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1.
BMC Bioinformatics ; 20(1): 418, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409293

RESUMO

BACKGROUND: Standard RNAseq methods using bulk RNA and recent single-cell RNAseq methods use DNA barcodes to identify samples and cells, and the barcoded cDNAs are pooled into a library pool before high throughput sequencing. In cases of single-cell and low-input RNAseq methods, the library is further amplified by PCR after the pooling. Preparation of hundreds or more samples for a large study often requires multiple library pools. However, sometimes correlation between expression profiles among the libraries is low and batch effect biases make integration of data between library pools difficult. RESULTS: We investigated 166 technical replicates in 14 RNAseq libraries made using the STRT method. The patterns of the library biases differed by genes, and uneven library yields were associated with library biases. The former bias was corrected using the NBGLM-LBC algorithm, which we present in the current study. The latter bias could not be corrected directly, but could be solved by omitting libraries with particularly low yields. A simulation experiment suggested that the library bias correction using NBGLM-LBC requires a consistent sample layout. The NBGLM-LBC correction method was applied to an expression profile for a cohort study of childhood acute respiratory illness, and the library biases were resolved. CONCLUSIONS: The R source code for the library bias correction named NBGLM-LBC is available at https://shka.github.io/NBGLM-LBC and https://shka.bitbucket.io/NBGLM-LBC . This method is applicable to correct the library biases in various studies that use highly multiplexed sequencing-based profiling methods with a consistent sample layout with samples to be compared (e.g., "cases" and "controls") equally distributed in each library.


Assuntos
Biblioteca Gênica , Análise de Sequência de RNA/métodos , Transcriptoma , Linhagem Celular , Análise por Conglomerados , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Componente Principal , RNA/química , RNA/metabolismo , Interface Usuário-Computador
2.
Yi Chuan ; 41(8): 754-760, 2019 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-31447426

RESUMO

Breeding by design is a new concept proposed in the beginning of the century. It refers to the breeding of varieties by crop design utilizing favorable alleles dispersed in different genetic resources in a genome. In the past 20 years, we have proposed a "three-step" strategy to carry out the research on breeding by design in rice. Firstly, we constructed a library of chromosomal single-segment substitution lines (SSSLs) by using of Huajingxian74 (HJX74), an elite xian (indica) variety from South China as the recipient and 43 accessions of seven species of rice AA genome as donors. The genes in the substituted segments of SSSLs were then detected. Breeding by design was conducted by selecting the favorable genes from the SSSL library. Our practice indicates that the SSSL library is a powerful platform for breeding by design and various "traits", "lines" and "varieties" of rice can be designed and bred by utilizing abundant genes in the SSSL library. Here, we introduce the platform of the HJX74-SSSL library and our work of breeding by design on the platform. It will provide a case study for crop design.


Assuntos
Biblioteca Gênica , Oryza/genética , Melhoramento Vegetal , China , Genes de Plantas
3.
Microbiol Res ; 228: 126306, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31422233

RESUMO

The mariner transposon family of Himar1 has been widely used for the random mutagenesis of bacteria to generate single insertions into the chromosome. Here, a versatile toolbox of mariner transposon derivatives was generated and applied to the functional genomics investigation of fish pathogen Edwardsiella piscicida. In this study, we combined the merits of the random mutagenesis of mariner transposon and common efficient reporter marker genes or regulatory elements, mCherry, gfp, luxAB, lacZ, sacBR, and PBAD and antibiotic resistance cassettes to construct a series of derivative transposon vectors, pMmch, pMKGR, pMCGR, pMXKGR, pMLKGR, pMSGR, and pMPR, based on the initial transposon pMar2xT7. The function and effectiveness of the modified transposons were verified by introducing them into E. piscicida EIB202. Based on the toolbox, a transposon insertion mutant library containing approximately 3.0 × 105 distinct mutants was constructed to explore the upstream regulators of esrB, the master regulator of the type III and type VI secretion systems (T3/T6SS) in E. piscicida. Following analysis by Con-ARTIST, ETAE_3474, annotated as fabR and involved in fatty acid metabolism, was screened out and identified as a novel regulator mediating T3SS and T6SS expression. In addition, the fabR mutants displayed critical virulence attenuation in turbot. Due to the broad-range host compatibility of mariner transposons, the newly built transposon toolbox can be applied for functional genomics studies in various bacteria.


Assuntos
Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , Edwardsiella/genética , Regulação Bacteriana da Expressão Gênica/genética , Testes Genéticos/métodos , Genoma Bacteriano/genética , Animais , Mapeamento Cromossômico , Farmacorresistência Bacteriana/genética , Ácidos Graxos/metabolismo , Doenças dos Peixes/microbiologia , Biblioteca Gênica , Genes Reporter/genética , Genômica/métodos , Mutagênese Insercional/métodos , Fatores de Transcrição/genética , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo VI/genética , Virulência , Fatores de Virulência/genética
4.
BMC Genomics ; 20(Suppl 7): 536, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31291895

RESUMO

BACKGROUND: Massively parallel reporter assays (MPRAs) enable high-throughput functional evaluation of various DNA regulatory elements and their mutant variants. The assays are based on construction of highly diverse plasmid libraries containing two variable fragments, a region of interest (a sequence under study; ROI) and a barcode (BC) used to uniquely tag each ROI, which are separated by a constant spacer sequence. The sequences of BC-ROI combinations present in the libraries may be either known a priori or not. In the latter case, it is necessary to identify these combinations before performing functional experiments. Typically, this is done by PCR amplification of the BC-ROI regions with flanking primers, followed by next-generation sequencing (NGS) of the products. However, chimeric DNA molecules formed on templates with identical spacer fragment during the amplification process may substantially hamper the identification of genuine BC-ROI combinations, and as a result lower the performance of the assays. RESULTS: To identify settings that minimize formation of chimeric products we tested a number of PCR amplification parameters, such as conventional and emulsion types of PCR, one- or two-round amplification strategies, amount of DNA template, number of PCR cycles, and the duration of the extension step. Using specific MPRA libraries as templates, we found that the two-round amplification of the BC-ROI regions with a very low initial template amount, an elongated extension step, and a specific number of PCR cycles result in as low as 0.30 and 0.32% of chimeric products for emulsion and conventional PCR approaches, respectively. CONCLUSIONS: We have identified PCR parameters that ensure synthesis of specific (non-chimeric) products from highly diverse MPRA plasmid libraries. In addition, we found that there is a negligible difference in performance of emulsion and conventional PCR approaches performed with the identified settings.


Assuntos
DNA/genética , Biblioteca Gênica , Plasmídeos , Reação em Cadeia da Polimerase/métodos , Primers do DNA , Sequenciamento de Nucleotídeos em Larga Escala , Moldes Genéticos
5.
Zhongguo Zhong Yao Za Zhi ; 44(12): 2421-2432, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31359707

RESUMO

With the development of various biotechnology,the research on molecular genetics of medicinal plants has gradually deepened. In this paper,the research system of molecular genetics of medicinal plants was proposed for the first time,which was elaborated from the aspects of genetic resources,genome,gene function and research methods. The application fields of medicinal plant mainly contain species identification,molecular breeding and biosynthesis. The research directions of molecular genetics of medicinal plants in genetic resources,model platform,synthetic biology and molecular breeding were put forward,which include 1 000 genome projects of medicinal plants,model species and mutant libraries,gene original libraries of heterologous synthetic systems,construction gene original library and specific chassis cells in heterologous synthesis system of active ingredient,breeding of new varieties of medicinal plants with high active ingredient and high resistance based on molecular markers andtransgenes.


Assuntos
Biologia Molecular/tendências , Plantas Medicinais/genética , Biotecnologia , Biblioteca Gênica , Marcadores Genéticos , Genoma de Planta , Melhoramento Vegetal , Pesquisa , Transgenes
6.
Nat Commun ; 10(1): 2948, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270316

RESUMO

CRISPR-Cas systems inherently multiplex through CRISPR arrays-whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Biblioteca Gênica , Técnicas Genéticas , RNA/biossíntese , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , DNA/genética , Endonucleases/metabolismo , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Plasmídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo
7.
Nat Commun ; 10(1): 2960, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273196

RESUMO

Clone collections of modified strains ("libraries") are a major resource for systematic studies with the yeast Saccharomyces cerevisiae. Construction of such libraries is time-consuming, costly and confined to the genetic background of a specific yeast strain. To overcome these limitations, we present CRISPR-Cas12a (Cpf1)-assisted tag library engineering (CASTLING) for multiplexed strain construction. CASTLING uses microarray-synthesized oligonucleotide pools and in vitro recombineering to program the genomic insertion of long DNA constructs via homologous recombination. One simple transformation yields pooled libraries with >90% of correctly tagged clones. Up to several hundred genes can be tagged in a single step and, on a genomic scale, approximately half of all genes are tagged with only ~10-fold oversampling. We report several parameters that affect tagging success and provide a quantitative targeted next-generation sequencing method to analyze such pooled collections. Thus, CASTLING unlocks avenues for increasing throughput in functional genomics and cell biology research.


Assuntos
Sistemas CRISPR-Cas/genética , Técnicas Genéticas , Saccharomyces cerevisiae/genética , Células Clonais , Biblioteca Gênica , Engenharia Genética , Genoma Fúngico , Proteínas de Fluorescência Verde/metabolismo , Proteínas Nucleares/metabolismo
8.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(6): 557-562, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31292061

RESUMO

Objective To construct a random mutagenesis library of 3E1D7, a chimerical antibody against c-mesenchymal epithelial transition factor (c-Met), using mammalian cell surface display. Methods Antibody genes with randomly mutated complementarity-determining region 3 (CDR3) part were inserted into the mammalian expression plasmid pSZI-CD to construct the random mutagenesis library using double enzyme digestion. Reconstructed plasmids were then cloned into CHO cells by transfection. The expression level of antibodies on the surface of CHO cells was checked by C6 PLUS flow cytometry. Results 3E1D7 random mutagenesis library was successfully constructed with a volume of 5.52×106 in diversity on gene level. Sequence analysis showed that all 20 clones randomly picked from the library coded for 20 different mutated amino acid sequences in open reading frames. After transfection, the expression of full-length antibodies on CHO cell surfaces could be detected by flow cytometry. Conclusion A random mutagenesis library of a certain anti-c-Met antibody has been successfully constructed with an exhibitable diversity of 5.52×106, which would be a useful platform for further screening of therapeutic antibodies.


Assuntos
Biblioteca Gênica , Vetores Genéticos , Mutagênese , Sequência de Aminoácidos , Animais , Anticorpos/química , Cricetinae , Cricetulus , Fases de Leitura Aberta , Plasmídeos/genética , Transfecção
9.
Gene ; 712: 143962, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31288057

RESUMO

Veratrum nigrum is protected plant of Melanthiaceae family, able to synthetize unique steroidal alkaloids important for pharmacy. Transcriptomes from leaves, stems and rhizomes of in vitro maintained V. nigrum plants were sequenced and annotated for genes and markers discovery. Sequencing of samples derived from the different organs resulted in a total of 108,511 contigs with a mean length of 596 bp. Transcripts derived from leaf and stalk were annotated at 28%, and 38% in Nr nucleotide database, respectively. The sequencing revealed 949 unigenes related with lipid metabolism, including 73 transcripts involved in steroids and genus-specific steroid alkaloids biosynthesis. Additionally, 3203 candidate SSRs markers we identified in unigenes with average density of one SSR locus every 6.2 kb sequence. Unraveling of biochemical machinery of the pathway responsible for steroidal alkaloids will open possibility to design and optimize biotechnological process. The transcriptomic data provide valuable resources for biochemical, molecular genetics, comparative transcriptomics, functional genomics, ecological and evolutionary studies of V. nigrum.


Assuntos
Alcaloides/biossíntese , Regulação da Expressão Gênica de Plantas , Esteroides/biossíntese , Transcriptoma , Veratrum/metabolismo , Mapeamento de Sequências Contíguas , DNA Complementar/metabolismo , Biblioteca Gênica , Ontologia Genética , Marcadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Repetições de Microssatélites , Anotação de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Análise de Sequência de RNA
10.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(6): 692-698, 2019 Jun 30.
Artigo em Chinês | MEDLINE | ID: mdl-31270048

RESUMO

OBJECTIVE: To optimize DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing (Native ChIP-seq) to obtain high-quality Native ChIP-seq data. METHODS: Human nasopharyngeal carcinoma HONE1 cell lysate was digested with MNase for release of the nucleosomes, and the histone-DNA complexes were immunoprecipitated with specific antibodies. The protein component in the precipitate was digested with proteinase K followed by DNA purification; the DNA library was constructed for sequence analysis. RESULTS: Compared with the conventional DNA library construction, Tn5 transposase method allowed direct enrichment of the target DNA after Tn5 fragmentation, which was simple, time-saving and more efficient. The IGV visualized map showed that the information obtained by the two library construction methods was consistent. The sequencing data obtained by the two methods revealed more signal enrichment with Tn5 transposase library construction than with the conventional approach. H3K4me3 ChIP results showed a good reproducibility after Tn5 transposase library construction with a signal-to-noise ratio above 50%. CONCLUSIONS: Tn5 transposase method improves the efficiency of DNA library construction and the results of subsequent sequence analysis, and is especially suitable for detecting histone modification in the DNA to provide a better technical option for epigenetic studies.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Imunoprecipitação da Cromatina , DNA , Biblioteca Gênica , Humanos , Reprodutibilidade dos Testes , Análise de Sequência de DNA
11.
Biochemistry (Mosc) ; 84(3): 250-262, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31221063

RESUMO

Clonal composition of human multipotent mesenchymal stromal cells (MMSCs) labeled with lentiviral vectors carrying genetic barcodes was studied. MMSCs were transduced with a cloned library of self-inactivating lentiviral vectors carrying 667 unique barcodes. At each cell culture passage, 120 cells were plated one cell per well in 96-well plates. The efficiency of cloning and labeling of the clonogenic cells was determined. DNA was extracted from the cell-derived colonies, and the barcodes were identified by Sanger sequencing. Also, DNA was extracted from the total MMSC population at each passage to analyze the diversity and representation of barcodes by deep sequencing using the Illumina platform. It was shown that the portion of MMSCs labeled with the lentiviral vectors remained stable in the passaged cells. Because of the high multiplicity of infection, the labeling procedure could decrease the proliferative potential of MMSCs. Identification of barcodes in individual cell clones confirmed the polyclonal character of the MMSC population. Clonal composition of MMSCs changed significantly with the passages due to the depletion of proliferative potential of most cells. Large clones were found at the first passage; at later passages, many small clones with a limited proliferative potential were detected in the population. The results of deep sequencing confirmed changes in the clonal composition of MMSCs. The polyclonal MMSC population contained only a small number of cells with a high proliferative potential, some of which could be stem cells. MMSCs with a high proliferative potential were detected more often in the earliest passages. In this regard, we would recommend to use MMSCs of early passages for regenerative medicine applications based on cell proliferation.


Assuntos
Evolução Clonal/genética , Células Clonais/metabolismo , Código de Barras de DNA Taxonômico , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Proliferação de Células , Células Cultivadas , Biblioteca Gênica , Humanos
12.
World J Microbiol Biotechnol ; 35(7): 97, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31222457

RESUMO

Endophytic Streptomyces sp. SSD49 inhibited eight pathogens, including the human opportunistic pathogenic microorganisms, the plant pathogenic fungi and bacteria. The growth of soybeans, tomatoes, peppers and Populus tomentosa seedings inoculated with SSD49 are remarkably promoted. Here, we constructed two P. tomentosa seedling microRNA (miRNA) libraries inoculated with (PS30d) and without SSD49 (PC30d) to explore the molecular regulatory roles in the plant response to the beneficial bacteria. Totals of 314 known and 144 novel miRNAs were identified, among which 27 known and 11 novel miRNA had significantly different expression. The targets of up-regulated miR160, miR156, ptc114 and down-regulated miR319 and other differential expressed miRNAs primarily regulated genes encoding transcription factors (auxin response factor, small auxin-up RNA, and GRAS proteins), disease resistance proteins, phytohormone oxidase, and response regulators, which could promote plant growth, influence disease resistance and miRNA biosynthesis in P. tomentosa. This is the first report on the genome-wide identification of biocontrol endophytic Streptomyces inoculation-responsive miRNAs using small RNA sequencing in P. tomentosa and these findings provide new insight into understanding the biocontrol effects of endophytic Streptomyces.


Assuntos
MicroRNAs/genética , Reguladores de Crescimento de Planta , Populus/genética , RNA de Plantas/isolamento & purificação , Streptomyces/metabolismo , Agentes de Controle Biológico , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Populus/metabolismo , Populus/microbiologia , RNA de Plantas/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/isolamento & purificação , Análise de Sequência de RNA
13.
Mol Biol (Mosk) ; 53(3): 513-523, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184617

RESUMO

The effects of modified deoxyuridine triphosphates (mod-dUTPs) with different substituents at the C5 position of the pyrimidine cycle on the kinetics of PCR with Taq and Vent (exo-) DNA polymerases are studied. Substituents in mod-dUTP include carboxamide group and groups that are part of the side chains of alanine, valine, leucine, phenylalanine, tryptophan, or tyrosine. For each mod-dUTP, the yields of the target product are measured with the full substitution of dTTP. A fragment of bacterial DNA with a certain nucleotide sequence and a synthetic combinatorial DNA library of random nucleotide sequences are used as templates for amplification. For each mod-dUTP-template-polymerase combination, the correlation between the amplification efficiencies and yields of the target product are investigated. PCR product accumulation curves are influenced by both the template used and the presence of a modified substrate. The catalytic activity of Taq polymerase is higher when mod-dUTPs with short aliphatic substituents are used and decreases when the derivatives with long aliphatic, phenyl, and indole substituents are utilized. Vent (exo-) polymerase is less sensitive to the chemical structure of mod-dUTP. The dynamic measuring of DNA accumulation may be useful for optimizing the temperature-time PCR profiles individually for each of the mod-dUTP. The derivatives may be used in combination with Vent (exo-) polymerase to obtain modified DNA sequences for the method of selection of modified aptamers (mod-SELEX).


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , DNA/química , Reação em Cadeia da Polimerase em Tempo Real , Biblioteca Gênica , Cinética , Técnica de Seleção de Aptâmeros
14.
Nat Commun ; 10(1): 2798, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243268

RESUMO

Dynamic combinatorial chemistry (DCC) has proven its potential in drug discovery speeding the identification of modulators of biological targets. However, the exchange chemistries typically take place under specific reaction conditions, with limited tools capable of operating under physiological parameters. Here we report a catalyzed protein-directed DCC working at low temperatures that allows the calcium sensor NCS-1 to find the best ligands in situ. Ultrafast NMR identifies the reaction intermediates of the acylhydrazone exchange, tracing the molecular assemblies and getting a real-time insight into the essence of DCC processes at physiological pH. Additionally, NMR, X-ray crystallography and computational methods are employed to elucidate structural and mechanistic aspects of the molecular recognition event. The DCC approach leads us to the identification of a compound stabilizing the NCS-1/Ric8a complex and whose therapeutic potential is proven in a Drosophila model of disease with synaptic alterations.


Assuntos
Cálcio/metabolismo , Biblioteca Gênica , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Animais , Catálise , Células Cultivadas , Técnicas de Química Combinatória , Drosophila/fisiologia , Imagem por Ressonância Magnética , Masculino , Membranas Artificiais , Camundongos , Proteínas Sensoras de Cálcio Neuronal/genética , Neurônios/metabolismo , Palmitoil-CoA Hidrolase , Permeabilidade , Conformação Proteica , Proteínas
15.
Nat Commun ; 10(1): 2880, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253799

RESUMO

Cell state-specific promoters constitute essential tools for basic research and biotechnology because they activate gene expression only under certain biological conditions. Synthetic Promoters with Enhanced Cell-State Specificity (SPECS) can be superior to native ones, but the design of such promoters is challenging and frequently requires gene regulation or transcriptome knowledge that is not readily available. Here, to overcome this challenge, we use a next-generation sequencing approach combined with machine learning to screen a synthetic promoter library with 6107 designs for high-performance SPECS for potentially any cell state. We demonstrate the identification of multiple SPECS that exhibit distinct spatiotemporal activity during the programmed differentiation of induced pluripotent stem cells (iPSCs), as well as SPECS for breast cancer and glioblastoma stem-like cells. We anticipate that this approach could be used to create SPECS for gene therapies that are activated in specific cell states, as well as to study natural transcriptional regulatory networks.


Assuntos
Aprendizado de Máquina , Regiões Promotoras Genéticas , Software , Neoplasias da Mama , Linhagem Celular Tumoral , Separação Celular/métodos , Feminino , Regulação da Expressão Gênica , Biblioteca Gênica , Glioblastoma , Humanos , Células-Tronco Pluripotentes Induzidas , Lentivirus , Células-Tronco Neoplásicas , Organoides , Elementos Reguladores de Transcrição
16.
Biochemistry (Mosc) ; 84(4): 380-389, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228929

RESUMO

MicroRNAs (miRNAs), a family of ∼22-nucleotide non-coding single-stranded RNA molecules, are considered as key post-transcriptional regulators of gene expression that regulate various biological processes in living organism. Many miRNAs have been identified in animals; however, few have been reported in Hynobiidae species. The present study is aimed to identify a full repertoire of miRNAs in Batrachuperus yenyuanensis (Yenyuan stream salamander), which would significantly increase our knowledge of miRNAs in amphibians. A small RNA library was constructed from B. yenyuanensis and sequenced using deep sequencing. As a result, 1,717,751 clean reads were obtained, representing 356 known and 80 novel miRNAs. Additionally, expression levels of eight randomly selected miRNAs in B. yenyuanensis were confirmed using the stem-loop quantitative real-time reverse transcription PCR. In addition, 13,972 targets were predicted for these identified miRNAs, although the physiological functions of many of these targets remain unknown. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis suggested that the predicted targets are involved in a variety of physiological regulatory functions in B. yenyuanensis. These results provide useful information for further research on the miRNAs involved in the growth and development of B. yenyuanensis, as well as adaptation of this species to its high-altitude habitats.


Assuntos
MicroRNAs/metabolismo , Urodelos/genética , Animais , Sequência de Bases , Biblioteca Gênica , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , MicroRNAs/análise , MicroRNAs/química , Reação em Cadeia da Polimerase , Análise de Sequência de RNA , Testículo/metabolismo , Urodelos/metabolismo
17.
Plant Physiol Biochem ; 141: 456-465, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31247428

RESUMO

Alfalfa (Medicago sativa L.) is an important perennial legume and used as a forage crop worldwide, and has extensive resistance to various abiotic stresses. Nitric oxide (NO) plays a critical role in response to external and internal cues to regulate plant growth and development. However, endogenous NO-mediated molecular mechanisms of drought tolerance in alfalfa is poorly understood. To get a deeper insight into the regulate pathway of NO, RNA-Seq was used to profile transcriptome changes of alfalfa seedlings, which were treated with NO scavenger under normal and drought conditions. A total of 1,025 and 3,461 differently-expressed genes (FDR < 0.0001; fold change ≥ 2) were observed while NO absence under normal and drought conditions, respectively. Based on GO enrich and KEGG pathway analysis, we found NO absence induced photosynthesis, carbon fixation in photosynthetic organisms and primary metabolism were significantly up-enriched. Most oxidoreductase, dehydrogenase, reductase and transferase genes were down-regulated in the above processes. Moreover, NO absence restrained chlorophyll biosynthesis and decreased different sugar content. Therefore, this work provides insights into the mechanism that NO-mediated enhanced photosynthesis and carbohydrate metabolism in alfalfa under drought stress.


Assuntos
Metabolismo dos Carboidratos , Secas , Medicago sativa/enzimologia , Medicago sativa/fisiologia , Óxido Nítrico/química , Fotossíntese , Parede Celular/metabolismo , Clorofila/química , Cloroplastos/metabolismo , Perfilação da Expressão Gênica , Biblioteca Gênica , Plântula/enzimologia , Plântula/fisiologia , Análise de Sequência de RNA , Amido/química , Estresse Fisiológico , Sacarose/química
18.
Microb Cell Fact ; 18(1): 107, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31196093

RESUMO

BACKGROUND: Microbial mutagenesis is an important avenue to acquire microbial strains with desirable traits for industry application. However, mutagens either chemical or physical used often leads narrow library pool due to high lethal rate. The T4 DNA ligase is one of the most widely utilized enzymes in modern molecular biology. Its contribution to repair chromosomal DNA damages, therefore cell survival during mutagenesis will be discussed. RESULTS: Expression of T4 DNA ligase in vivo could substantially increase cell survival to ionizing radiation in multiple species. A T4 mediated survival-coupled mutagenesis approach was proposed. When polyhydroxybutyrate (PHB)-producing E. coli with T4 DNA ligase expressed in vivo was subjected to ionizing radiation, mutants with improved PHB production were acquired quickly owing to a large viable mutant library generated. Draft genome sequence analysis showed that the mutants obtained possess not only single nucleotide variation (SNV) but also DNA fragment deletion, indicating that T4 DNA ligase in vivo may contribute to the repair of DNA double strand breaks. CONCLUSIONS: Expression of T4 DNA ligase in vivo could notably enhance microbial survival to excess chromosomal damages caused by various mutagens. Potential application of T4 DNA ligase in microbial mutagenesis was explored by mutating and screening PHB producing E. coli XLPHB strain. When applied to atmospheric and room temperature plasma (ARTP) microbial mutagenesis, large survival pool was obtained. Mutants available for subsequent screening for desirable features. The use of T4 DNA ligase we were able to quickly improve the PHB production by generating a larger viable mutants pool. This method is a universal strategy can be employed in wide range of bacteria. It indicated that traditional random mutagenesis became more powerful in combine with modern genetic molecular biology and has exciting prospect.


Assuntos
DNA Ligases/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Proteínas Virais/genética , Bacteriófagos/enzimologia , DNA Ligases/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Biblioteca Gênica , Viabilidade Microbiana , Mutagênese , Poli-Hidroxialcanoatos/biossíntese , Proteínas Virais/metabolismo
19.
Vet Microbiol ; 233: 28-38, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176409

RESUMO

The anti-phagocytic abilities of bacteria often affect bacterial pathogenicity. Here, random mutant library of Streptococcus equi subsp. zooepidemicus (SEZ) was constructed using transposon mutagenesis. After careful screening, 30 transposon mutants with different transposon insertion sites were identified by conducting quantitative phagocytosis and insertion-site confirmation assays, whose anti-phagocytic abilities were significantly reduced relative to the wild-type strain. Insertion sites of 19 strains were monocistronic, including genes coding membrane proteins, transporters, and enzymes with unknown pathological function, such as sadM, adhP, purD, guaA, alpha-galactosidase coding gene, ABC transporter permease coding gene, metallo-beta-lactamase coding gene, and three secreted enzyme coding genes spuZ, slaB, and endoS, as well as known virulence factor coding genes, such as hasA and szM. The insertion sites of another 11 strains were polycistronic. We focused on four monocistronic-mutant strains: MhtpZ, MspuZ, MslaB, and MendoS. The anti-phagocytic abilities of not only the mutants that were precoincubated with the recombinant proteins, but also the complement strains were significantly more pronounced than those of all four corresponding mutants. The polyclonal antiserum against SlaB or EndoS also significantly decreased the anti-phagocytic capacity of wild-type SEZ. All four mutants exhibited significantly decreased viability in whole blood and reduced lethality in mice relative to the wild-type strain. Thus, we identified a variety of new anti-phagocytic factors, particularly multiple SEZ secreted enzymes. These factors are instrumental in the phagocytic resistance of SEZ in the absence of opsonin. Our results provide a framework for further studies of SEZ pathogenesis and relevant vaccine development for novel potential targets.


Assuntos
Genes Bacterianos , Óperon , Fagócitos/microbiologia , Fagocitose , Streptococcus equi/genética , Animais , Elementos de DNA Transponíveis , Feminino , Biblioteca Gênica , Camundongos , Camundongos Endogâmicos ICR , Mutagênese , Mutação , Células RAW 264.7 , Streptococcus equi/patogenicidade , Fatores de Virulência/genética
20.
BMC Plant Biol ; 19(1): 247, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31185902

RESUMO

BACKGROUND: MiRNAs (microRNA) are 18-24 nt endogenous noncoding RNAs that regulate gene expression at the post-transcriptional level, including tissue-specific, developmental timing and evolutionary conservation gene expression. RESULTS: This study used high-throughput sequencing technology for the first time in Larix olgensis, predicted 78 miRNAs, including 12,229,003 reads sRNA, screened differentially expressed miRNAs. Predicting target genes was helpful for understanding the miRNA regulation function and obtained 333 corresponding target genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation were analysed, mostly including nucleic acid binding, plant hormone signal transduction, pantothenate and CoA biosynthesis, and cellulose synthase. This study will lay the foundation for clarifying the complex miRNA-mediated regulatory network for growth and development. In view of this, spatio-temporal expression of miR396, miR950, miR164, miR166 and miR160 were analysed in Larix olgensis during the growth stages of not lignified, beginning of lignification, and completely lignified in different tissues (root, stem, and leaf) by quantitative real-time PCR (qRT-PCR). There were differences in the expression of miRNAs in roots, stems and leaves in the same growth period. At 60 days, miR160, miR166 and miR396-2 exhibited the highest expression in leaves. At 120 days, most miRNAs in roots and stems decreased significantly. At 180 days, miRNAs were abundantly expressed in roots and stems. Meanwhile, analysis of the expression of miRNAs in leaves revealed that miR396-2 was reduced as time went on, whereas other miRNAs increased initially and then decreased. On the other hand, in the stems, miR166-1 was increase, whereas other miRNAs, especially miR160, miR164, miR396 and miR950-1, first decreased and then increased. Similarly, in the roots, miR950-2 first decreased and then increased, whereas other miRNAs exhibited a trend of continuous increase. CONCLUSIONS: The present investigation included rapid isolation and identification of miRNAs in Larix olgensis through construction of a sRNA library using Solexa and predicted 78 novel miRNAs, which showed differential expression levels in different tissues and stages. These results provided a theoretical basis for further revealing the genetic regulation mechanism of miRNA in the growth and development of conifers and the verification of function in target genes.


Assuntos
Regulação da Expressão Gênica de Plantas , Larix/genética , MicroRNAs/genética , RNA de Plantas/genética , Perfilação da Expressão Gênica , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Larix/metabolismo , MicroRNAs/metabolismo , RNA de Plantas/metabolismo
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