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1.
Anticancer Res ; 39(9): 4637-4642, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519561

RESUMO

AIM: The aim of this study was to characterize the role of Alport syndrome, mental retardation, midface hypoplasia, and elliptocytosis chromosomal region gene 1 (AMMECR1) in human lung cancer cell lines. MATERIALS AND METHODS: AMMECR1 gene expression was evaluated in four lung cell lines, with A549 then selected for further in-depth examination. To characterize the role of AMMECR1, silencing was achieved utilizing lentivirus-mediated RNA interference, and confirmed by quantitative real-time polymerase chain reaction and western blotting. The impact of AMMECR1 silencing on cellular proliferation was assessed using Celigo-based and MTT assays. Apoptosis was determined using the annexin V-allophycocyanin single staining method. Cell-cycle arrest was assessed by flow cytometry. Finally, colony formation was assessed using Giemsa staining. RESULTS: In A549 cells, AMMECR1 silencing was found to significantly suppress cell proliferation, reduce colony formation, promote apoptosis, and arrest cells in the S and G2/M phases. CONCLUSION: AMMECR1 plays a critical role in cell proliferation, cell-cycle progression, and apoptosis of human lung cancer cells, and may serve as a potential therapeutic target for non-small-cell lung cancer.


Assuntos
Apoptose/genética , Ciclo Celular/genética , Neoplasias Pulmonares/genética , Proteínas/genética , Células A549 , Linhagem Celular Tumoral , Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Neoplasias Pulmonares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas/metabolismo , RNA Mensageiro/genética
2.
Anticancer Res ; 39(9): 4853-4864, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519588

RESUMO

BACKGROUND/AIM: Colorectal cancer (CRC) is the leading cause of cancer mortality worldwide. Its poor prognosis can be ascribed primarily to high recurrence rates. Accordingly, the aim of this study was to identify novel prognostic biomarkers and therapeutic targets for management of CRC. MATERIALS AND METHODS: To develop prognostic biomarkers, we performed RNA-seq analysis and real-time RT-PCR in primary cancer tissues with or without systemic recurrence. To characterize the molecular functions of the encoded proteins, CRC cells underexpressing or overexpressing the candidate genes were established and appropriate cell-based assays were applied. RESULTS: ITGB1 and RHOC mRNA levels were up-regulated in the recurrence group of CRC patients. Overexpression of ITGB1 or RHOC stimulated CRC cell proliferation, invasion and migration, whereas the opposite effects were observed in cells underexpressing either protein. Five-year recurrence-free survival rates were significantly higher in the ITGB1- and RHOC-underexpression groups than those in the overexpression. CONCLUSION: ITGB1 and RHOC are potential predictors of recurrence and therapeutic targets for CRC, possibly predicting a high-risk group of stage II patients.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Integrina beta1/metabolismo , Proteína de Ligação a GTP rhoC/metabolismo , Idoso , Biomarcadores Tumorais , Proliferação de Células , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , RNA Interferente Pequeno/genética , Recidiva , Análise de Sobrevida
3.
World J Microbiol Biotechnol ; 35(8): 128, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375920

RESUMO

Large patch disease, caused by Rhizoctonia solani AG2-2, is the most devastating disease in Zoysiagrass (Zoysia japonica). Current large patch disease control strategies rely primarily upon the use of chemical pesticides. Streptomyces sp. S8 is known to possess exceptional antagonistic properties that could potentially suppress the large patch pathogen found at turfgrass plantations. This study aims to demonstrate the feasibility of using the strain as a biological control mechanism. Sequencing of the S8 strain genome revealed a valinomycin biosynthesis gene cluster. This cluster is composed of the vlm1 and vlm2 genes, which are known to produce antifungal compounds. In order to verify this finding for the large patch pathogen, a valinomycin biosynthesis knockout mutant was created via the CRISPR/Cas9 system. The mutant lost antifungal activity against the large patch pathogen. Consequently, it is anticipated that eco-friendly microbial preparations derived from the S8 strain can be utilized to biologically control large patch disease.


Assuntos
Antifúngicos/metabolismo , Antifúngicos/farmacologia , Rhizoctonia/efeitos dos fármacos , Streptomyces/metabolismo , Valinomicina/metabolismo , Valinomicina/farmacologia , Vias Biossintéticas/genética , Técnicas de Inativação de Genes , Genoma Bacteriano , Família Multigênica , Controle Biológico de Vetores/métodos , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Poaceae/microbiologia , Rhizoctonia/crescimento & desenvolvimento , Análise de Sequência de DNA , Streptomyces/genética
4.
Sheng Wu Gong Cheng Xue Bao ; 35(8): 1382-1390, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31441609

RESUMO

Mitochondrial dynamics, the processes of mitochondrial fusion and fission maintain homeostasis, are precisely regulated by fusion/fission-related proteins, and play an important physiological role in mitochondrial metabolism, quality and function. The aberrant changes of these proteins can trigger mitochondrial dynamics imbalance, which cause mitochondrial dysfunctions and result various disease states. This article focuses on gene knockout technology, and reviews the role and application progress of genes encoding for fusion and fission knockout mice in insulin resistance researches, in order to lay a foundation for future studies on signal transduction mechanism of mitochondrial dynamics imbalance in insulin resistance.


Assuntos
Resistência à Insulina , Dinâmica Mitocondrial , Animais , Técnicas de Inativação de Genes , Camundongos , Mitocôndrias , Proteínas Mitocondriais
5.
Microbiol Res ; 227: 126297, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31421711

RESUMO

Many plant growth promoting rhizobacteria such as Bacillus velezensis GJ11 can produce acetoin to trigger induced systemic resistance (ISR) in plants. For improving acetoin production, the mutant strains were respectively constructed by knockout of the gene of bdh (2,3-butanediol dehydrogenase) and gdh (glycerol dehydrogenase) in GJ11, but only GJ11Δbdh produced a high level of acetoin triggering strong ISR against Pseudomonas syringae infection in plants. GJ11Δbdh could induce H2O2 accumulation in plants by producing a high level of acetoin. H2O2 was necessary for triggering ISR against the pathogen infection because after scavenging H2O2 with ascorbic acid or catalase, the inhibition role to pathogen infection induced by acetoin almost disappeared in plants. Further investigation found the plants treated with GJ11Δbdh in an obvious "priming" state, in which the mild immune response was observed such as a slight increase of H2O2 production, callose deposition, and enzymes activity related with defence response (e.g. POD, PAL and PPO). The plants in "priming" could rapidly respond to the pathogen infection accompanying with a significant increase of H2O2 production, callose deposition, and enzymes activity. Collectively, this study provides new insight into the role of acetoin as a strong elicitor of defense response, and ascribes a new approach to construct the mutant strains with high production of acetoin for triggering stronger ISR against pathogens infection in plants.


Assuntos
Acetoína/metabolismo , Arabidopsis/genética , Bacillus/genética , Bacillus/metabolismo , Resistência à Doença/genética , Imunidade Vegetal/genética , Oxirredutases do Álcool/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Ácido Ascórbico/metabolismo , Catalase/metabolismo , Resistência à Doença/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Genes de Plantas/genética , Peróxido de Hidrogênio/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/fisiologia , Pseudomonas syringae/patogenicidade , Desidrogenase do Álcool de Açúcar/genética
6.
BMC Plant Biol ; 19(1): 354, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31412779

RESUMO

BACKGROUND: High temperature is a major environmental stress that limits plant growth and agriculture productivity. Mitogen-activated protein kinases (MAPKs) are highly conserved serine and threonine protein kinases that participate in response to diverse environmental stresses in plants. A total of 16 putative SlMAPK genes are identified in tomato, and SlMAPK3 is one of the most extensively studied SlMAPKs. However, the role of SlMAPK3 in response to heat stress is not clearly understood in tomato plants. In this study, we performed functional analysis of SlMAPK3 for its possible role in response to heat stress. RESULTS: qRT-PCR analyses revealed that SlMAPK3 relative expression was depressed by heat stress. Here, wild-type (WT) tomato plants and CRISPR/Cas9-mediated slmapk3 mutant lines (L8 and L13) were used to investigate the function of SlMAPK3 in response to heat stress. Compared with WT plants, slmapk3 mutants exhibited less severe wilting and less membrane damage, showed lower reactive oxygen species (ROS) contents, and presented higher both activities and transcript levels of antioxidant enzymes, as well as elevated expressions of genes encoding heat stress transcription factors (HSFs) and heat shock proteins (HSPs). CONCLUSIONS: CRISPR/Cas9-mediated slmapk3 mutants exhibited more tolerance to heat stress than WT plants, suggesting that SlMAPK3 was a negative regulator of thermotolerance. Moreover, antioxidant enzymes and HSPs/HSFs genes expression were involved in SlMAPK3-mediated heat stress response in tomato plants.


Assuntos
Resposta ao Choque Térmico/genética , Lycopersicon esculentum/genética , Proteínas de Plantas/genética , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/metabolismo , Técnicas de Inativação de Genes , Homeostase , Lycopersicon esculentum/metabolismo , Proteínas de Plantas/metabolismo
7.
Sheng Li Xue Bao ; 71(4): 588-596, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31440756

RESUMO

The aim of the study was to establish Ace2 (angiotensin-converting enzyme 2) knockout mouse model with CRISPR/Cas9 gene targeting technology. A vector targeting Ace2 gene knockout was constructed with the primers of single-guide RNA (gRNA), and then transcribed gRNA/Cas9 mRNA was micro-injected into the mouse zygote. The deletion of exons 3 to 18 of Ace2 gene in mice was detected and identified by PCR and gene sequencing. The Ace2 gene knock-out mice were bred and copulated. Ace2 protein and mRNA expression were detected by Western blot and qRT-PCR in F3 progeny knock-out male mice. The gRNA expression vector was successfully constructed and transcribed in vitro, and active gRNA and Cas9 mRNA were injected directly into zygote. The deletion of exons 3 to 18 of Ace2 gene in six positive founder mice as the F0 generation were confirmed by PCR and gene sequencing. Six founder mice were mated with wild-type mice, then achieved F1 generation were mated and produced F2 generation. The female positive mouse of F2 was selected to mate with wild-type mice and produce Ace2-/Y mice of F3 generation. Ace2 mRNA and protein were not detected in tissues of these Ace2-/Y mice. In conclusion, a mouse model with Ace2 deficiency has been successfully established with CRISPR/Cas9 technique, which shall lay a foundation for future investigation of Ace2.


Assuntos
Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Camundongos Knockout , RNA Guia/genética , Animais , Feminino , Marcação de Genes , Masculino , Camundongos
8.
Sheng Wu Gong Cheng Xue Bao ; 35(7): 1247-1255, 2019 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-31328481

RESUMO

L-tyrosine is one of three aromatic amino acids that are widely used in food, pharmaceutical and chemical industries. The transport system engineering provides an important research strategy for the metabolic engineering of Escherichia coli to breed L-tyrosine producing strain. The intracellular transport of L-tyrosine in E. coli is mainly regulated by two distinct permeases encoded by aroP and tyrP genes. The aroP and tyrP gene knockout mutants were constructed by CRISPR-Cas technique on the basis of L-tyrosine producing strain HGXP, and the effects of regulating transport system on L-tyrosine production were investigated by fermentation experiments. The fermentation results showed that the aroP and tyrP knockout mutants produced 3.74 and 3.45 g/L L-tyrosine, respectively, which were 19% and 10% higher than that of the original strain. The optimum induction temperature was determined to be 38 °C. Fed-batch fermentation was carried out on a 3-L fermentor. The L-tyrosine yields of aroP and tyrP knockout mutants were further increased to 44.5 and 35.1 g/L, respectively, which were 57% and 24% higher than that of the original strain. The research results are of great reference value for metabolic engineering of E. coli to produce L-tyrosine.


Assuntos
Escherichia coli , Proteínas de Escherichia coli , Técnicas de Inativação de Genes , Engenharia Metabólica , Tirosina
9.
Cell Mol Biol Lett ; 24: 44, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31285745

RESUMO

Background: Deer antler is the only mammalian organ that can be completely regenerated every year. Its periodic regeneration is regulated by multiple factors, including transforming growth factor ß (TGF-ß). This widely distributed multi-functional growth factor can control the proliferation and differentiation of many types of cell, and it may play a crucial regulatory role in antler regeneration. This study explored the role of TGF-ß1 during the rapid growth of sika deer antler. Methods: Three CRISPR-Cas9 knockout vectors targeting the TGF-ß1 gene of sika deer were constructed and packaged with a lentiviral system. The expression level of TGF-ß1 protein in the knockout cell line was determined using western blot, the proliferation and migration of cartilage cells in vitro were respectively determined using EdU and the cell scratch test, and the expression levels of TGF-ß pathway-related genes were determined using a PCR array. Results: Of the three gRNAs designed, pBOBI-gRNA2 had the best knockout effect. Knockout of TGF-ß1 gene inhibits the proliferation of cartilage cells and enhances their migration in vitro. TGF-ß signaling pathway-related genes undergo significant changes, so we speculate that when the TGF-ß pathway is blocked, the BMP signaling pathway mediated by BMP4 may play a key role. Conclusions: TGF-ß1 is a newly identified regulatory factor of rapid growth in sika deer antler.


Assuntos
Cartilagem/metabolismo , Proliferação de Células , Cervos/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Animais , Animais Geneticamente Modificados , Chifres de Veado , Sistemas CRISPR-Cas , Cartilagem/fisiologia , Linhagem Celular , Cervos/genética , Cervos/fisiologia , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Masculino , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/fisiologia
10.
Microbiol Res ; 226: 10-18, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31284939

RESUMO

Microbial oxidation of antimonite [Sb(III)] to antimonate [Sb(V)] is a detoxification process which contributes to Sb(III) resistance. Antimonite oxidase AnoA is essential for Sb(III) oxidation, however, the regulation mechanism is still unknown. Recently, we found that the expressions of phosphate transporters were induced by Sb(III) using proteomics analysis in Agrobacterium tumefaciens GW4, thus, we predicted that the phosphate regulator PhoB may regulate bacterial Sb(III) oxidation and resistance. In this study, comprehensive analyses were performed and the results showed that (1) Genomic analysis revealed two phoB (named as phoB1 and phoB2) and one phoR gene in strain GW4; (2) Reporter gene assay showed that both phoB1 and phoB2 were induced in low phosphate condition (50 µM), but only phoB2 was induced by Sb(III); (3) Genes knock-out/complementation, Sb(III) oxidation and Sb(III) resistance tests showed that deletion of phoB2 significantly inhibited the expression of anoA and decreased bacterial Sb(III) oxidation efficiency and Sb(III) resistant. In contrast, deletion of phoB1 did not obviously affect anoA's expression level and Sb(III) oxidation/resistance; (4) A putative Pho motif was predicted in several A. tumefaciens strains and electrophoretic mobility shift assay (EMSA) showed that PhoB2 could bind with the promoter sequence of anoA; (5) Site-directed mutagenesis and short fragment EMSA revealed the exact DNA binding sequence for the protein-DNA interaction. These results showed that PhoB2 positively regulates Sb(III) oxidation and PhoB2 is also associated with Sb(III) resistance. Such regulation mechanism may provide a great contribution for bacterial survival in the environment with Sb and for bioremediation application.


Assuntos
Agrobacterium tumefaciens/metabolismo , Antimônio/metabolismo , Proteínas de Bactérias/metabolismo , Fosfatos/metabolismo , Agrobacterium tumefaciens/genética , Arsenitos/metabolismo , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Farmacorresistência Bacteriana/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Mutagênese Sítio-Dirigida , Oxirredução , Proteínas de Transporte de Fosfato/metabolismo , Proteômica
11.
World J Microbiol Biotechnol ; 35(7): 111, 2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31280424

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) immune systems in bacteria have been used as tools for genome engineering. Thus far, the CRISPR-Cas system has been used in various yeast, bacterial, and mammalian cells. Saccharomyces cerevisiae is a nonpathogenic yeast, classified under "generally recognized as safe", and has long been used to produce consumables such as alcohol or bread. Additionally, recombinant cells of S. cerevisiae have been constructed and used to produce various bio-based chemicals. Some types of CRISPR-Cas system for genetic manipulation have been constructed during the early developmental stages of the CRISPR-Cas system and have been mainly used for gene knock-in and knock-out manipulations. Thereafter, these systems have been used for various novel purposes such as metabolic engineering and tolerance engineering. In this review, we have summarized different aspects of the CRISPR-Cas in the yeast S. cerevisiae, from its basic principles to various applications. This review describes the CRISPR system in S. cerevisiae based on the differences in its origin and efficiency followed by its basic applications; for example, its involvement in gene knock-in and knock-out has been outlined. Finally, advanced applications of the CRISPR system in the bioproduction of useful chemicals have been summarized.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Edição de Genes/métodos , Regulação Fúngica da Expressão Gênica , Técnicas de Introdução de Genes/métodos , Técnicas de Inativação de Genes/métodos , Saccharomyces cerevisiae/genética
12.
Sheng Wu Gong Cheng Xue Bao ; 35(5): 784-794, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31222997

RESUMO

The establishment and development of gene knockout mice have provided powerful support for the study of gene function and the treatment of human diseases. Gene targeting and gene trap are two techniques for generating gene knockout mice from embryonic stem cells. Gene targeting replaces endogenous knockout gene by homologous recombination. There are two ways to knock out target genes: promoter trap and polyA trap. In recent years, many new gene knockout techniques have been developed, including Cre/loxP system, CRISP/Cas9 system, latest ZFN technology and TALEN technology. This article focuses on the several new knockout mouse techniques.


Assuntos
Técnicas de Inativação de Genes , Camundongos Knockout , Animais , Modelos Animais de Doenças , Células-Tronco Embrionárias , Técnicas de Inativação de Genes/tendências , Marcação de Genes/tendências , Recombinação Homóloga , Humanos , Camundongos
13.
Sheng Wu Gong Cheng Xue Bao ; 35(5): 910-918, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31223009

RESUMO

Parthenogenetic embryonic stem cells (pESCs) derived from bi-maternal genomes do not have competency of tetraploid complementation, due to lacking of paternal imprinting genes. To make pESCs possess fully development potentials and similar pluripotency to zygote-derived ESCs, we knocked out one allelic gene of the two essential maternal imprinting genes (H19 and IG) in their differentially methylated regions (DMR) via CRISPR/Cas9 system and obtained double knock out (DKO) pESCs. Maternal pESCs had similar morphology, expression levels of pluripotent makers and in vitro neural differentiation potentials to zygotes-derived ESCs. Besides that, DKO pESCs could contribute to full-term fetuses through tetraploid complementation, proving that they held fully development potentials. Derivation of DKO pESCs provided a type of major histocompatibility complex (MHC) matched pluripotent stem cells, which would benefit research in regenerative medicine.


Assuntos
Células-Tronco Embrionárias , Partenogênese , Células-Tronco Pluripotentes , Tetraploidia , Animais , Técnicas de Inativação de Genes , Impressão Genômica , Camundongos , Medicina Regenerativa
14.
Oncology ; 97(3): 164-172, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31195398

RESUMO

BACKGROUND: MicroRNAs are a class of small noncoding RNAs that play an important role in progression and drug resistance in cancer. Several reports have shown that miR-130b modulates cell growth and drug resistance in some cancers. However, the expression and biological role of miR-130b in renal cell carcinoma (RCC) remain poorly understood. This study aimed to examine the expression and functional role of miR-130b and to analyze the association between miR-130b and sunitinib resistance in RCC. METHODS: The expression of miR-130b in 32 RCC tissues and their corresponding normal kidney tissues was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). We performed a 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay in RCC cell lines transfected with miR-130b inhibitor or miR-130b mimics. We evaluated the relationship between miR-130b and PTEN and also analyzed the effect of miR-130b on sunitinib resistance. RESULTS: qRT-PCR analysis showed that the expression of miR-130b was higher in RCC tissues than in corresponding normal kidney tissues. The MTT assay revealed that miR-130b modulated cell growth. qRT-PCR revealed an inverse correlation between miR-130b and PTEN in RCC. Western blotting demonstrated that miR-130b regulated the expression of PTEN in the RCC cell line. Additionally, miR-130b was associated with sunitinib resistance through regulation of PTEN. We established the sunitinib-resistant Caki-1 (Caki-1-SR) cells and observed that the expression of miR-130b was elevated in Caki-1-SR cells compared with parental Caki-1 cells. Knockdown of miR-130b improved sunitinib resistance in Caki-1-SR cells. CONCLUSION: The expression of miR-130b was upregulated in RCC. miR-130b promoted cell growth and was associated with sunitinib resistance through regulating PTEN expression. Collectively, these results suggest that miR-130b may play an oncogenic role and be a promising therapeutic target.


Assuntos
Carcinoma de Células Renais/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , MicroRNAs/genética , PTEN Fosfo-Hidrolase/genética , Sunitinibe/farmacologia , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Técnicas de Inativação de Genes , Humanos , Neoplasias Renais/metabolismo , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , PTEN Fosfo-Hidrolase/metabolismo
15.
Genes Dev ; 33(13-14): 782-798, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171699

RESUMO

Mouse embryonic stem cell (ESC) cultures contain a rare cell population of "2C-like" cells resembling two-cell embryos, the key stage of zygotic genome activation (ZGA). Little is known about positive regulators of the 2C-like state and two-cell stage embryos. Here we show that GADD45 (growth arrest and DNA damage 45) proteins, regulators of TET (TET methylcytosine dioxygenase)-mediated DNA demethylation, promote both states. Methylome analysis of Gadd45a,b,g triple-knockout (TKO) ESCs reveal locus-specific DNA hypermethylation of ∼7000 sites, which are enriched for enhancers and loci undergoing TET-TDG (thymine DNA glycosylase)-mediated demethylation. Gene expression is misregulated in TKOs, notably upon differentiation, and displays signatures of DNMT (DNA methyltransferase) and TET targets. TKOs manifest impaired transition into the 2C-like state and exhibit DNA hypermethylation and down-regulation of 2C-like state-specific genes. Gadd45a,b double-mutant mouse embryos display embryonic sublethality, deregulated ZGA gene expression, and developmental arrest. Our study reveals an unexpected role of GADD45 proteins in embryonic two-cell stage regulation.


Assuntos
Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Desmetilação do DNA , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animais , Células Cultivadas , Técnicas de Inativação de Genes , Camundongos
16.
Nat Plants ; 5(6): 581-588, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31182842

RESUMO

Chloroplasts are integral to sensing biotic and abiotic stress in plants, but their role in transducing Ca2+-mediated stress signals remains poorly understood1,2. Here we identify cMCU, a member of the mitochondrial calcium uniporter (MCU) family, as an ion channel mediating Ca2+ flux into chloroplasts in vivo. Using a toolkit of aequorin reporters targeted to chloroplast stroma and the cytosol in cMCU wild-type and knockout lines, we provide evidence that stress-stimulus-specific Ca2+ dynamics in the chloroplast stroma correlate with expression of the channel. Fast downstream signalling events triggered by osmotic stress, involving activation of the mitogen-activated protein kinases (MAPK) MAPK3 and MAPK6, and the transcription factors MYB60 and ethylene-response factor 6 (ERF6), are influenced by cMCU activity. Relative to wild-type plants, cMCU knockouts display increased resistance to long-term water deficit and improved recovery on rewatering. Modulation of stromal Ca2+ in specific processing of stress signals identifies cMCU as a component of plant environmental sensing.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Canais de Cálcio/metabolismo , Cloroplastos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cálcio/metabolismo , Canais de Cálcio/genética , Cloroplastos/genética , Escherichia coli , Técnicas de Inativação de Genes , Sistema de Sinalização das MAP Quinases , Pressão Osmótica
17.
Nat Plants ; 5(6): 575-580, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31182848

RESUMO

Doubled haploid (DH) breeding based on in vivo haploid induction has led to a new approach for maize breeding1. All modern haploid inducers used in DH breeding are derived from the haploid inducer line Stock6. Two key quantitative trait loci, qhir1 and qhir8, lead to high-frequency haploid induction2. Mutation of the gene MTL/ZmPLA1/NLD in qhir1 could generate a ~2% haploid induction rate (HIR)3-5; nevertheless, this mutation is insufficient for modern haploid inducers whose average HIR is ~10%6. Therefore, cloning of the gene underlying qhir8 is important for illuminating the genetic basis of haploid induction. Here, we present the discovery that mutation of a non-Stock6-originating gene in qhir8, namely, ZmDMP, enhances and triggers haploid induction. ZmDMP was identified by map-based cloning and further verified by CRISPR-Cas9-mediated knockout experiments. A single-nucleotide change in ZmDMP leads to a 2-3-fold increase in the HIR. ZmDMP knockout triggered haploid induction with a HIR of 0.1-0.3% and exhibited a greater ability to increase the HIR by 5-6-fold in the presence of mtl/zmpla1/nld. ZmDMP was highly expressed during the late stage of pollen development and localized to the plasma membrane. These findings provide important approaches for studying the molecular mechanism of haploid induction and improving DH breeding efficiency in maize.


Assuntos
Haploidia , Proteínas de Membrana/genética , Proteínas de Plantas/genética , Zea mays/genética , Mapeamento Cromossômico , Cromossomos de Plantas , Técnicas de Inativação de Genes , Mutação
18.
BMC Plant Biol ; 19(1): 268, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221084

RESUMO

BACKGROUND: Research on plant amino acid transporters was mainly performed in Arabidopsis, while our understanding of them is generally scant in rice. OsLHT1 (Lysine/Histidine transporter) has been previously reported as a histidine transporter in yeast, but its substrate profile and function in planta are unclear. The aims of this study are to analyze the substrate selectivity of OsLHT1 and influence of its disruption on rice growth and fecundity. RESULTS: Substrate selectivity of OsLHT1 was analyzed in Xenopus oocytes using the two-electrode voltage clamp technique. The results showed that OsLHT1 could transport a broad spectrum of amino acids, including basic, neutral and acidic amino acids, and exhibited a preference for neutral and acidic amino acids. Two oslht1 mutants were generated using CRISPR/Cas9 genome-editing technology, and the loss-of-function of OsLHT1 inhibited rice root and shoot growth, thereby markedly reducing grain yields. QRT-PCR analysis indicated that OsLHT1 was expressed in various rice organs, including root, stem, flag leaf, flag leaf sheath and young panicle. Transient expression in rice protoplast suggested OsLHT1 was localized to the plasma membrane, which is consistent with its function as an amino acid transporter. CONCLUSIONS: Our results indicated that OsLHT1 is an amino acid transporter with wide substrate specificity and with preference for neutral and acidic amino acids, and disruption of OsLHT1 function markedly inhibited rice growth and fecundity.


Assuntos
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/genética , Aminoácidos/metabolismo , Animais , Sítios de Ligação , Técnicas de Inativação de Genes , Filogenia , Proteínas de Plantas/genética , Xenopus
19.
BMC Plant Biol ; 19(1): 274, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234787

RESUMO

BACKGROUND: miRNAs are major regulators of gene expression and have proven their role in understanding the genetic regulation of biosynthetic pathways. Stevioside and rebaudioside-A, the two most abundant and sweetest compounds found in leaf extract of Stevia rebaudiana, have been used for many years in treatment of diabetes. It has been found that the crude extract is more potent than the purified extract. Stevioside, being accumulated in higher concentration, imparts licorice like aftertaste. Thus, in order to make the sweetener more potent and palatable, there is a need to increase the intrinsic concentration of steviol glycosides and to alter the ratio of rebaudioside-A to stevioside. Doing so would significantly increase the quality of the sweeteners, and the potential to be used on a wider scale. To do so, in previous report, miRNAs associated with genes of steviol glycosides biosynthetic pathway were identified in S. rebaudiana. In continuation to that in this study, the two miRNAs (miR319g and miRStv_11) targeting key genes of steviol glycosides biosynthetic pathway were modulated and their impact was evaluated on steviol glycosides contents. RESULTS: The over-expression results showed that miRStv_11 induced, while miR319g had repressive action on its target genes. The knock-down constructs for miR319g and miRStv_11 were then prepared and it was demonstrated that the expression of anti-miR319g produced inhibitory effect on its target miRNA, resulting in enhanced expression of its target genes. On the other hand, anti-miRStv_11 resulted in down-regulation of miRStv_11 and its target gene. Further miRStv_11 and anti-miR319gwere co-expressed which resulted in significant increase in stevioside (24.5%) and rebaudioside-A (51%) contents. CONCLUSION: In conclusion, the role of miR319g and miRStv_11 was successfully validated in steviol gycosides biosynthetic pathway gene regulation and their effect on steviol gycosides contents. In this study, we found the positively correlated miRNA-mRNA interaction network in plants, where miRStv_11 enhanced the expression of KAH gene. miRNAs knock-down was also successfully achieved using antisense precursors. Overall, this study thus reveals more complex nature and fundamental importance of miRNAs in biosynthetic pathway related gene networks and hence, these miRNAs can be successfully employed to enhance the ratio of rebaudioside-A to stevioside, thus enhancing the sweetening indices of this plant and making it more palatable.


Assuntos
Diterpenos de Caurano/biossíntese , Glucosídeos/biossíntese , MicroRNAs/metabolismo , RNA de Plantas/metabolismo , Stevia/metabolismo , Diterpenos de Caurano/química , Diterpenos de Caurano/genética , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Inativação Gênica , Glucosídeos/química , Glucosídeos/genética , MicroRNAs/genética , Folhas de Planta/química , Regiões Promotoras Genéticas , RNA de Plantas/genética , Stevia/genética , Edulcorantes/química
20.
Microbiol Res ; 223-225: 44-50, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178050

RESUMO

Classic genome editing tools including ZFN, TALEN, and CRISPR/Cas9 rely on DNA double-strand breaks for genome editing. To prevent the potential hazard caused by double-strand breaks (DSBs), a series of single base editing tools that convert cytidine (C) to thymine (T) without DSBs have been developed extensively in multiple species. Herein, we report for the first time that C was converted to T with a high frequency in the filamentous fungi Aspergillus niger by fusing cytidine deaminase and Cas9 nickase. Using the CRISPR/Cas9-dependent base editor and inducing nonsense mutations via single base editing, we inactivated the uridine auxotroph gene pyrG and the pigment gene fwnA with an efficiency of 47.36%-100% in A.niger. At the same time, the single-base editing results of the non-phenotypic gene prtT showed an efficiency of 60%. The editable window reached 8 bases (from C2 to C9 in the protospacer) in A. niger. Overall, we successfully constructed a single base editing system in A. niger. This system provides a more convenient tool for investigating gene function in A. niger, and provides a new tool for genetic modification in filamentous fungi.


Assuntos
Aspergillus niger/genética , Sistemas CRISPR-Cas , Citidina Desaminase/genética , Edição de Genes/métodos , Aspergillus niger/enzimologia , Sequência de Bases , Desoxirribonuclease I/genética , Proteínas Fúngicas/genética , Técnicas de Inativação de Genes , Genes Fúngicos/genética , Mutagênese
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