolismo , Desinfetantes/farmacologia , Salmonella enterica/genética , Salmonella enteritidis/genética , Sorogrupo , Hipoclorito de Sódio/farmacologia , Transcriptoma
13.
Methods Mol Biol ; 2477: 71-77, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524112

RESUMO

Direct RNA sequencing (dRNA-seq) simultaneously enables the detection of RNA modifications and characterization of full-length transcripts. In principle, full-length native RNA molecule is translocated through the nanopore by a motor protein while a sensor measures ionic current shifts. Then, the current shifts are interpreted by an algorithm that turn out to RNA sequence. Currently, the standard protocol of dRNA-seq provided by Oxford Nanopore Technologies (ONT) allows to directly ligate and sequence only polyadenylated RNA (poly(A) RNA). Here, we describe a method of dRNA-seq that can be applied for both poly(A) RNA and non-poly(A) tailed-RNA.


Assuntos
Sequenciamento por Nanoporos , Nanoporos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/genética , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , Transcriptoma
14.
Methods Mol Biol ; 2477: 79-89, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524113

RESUMO

Computational approaches are the main approaches used in genome annotation. However, accuracy is low. Untranslated regions are not identified, complex isoforms are not predicted correctly and discovery rate of noncoding RNA is low. RNA-seq has revolutionized transcriptome reconstruction over the last decade. However, fragmentation included in cDNA sequencing leads to information loss, requiring transcripts to be assembled and reconstructed, thus affecting the accuracy of reconstructed transcriptome. Recently, long-read sequencing has been introduced with technologies such as Oxford Nanopore sequencing. cDNA is sequenced directly without fragmentation producing long reads that don't need to be assembled keeping the transcript structure intact and increasing the accuracy of transcriptome reconstruction.Here we present a protocol and a pipeline to reconstruct the transcriptome of compact genomes including yeasts. It involves generating full-length cDNA and using Oxford Nanopore ligation-based sequencing kit to sequence multiple samples in the same run. The pipeline (1) strands the generated long reads, (2) corrects the reads by mapping them to the reference genome, (3) identifies transcripts including 5'UTR and 3'UTR, (4) profiles the isoforms, filtering out artifacts resulting from low accuracy in sequencing, and (5) improves accuracy of provided annotations. Using long reads improves the accuracy of transcriptome reconstruction and helps in discovering a significant number of novel RNAs.


Assuntos
Sequenciamento por Nanoporos , DNA Complementar/genética , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Isoformas de Proteínas/genética , Saccharomyces cerevisiae/genética , Transcriptoma
15.
Methods Mol Biol ; 2477: 91-103, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524114

RESUMO

In order to perform a well-balanced comparative transcriptomic analysis, the reference genome and annotations for all species included in the comparison must be of a similar quality and completeness. Frequently, comparative transcriptomic analyses include non-model organisms whose annotations are not as well curated; this inequality can lead to biases.To avoid potential biases stemming from incomplete annotations, a comparative transcriptomic analysis can incorporate de novo transcriptome assemblies for each species, which reduces this disparity. This chapter covers all of the steps which are necessary to run a comparative transcriptomic analysis with de novo transcriptome assemblies, from the first step of the experimental design to the sequencing, and ultimately the bioinformatic analysis.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , RNA , Biologia Computacional , Análise de Sequência de RNA , Transcriptoma
16.
BMC Genomics ; 23(1): 349, 2022 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-35524177

RESUMO

BACKGROUND: Real-time quantitative PCR is a widely used method for gene expression analyses in various organisms. Its accuracy mainly relies on the correct selection of reference genes. Any experimental plan involving real-time PCR needs to evaluate the characteristics of the samples to be examined and the relative stability of reference genes. Most studies in mollusks rely on reference genes commonly used in vertebrates. RESULTS: In this study, we focused on the transcriptome of the bivalve mollusk Mytilus galloprovincialis in physiological state to identify suitable reference genes in several adult tissues. Candidate genes with highly stable expression across 51 RNA-seq datasets from multiple tissues were selected through genome-wide bioinformatics analysis. This approach led to the identification of three genes (Rpl14, Rpl32 and Rpl34), whose suitability was evaluated together with 7 other reference genes commonly reported in literature (Act, Cyp-A, Ef1α, Gapdh, 18S, 28S and Rps4). The stability analyses performed with geNorm, NormFinder and Bestkeeper identified specific either single or pairs of genes suitable as references for gene expression analyses in specific tissues and revealed the Act/Cyp-A pair as the most appropriate to analyze gene expression across different tissues. CONCLUSION: Mytilus galloprovincialis is a model system increasingly used in ecotoxicology and molecular studies. Our transcriptome-wide approach represents the first comprehensive investigation aimed at the identification of suitable reference genes for expression studies in this species.


Assuntos
Perfilação da Expressão Gênica , Mytilus , Animais , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Mytilus/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Padrões de Referência , Transcriptoma
17.
Parasit Vectors ; 15(1): 159, 2022 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-35524281

RESUMO

BACKGROUND: Ivermectin (IVM) is one of the most important and widely used anthelmintics in veterinary medicine. However, its efficacy is increasingly compromised by widespread resistance, and the exact mechanism of IVM resistance remains unclear for most parasitic nematodes, including Haemonchus contortus, a blood-sucking parasitic nematode of small ruminants. METHODS: In this study, an H. contortus IVM-resistant strain from Zhaosu, Xinjiang, China, was isolated and assessed by the control test, faecal egg count reduction test (FECRT) and the larval development assay (LDA). Subsequently, comparative analyses on the transcriptomics of IVM-susceptible and IVM-resistant adult worms of this parasite were carried out using RNA sequencing (RNA-seq) and bioinformatics. RESULTS: In total, 543 (416 known, 127 novel) and 359 (309 known, 50 novel) differentially expressed genes (DEGs) were identified in male and female adult worms of the resistant strain compared with those of the susceptible strain, respectively. In addition to several previously known candidate genes which were supposed to be associated with IVM resistance and whose functions were involved in receptor activity, transport, and detoxification, we found some new potential target genes, including those related to lipid metabolism, structural constituent of cuticle, and important pathways such as antigen processing and presentation, lysosome, autophagy, apoptosis, and NOD1-like receptor signalling pathways. Finally, the results of quantitative real-time polymerase chain reaction confirmed that the transcriptional profiles of selected DEGs (male: 8 genes, female: 10 genes) were consistent with those obtained by the RNA-seq. CONCLUSIONS: Our results indicate that IVM has multiple effects, including both neuromuscular and non-neuromuscular targets, and provide valuable information for further studies on the IVM resistance mechanism in H. contortus.


Assuntos
Anti-Helmínticos , Hemoncose , Haemonchus , Doenças dos Ovinos , Animais , Anti-Helmínticos/farmacologia , Anti-Helmínticos/uso terapêutico , Resistência a Medicamentos/genética , Feminino , Hemoncose/parasitologia , Haemonchus/genética , Ivermectina/farmacologia , Ivermectina/uso terapêutico , Masculino , Ovinos/genética , Doenças dos Ovinos/parasitologia , Transcriptoma
18.
BMC Plant Biol ; 22(1): 233, 2022 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-35525915

RESUMO

BACKGROUND: Soil salinization is a threat to food security. China is rich in saline land resources for potential and current utilization. The cultivation and promotion of salt-tolerant rice varieties can greatly improve the utilization of this saline land. The super hybrid rice Chaoyouqianhao (CY1000) is one of the most salt-tolerant rice varieties and is widely used, but the molecular mechanism underlying its salt tolerance is not clear. RESULTS: In this study, the characteristics of CY1000 and its parents were evaluated in the field and laboratory. The results showed that aboveground parts of CY1000 were barely influenced by salt stress, while the roots were less affected than those of its parents. A comparative transcriptomic strategy was used to analyze the differences in the response to salt stress among the male and female parents of CY1000 at the seedling stage and the model indica rice 93-11. We found that the salt tolerance of CY1000 was mainly inherited from its male parent R900, and its female parent GX24S showed hardly any salt tolerance. To adapt to salt stress, CY1000 and R900 upregulated the expression of genes associated with soluble component synthesis and cell wall synthesis and other related genes and downregulated the expression of most genes related to growth material acquisition and consumption. In CY1000 and R900, the expression of genes encoding some novel key proteins in the ubiquitination pathway was significantly upregulated. After treatment with MG-132, the salt tolerance of CY1000 and R900 was significantly decreased and was almost the same as that of the wild type after salt stress treatment, indicating that ubiquitination played an important role in the salt tolerance mechanism of CY1000. At the same time, we found that some transcription factors were also involved in the salt stress response, with some transcription factors responding only in hybrid CY1000, suggesting that salt tolerance heterosis might be regulated by transcription factors in rice. CONCLUSION: Our results revealed that the ubiquitination pathway is important for salt tolerance in rice, and several novel candidate genes were identified to reveal a novel salt tolerance regulation network. Additionally, our work will help clarify the mechanism of heterosis in rice. Further exploration of the molecular mechanism underlying the salt tolerance of CY1000 can provide a theoretical basis for breeding new salt-tolerant rice varieties.


Assuntos
Oryza , Regulação da Expressão Gênica de Plantas , Oryza/metabolismo , Melhoramento Vegetal , Estresse Salino , Fatores de Transcrição/genética , Transcriptoma
19.
BMC Genomics ; 23(1): 351, 2022 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-35525921

RESUMO

BACKGROUND: Immune status in the tumor microenvironment is an important determinant of cancer progression and patient prognosis. Although a higher immune activity is often associated with a better prognosis, this trend is not absolute and differs across cancer types. We aimed to give insights into why some cancers do not show better survival despite higher immunity by assessing the relationship between different biological factors, including cytotoxicity, and patient prognosis in various cancer types using RNA-seq data collected by The Cancer Genome Atlas. RESULTS: Results showed that a higher immune activity was associated with worse overall survival in patients with uveal melanoma and low-grade glioma, which are cancers of immune-privileged sites. In these cancers, epithelial or endothelial mesenchymal transition and inflammatory state as well as immune activation had a notable negative correlation with patient survival. Further analysis using additional single-cell data of uveal melanoma and glioma revealed that epithelial or endothelial mesenchymal transition was mainly induced in retinal pigment cells or endothelial cells that comprise the blood-retinal and blood-brain barriers, which are unique structures of the eye and central nervous system, respectively. Inflammation was mainly promoted by macrophages, and their infiltration increased significantly in response to immune activation. Furthermore, we found the expression of inflammatory chemokines, particularly CCL5, was strongly correlated with immune activity and associated with poor survival, particularly in these cancers, suggesting that these inflammatory mediators are potential molecular targets for therapeutics. CONCLUSIONS: In uveal melanoma and low-grade glioma, inflammation from macrophages and epithelial or endothelial mesenchymal transition are particularly associated with a poor prognosis. This implies that they loosen the structures of the blood barrier and impair homeostasis and further recruit immune cells, which could result in a feedback loop of additional inflammatory effects leading to runaway conditions.


Assuntos
Glioma , Transcriptoma , Células Endoteliais , Glioma/genética , Humanos , Inflamação , Melanoma , Prognóstico , Microambiente Tumoral/genética , Neoplasias Uveais
20.
Biochemistry (Mosc) ; 87(3): 269-293, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35526848

RESUMO

A unique set of features and characteristics of species of the Cnidaria phylum is the one reason that makes them a model for a various studies. The plasticity of a life cycle and the processes of cell differentiation and development of an integral multicellular organism associated with it are of a specific scientific interest. A new stage of development of molecular genetic methods, including methods for high-throughput genome, transcriptome, and epigenome sequencing, both at the level of the whole organism and at the level of individual cells, makes it possible to obtain a detailed picture of the development of these animals. This review examines some modern approaches and advances in the reconstruction of the processes of ontogenesis of cnidarians by studying the regulatory signal transduction pathways and their interactions.


Assuntos
Cnidários , Animais , Cnidários/genética , Cnidários/metabolismo , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Transdução de Sinais , Transcriptoma
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA