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1.
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
2.
Bioengineered ; 10(1): 335-344, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31322471

RESUMO

Selenium-enriched yeast can transform toxic inorganic selenium into absorbable organic selenium, which is of great significance for human health and pharmaceutical industry. A yeast Rhodotorula glutinis X-20 we obtained before has good selenium-enriched ability, but its selenium content is still low for industrial application. In this study, strategies of process optimization and transport regulation of selenium were thus employed to further improve the cell growth and selenium enrichment. Through engineering phosphate transporters from Saccharomyces cerevisiae into R. glutinis X-20, the selenium content was increased by 21.1%. Through using mixed carbon culture (20 g L-1, glycerol: glucose 3:7), both biomass and selenium content were finally increased to 5.3 g L-1 and 5349.6 µg g-1 (cell dry weight, DWC), which were 1.14 folds and 6.77 folds compared to their original values, respectively. Our results indicate that high selenium-enrichment ability and biomass production can be achieved through combining process optimization and regulation of selenium transport.


Assuntos
Engenharia Metabólica/métodos , Fosfatos/metabolismo , Rhodotorula/genética , Saccharomyces cerevisiae/genética , Selênio/metabolismo , Transgenes , Transporte Biológico , Biomassa , Meios de Cultura/química , Meios de Cultura/farmacologia , Fermentação , Expressão Gênica , Glucose/química , Glucose/metabolismo , Glicerol/química , Glicerol/metabolismo , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Simportadores de Próton-Fosfato/genética , Simportadores de Próton-Fosfato/metabolismo , Rhodotorula/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismo
3.
Nat Med ; 25(8): 1266-1273, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285633

RESUMO

The ability to safely control transgene expression with simple synthetic gene switches is critical for effective gene- and cell-based therapies. In the present study, the signaling pathway controlled by human transient receptor potential (TRP) melastatin 8 (hTRPM8), a TRP channel family member1, is harnessed to control transgene expression. Human TRPM8 signaling is stimulated by menthol, an innocuous, natural, cooling compound, or by exposure to a cool environment (15-18 °C). By functionally linking hTRPM8-induced signaling to a synthetic promoter containing elements that bind nuclear factor of activated T cells, a synthetic gene circuit was designed that can be adjusted by exposure to either a cool environment or menthol. It was shown that this gene switch is functional in various cell types and human primary cells, as well as in mice implanted with engineered cells. In response to transdermal delivery of menthol, microencapsulated cell implants harboring this gene circuit, coupled to expression of either of two therapeutic proteins, insulin or a modified, activin type IIB, receptor ligand trap protein (mActRIIBECD-hFc), could alleviate hyperglycemia in alloxan-treated mice (a model of type 1 diabetes) or reverse muscle atrophy in dexamethasone-treated mice (a model of muscle wasting), respectively. This fully human-derived orthogonal transgene switch should be amenable to a wide range of clinical applications.


Assuntos
Receptores de Activinas Tipo II/sangue , Insulina/biossíntese , Canais de Cátion TRPM/fisiologia , Transgenes , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/uso terapêutico , Fosfatase Alcalina/genética , Animais , Temperatura Baixa , Diabetes Mellitus Tipo 1/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Insulina/genética , Insulina/uso terapêutico , Mentol/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Distrofias Musculares/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos
4.
Nature ; 571(7763): 107-111, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31217582

RESUMO

Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants1,2. Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes.


Assuntos
Diarreia/congênito , Diarreia/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes , Intestinos/fisiologia , Deleção de Sequência/genética , Animais , Cromossomos Humanos Par 16/genética , Modelos Animais de Doenças , Feminino , Genes Reporter , Loci Gênicos/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Linhagem , Fenótipo , Ativação Transcricional , Transcriptoma/genética , Transgenes/genética
5.
Nat Commun ; 10(1): 2788, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243277

RESUMO

Many potentially therapeutic molecules have been identified for treating Duchenne muscular dystrophy. However, targeting those molecules only to sites of active pathology is an obstacle to their clinical use. Because dystrophic muscles become extensively inflamed, we tested whether expressing a therapeutic transgene in leukocyte progenitors that invade muscle would provide selective, timely delivery to diseased muscle. We designed a transgene in which leukemia inhibitory factor (LIF) is under control of a leukocyte-specific promoter and transplanted transgenic cells into dystrophic mice. Transplantation diminishes pathology, reduces Th2 cytokines in muscle and biases macrophages away from a CD163+/CD206+ phenotype that promotes fibrosis. Transgenic cells also abrogate TGFß signaling, reduce fibro/adipogenic progenitor cells and reduce fibrogenesis of muscle cells. These findings indicate that leukocytes expressing a LIF transgene reduce fibrosis by suppressing type 2 immunity and highlight a novel application by which immune cells can be genetically modified as potential therapeutics to treat muscle disease.


Assuntos
Terapia Genética , Fator Inibidor de Leucemia/metabolismo , Distrofia Muscular Animal/terapia , Animais , Células da Medula Óssea/metabolismo , Regulação da Expressão Gênica , Fator Inibidor de Leucemia/genética , Masculino , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/patologia , Distribuição Aleatória , Organismos Livres de Patógenos Específicos , Transgenes
6.
Plant Physiol Biochem ; 141: 231-239, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31195253

RESUMO

Anther/pollen development is a highly programmed process in flowering plants. However, the molecular mechanism of regulating anther/pollen development is still largely unclear so far. Here, we report a cotton WRKY transcription factor (GhWRKY22) that functions in anther/pollen development. Quantitative RT-PCR and GUS activity analyses revealed that GhWRKY22 is predominantly expressed in the late developing anther/pollen of cotton. The transgenic Arabidopsis plants expressing GhWRKY22 displayed the male fertility defect with the fewer viable pollen grains. Expression of the genes involved in jasmonate (JA) biosynthesis was up-regulated, whereas expression of the JA-repressors (JAZ1 and JAZ8) was down-regulated in the transgenic Arabidopsis plants expressing GhWRKY22, compared with those in wild type. Yeast one-hybrid and ChIP-qPCR assays demonstrated that GhWRKY22 modulated the expression of JAZ genes by directly binding to their promoters for regulating anther/pollen development. Yeast two-hybrid assay indicated that GhMYB24 could interact with GhJAZ8-A and GhJAZ13-A. Furthermore, expression of AtMYB24, AtPAL2 and AtANS2 was enhanced in the transgenic Arabidopsis plants, owing to GhWRKY22 overexpression. Taking the data together, our results suggest that GhWRKY22 acts as a transcriptional repressor to regulate anther/pollen development possibly by modulating the expression of the JAZ genes.


Assuntos
Gossypium/metabolismo , Pólen/fisiologia , Fatores de Transcrição/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Ciclopentanos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Hipocótilo/metabolismo , Oxilipinas/metabolismo , Fenótipo , Infertilidade das Plantas/genética , Proteínas de Plantas/metabolismo , Caules de Planta/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Sementes/metabolismo , Ativação Transcricional , Transgenes , Técnicas do Sistema de Duplo-Híbrido
7.
Genome Biol ; 20(1): 120, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186073

RESUMO

BACKGROUND: The three-dimensional (3D) organization of chromosomes is linked to epigenetic regulation and transcriptional activity. However, only few functional features of 3D chromatin architecture have been described to date. The KNOT is a 3D chromatin structure in Arabidopsis, comprising 10 interacting genomic regions termed KNOT ENGAGED ELEMENTs (KEEs). KEEs are enriched in transposable elements and associated small RNAs, suggesting a function in transposon biology. RESULTS: Here, we report the KNOT's involvement in regulating invasive DNA elements. Transgenes can specifically interact with the KNOT, leading to perturbations of 3D nuclear organization, which correlates with the transgene's expression: high KNOT interaction frequencies are associated with transgene silencing. KNOT-linked silencing (KLS) cannot readily be connected to canonical silencing mechanisms, such as RNA-directed DNA methylation and post-transcriptional gene silencing, as both cytosine methylation and small RNA abundance do not correlate with KLS. Furthermore, KLS exhibits paramutation-like behavior, as silenced transgenes can lead to the silencing of active transgenes in trans. CONCLUSION: Transgene silencing can be connected to a specific feature of Arabidopsis 3D nuclear organization, namely the KNOT. KLS likely acts either independent of or prior to canonical silencing mechanisms, such that its characterization not only contributes to our understanding of chromosome folding but also provides valuable insights into how genomes are defended against invasive DNA elements.


Assuntos
Arabidopsis/genética , Inativação Gênica , Genoma de Planta , Conformação de Ácido Nucleico , Transgenes , Elementos de DNA Transponíveis
8.
Neuron ; 103(1): 21-38.e5, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31147152

RESUMO

Understanding brain-wide neuronal dynamics requires a detailed map of the underlying circuit architecture. We built an interactive cellular-resolution atlas of the zebrafish brain at 6 days post-fertilization (dpf) based on the reconstructions of over 2,000 individually GFP-labeled neurons. We clustered our dataset in "morphotypes," establishing a unique database of quantitatively described neuronal morphologies together with their spatial coordinates in vivo. Over 100 transgene expression patterns were imaged separately and co-registered with the single-neuron atlas. By annotating 72 non-overlapping brain regions, we generated from our dataset an inter-areal wiring diagram of the larval brain, which serves as ground truth for synapse-scale, electron microscopic reconstructions. Interrogating our atlas by "virtual tract tracing" has already revealed previously unknown wiring principles in the tectum and the cerebellum. In conclusion, we present here an evolving computational resource and visualization tool, which will be essential to map function to structure in a vertebrate brain. VIDEO ABSTRACT.


Assuntos
Atlas como Assunto , Encéfalo/anatomia & histologia , Encéfalo/citologia , Peixe-Zebra/anatomia & histologia , Animais , Encéfalo/ultraestrutura , Mapeamento Encefálico , Cerebelo/anatomia & histologia , Conectoma , Expressão Gênica , Proteínas de Fluorescência Verde , Larva/anatomia & histologia , Larva/citologia , Neurônios/ultraestrutura , Transgenes , Vias Visuais/anatomia & histologia
9.
Plant Mol Biol ; 100(4-5): 481-494, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31073810

RESUMO

KEY MESSAGE: Modification of the poplar defense pathway through pathogen-induced expression of an amphibian host defense peptide modulates plant innate immunity and confers robust and reliable resistance against a major poplar pathogen, Septoria musiva. Host defense peptides (HDPs), also known as cationic antimicrobial peptides, represent a diverse group of small membrane-active molecules that are part of the innate defense system of their hosts against pathogen invasion. Here we describe a strategy for development of poplar plants with enhanced HDP production and resistance to the commercially significant fungal pathogen Septoria musiva. The naturally occurring linear amphipathic α-helical HDP dermaseptin B1, which has 31 residues and originated from the skin secretion of arboreal frogs, was N-terminally modified (MsrA2) and evaluated in vitro for antifungal activity and phytotoxicity. The MsrA2 peptide inhibited germination of S. musiva conidia at physiologically relevant low micromolar concentrations that were non-toxic to poplar protoplasts. The nucleotide sequence of MsrA2, optimized for expression in plants, was introduced into the commercial hybrid poplar Populus nigra L. × P. maximowiczii A. Henry (NM6) via Agrobacterium-mediated transformation. Transgene expression was regulated by the pathogen-inducible poplar promoter win3.12T, a part of the poplar innate defense system. Most importantly, the induced accumulation of MsrA2 peptide in poplar leaves was sufficient to confer resistance against S. musiva. The antifungal resistance of plants with high MsrA2 expression and MsrA2 accumulation was strong and reproducible, and without deleterious effects on plant growth and development. These results provide an insight into development of new technologies for engineering durable disease resistance against major pathogens of poplar and other plants.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Ascomicetos/imunologia , Resistência à Doença/genética , Populus/imunologia , Genoma de Planta , Plantas Geneticamente Modificadas/imunologia , Populus/genética , Populus/microbiologia , Regiões Promotoras Genéticas , Transformação Genética , Transgenes
10.
BMC Genomics ; 20(1): 336, 2019 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-31053056

RESUMO

BACKGROUND: Triploid coho salmon are excellent models for studying gene dosage and the effects of increased cell volume on gene expression. Triploids have an additional haploid genome in each cell and have fewer but larger cells than diploid coho salmon to accommodate the increased genome size. Studying gene expression in triploid coho salmon provides insight into how gene expression may have been affected after the salmonid-specific genome duplication which occurred some 90 MYA. Triploid coho salmon are sterile and consequently can live longer and grow larger than diploid congeners in many semelparous species (spawning only once) because they never reach maturity and post-spawning mortality is averted. Triploid fishes are also of interest to the commercial sector (larger fish are more valuable) and to fisheries management since sterile fish can potentially minimize negative impacts of escaped fish in the wild. RESULTS: The vast majority of genes in liver tissue had similar expression levels between diploid and triploid coho salmon, indicating that the same amount of mRNA transcripts were being produced per gene copy (positive gene dosage effects) within a larger volume cell. Several genes related to nutrition and compensatory growth were differentially expressed between diploid and triploid salmon, indicating that some loci are sensitive to cell size and/or DNA content per cell. To examine how robust expression between ploidies is under different conditions, a genetic/metabolic modifier in the form of different doses of a growth hormone transgene was used to assess gene expression under conditions that the genome has not naturally experienced or adapted to. While many (up to 1400) genes were differentially expressed between non-transgenic and transgenic fish, relatively few genes were differentially expressed between diploids and triploids with similar doses of the transgene. These observations indicate that the small effect of ploidy on gene expression is robust to large changes in physiological state. CONCLUSIONS: These findings are of interest from a gene regulatory perspective, but also valuable for understanding phenotypic effects in triploids, transgenics, and triploid transgenics that could affect their utility in culture conditions and their fitness and potential consequences of release into nature.


Assuntos
Animais Geneticamente Modificados/genética , Diploide , Regulação da Expressão Gênica , Hormônio do Crescimento/administração & dosagem , Fígado/metabolismo , Oncorhynchus kisutch/genética , Triploidia , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Hormônio do Crescimento/genética , Oncorhynchus kisutch/crescimento & desenvolvimento , Oncorhynchus kisutch/metabolismo , Transgenes
11.
Plant Physiol Biochem ; 141: 40-50, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31128562

RESUMO

Agrobacterium-mediated soybean transformation has been greatly improved in recent years, however the transformation efficiency is still low and highly genotype-dependent when compared to other species. Here, we characterized seventeen soybean genotypes based on their genetic transformation efficiencies, i.e., high and low, during Agrobacterium-mediated transformation. To reveal the molecular basis of this transformation difference, we constructed a highly efficient transient transgene expression system using soybean cotyledon protoplasts and then assess the methylation levels of promoter and coding regions of an EYFP (enhanced yellow fluorescent protein) gene introduced into the protoplast cultures of various soybean genotypes using BSP (bisulfite sequencing PCR). Increased methylation was found to be associated with the considerably decreased transfection efficiency (as percentage of EYFP fluorescent protoplasts) in low-efficacy genotypes as compared with those in high-efficacy on three DAT (day after transfection). 5-Azacytidine (5-Azac), a demethylating reagent commonly applied in epigenetic researches, significantly improved the transient transfection efficiency and transgene expression level in low-efficiency genotypes. Furthermore, the shoot regeneration efficiency in low-efficiency genotypes was substantially increased by 5-Azac treatment in an Agrobacterium-mediated soybean transformation system. Taken together, we concluded that lower methylation level in transgene contributed to enhanced shoot regeneration in Agrobacterium-mediated soybean transformation.


Assuntos
Agrobacterium tumefaciens/genética , Azacitidina/farmacologia , Técnicas de Transferência de Genes , Brotos de Planta/efeitos dos fármacos , Soja/efeitos dos fármacos , Cotilédone/genética , Metilação de DNA , Fabaceae/genética , Genes de Plantas , Genótipo , Metilação , Peptídeo Hidrolases/metabolismo , Plantas Geneticamente Modificadas/genética , Protoplastos , Regeneração , Análise de Sequência de DNA , Soja/genética , Transfecção , Transformação Genética , Transgenes
12.
Plant Cell Rep ; 38(9): 1039-1051, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31144112

RESUMO

KEY MESSAGE: Overexpression of FvC5SD improves drought tolerance in soybean. Drought stress is one of the most important abiotic stress factors that influence soybean crop quality and yield. Therefore, the creation of drought-tolerant soybean germplasm resources through genetic engineering technology is effective in alleviating drought stress. FvC5SD is a type of C-5 sterol desaturase gene that is obtained from the edible fungus Flammulina velutipes. This gene has good tolerance to the effects of stresses, including drought and low temperature, in yeast cells and tomato. In this study, we introduced the FvC5SD gene into the soybean variety Shennong9 through the Agrobacterium-mediated transformation of soybean to identify drought-tolerant transgenic soybean varieties. PCR, RT-PCR, and Southern blot analysis results showed that T-DNA was inserted into the soybean genome and stably inherited by the progeny. The ectopic expression of FvC5SD under the control of a CaMV 35S promoter in transgenic soybean plants enhanced the plant's tolerance to dehydration and drought. Under drought conditions, the transgenic plants accumulated lower levels of reactive oxygen species and exhibited higher activities and expression levels of enzymes and cell than wild-type soybean. iTRAQ analysis of the comparative proteomics showed that some exogenous genes coding either functional or regulatory proteins were induced in the transgenic lines under drought stress. FvC5SD overexpression can serve as a direct and efficient target in improving drought tolerance in soybean and may be an important biotechnological strategy for trait improvement in soybean and other crops.


Assuntos
Flammulina/genética , Depuradores de Radicais Livres/metabolismo , Regulação da Expressão Gênica de Plantas , Oxirredutases/genética , Espécies Reativas de Oxigênio/metabolismo , Secas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Oxirredutases/metabolismo , Plantas Geneticamente Modificadas , Soja/genética , Estresse Fisiológico , Transgenes
13.
Nat Plants ; 5(5): 453-454, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31040445
14.
Nat Plants ; 5(5): 505-511, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036912

RESUMO

The engineering of plant genomes presents exciting opportunities to modify agronomic traits and to produce high-value products in plants. Expression of foreign proteins from transgenes in the chloroplast genome offers advantages that include the capacity for prodigious protein output, the lack of transgene silencing and the ability to express multicomponent pathways from polycistronic mRNA. However, there remains a need for robust methods to regulate plastid transgene expression. We designed orthogonal activators that boost the expression of chloroplast transgenes harbouring cognate cis-elements. Our system exploits the programmable RNA sequence specificity of pentatricopeptide repeat proteins and their native functions as activators of chloroplast gene expression. When expressed from nuclear transgenes, the engineered proteins stimulate the expression of plastid transgenes by up to ~40-fold, with maximal protein abundance approaching that of Rubisco. This strategy provides a means to regulate and optimize the expression of foreign genes in chloroplasts and to avoid deleterious effects of their products on plant growth.


Assuntos
Proteínas de Arabidopsis/genética , Cloroplastos/genética , Regulação da Expressão Gênica de Plantas/genética , Genes de Troca/genética , Engenharia de Proteínas , Transgenes/genética , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Engenharia de Proteínas/métodos , Proteínas de Ligação a RNA/genética
15.
Nat Plants ; 5(5): 486-490, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036913

RESUMO

Non-green plastids are desirable for the expression of recombinant proteins in edible plant parts to enhance the nutritional value of tubers or fruits, or to deliver pharmaceuticals. However, plastid transgenes are expressed at extremely low levels in the amyloplasts of storage organs such as tubers1-3. Here, we report a regulatory system comprising a variant of the maize RNA-binding protein PPR10 and a cognate binding site upstream of a plastid transgene that encodes green fluorescent protein (GFP). The binding site is not recognized by the resident potato PPR10 protein, restricting GFP protein accumulation to low levels in leaves. When the PPR10 variant is expressed from the tuber-specific patatin promoter, GFP accumulates up to 1.3% of the total soluble protein, a 60-fold increase compared with previous studies2 (0.02%). This regulatory system enables an increase in transgene expression in non-photosynthetic plastids without interfering with chloroplast gene expression in leaves.


Assuntos
Proteínas de Plantas/genética , Plastídeos/genética , Engenharia de Proteínas/métodos , Proteínas de Ligação a RNA/genética , Transgenes/genética , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Fluorescência Verde/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Solanum tuberosum/genética , Zea mays/genética , Zea mays/metabolismo
16.
Nat Plants ; 5(5): 539-550, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31076735

RESUMO

Post-transcriptional gene silencing (PTGS) is a major mechanism regulating gene expression in higher eukaryotes. To identify novel players in PTGS, a forward genetics screen was performed on an Arabidopsis thaliana line overexpressing a strong growth-repressive gene, ETHYLENE RESPONSE FACTOR6 (ERF6). We identified six independent ethyl-methanesulfonate mutants rescuing the dwarfism of ERF6-overexpressing plants as a result of transgene silencing. Among the causative genes, ETHYLENE-INSENSITIVE5, SUPERKILLER2 and HASTY1 have previously been reported to inhibit PTGS. Notably, the three other causative genes have not, to date, been related to PTGS: UTP:RNA-URIDYLYLTRANSFERASE1 (URT1), C-TERMINAL DOMAIN PHOSPHATASE-LIKE3 (CPL3) and RESURRECTION1 (RST1). We show that these genes may participate in protecting the 3' end of transgene transcripts. We present a model in which URT1, CPL3 and RST1 are classified as PTGS suppressors, as compromisation of these genes provokes the accumulation of aberrant transcripts which, in turn, trigger the production of small interfering RNAs, initiating RNA silencing.


Assuntos
Proteínas de Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Membrana/fisiologia , Fosfoproteínas Fosfatases/fisiologia , Interferência de RNA , RNA Nucleotidiltransferases/fisiologia , RNA de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , RNA de Plantas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transgenes/genética
17.
J Zhejiang Univ Sci B ; 20(4): 322-331, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30932377

RESUMO

We investigated the microRNA172 (miR172)-mediated regulatory network for the perception of changes in external and endogenous signals to identify a universally applicable floral regulation system in ornamental plants, manipulation of which could be economically beneficial. Transgenic gloxinia plants, in which miR172 was either overexpressed or suppressed, were generated using Agrobacterium-mediated transformation. They were used to study the effect of altering the expression of this miRNA on time of flowering and to identify its mRNA target. Early or late flowering was observed in transgenic plants in which miR172 was overexpressed or suppressed, respectively. A full-length complementary DNA (cDNA) of gloxinia (Sinningia speciosa) APETALA2-like (SsAP2-like) was identified as a target of miR172. The altered expression levels of miR172 caused up- or down-regulation of SsAP2-like during flower development, which affected the time of flowering. Quantitative real-time reverse transcription PCR analysis of different gloxinia tissues revealed that the accumulation of SsAP2-like was negatively correlated with the expression of miR172a, whereas the expression pattern of miR172a was negatively correlated with that of miR156a. Our results suggest that transgenic manipulation of miR172 could be used as a universal strategy for regulating time of flowering in ornamental plants.


Assuntos
Proteínas de Arabidopsis/genética , Flores/fisiologia , Proteínas de Homeodomínio/genética , Lamiales/fisiologia , MicroRNAs/genética , Proteínas Nucleares/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clonagem Molecular , DNA Complementar/metabolismo , Flores/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/metabolismo , Lamiales/genética , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Transgenes
18.
PLoS Genet ; 15(4): e1007847, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30998684

RESUMO

The embryonic cuticle is necessary for normal seed development and seedling establishment in Arabidopsis. Although mutants with defective embryonic cuticles have been identified, neither the deposition of cuticle material, nor its regulation, has been described during embryogenesis. Here we use electron microscopy, cuticle staining and permeability assays to show that cuticle deposition initiates de novo in patches on globular embryos. By combining these techniques with genetics and gene expression analysis, we show that successful patch coalescence to form a continuous cuticle requires a signalling involving the endosperm-specific subtilisin protease ALE1 and the receptor kinases GSO1 and GSO2, which are expressed in the developing embryonic epidermis. Transcriptome analysis shows that this pathway regulates stress-related gene expression in seeds. Consistent with these findings we show genetically, and through activity analysis, that the stress-associated MPK6 protein acts downstream of GSO1 and GSO2 in the developing embryo. We propose that a stress-related signalling pathway has been hijacked in some angiosperm seeds through the recruitment of endosperm-specific components. Our work reveals the presence of an inter-compartmental dialogue between the endosperm and embryo that ensures the formation of an intact and functional cuticle around the developing embryo through an "auto-immune" type interaction.


Assuntos
Arabidopsis/embriologia , Arabidopsis/fisiologia , Desenvolvimento Embrionário , Desenvolvimento Vegetal , Transdução de Sinais , Estresse Fisiológico , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Desenvolvimento Embrionário/genética , Endosperma/embriologia , Endosperma/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fenótipo , Desenvolvimento Vegetal/genética , Plantas Geneticamente Modificadas , Sementes/genética , Estresse Fisiológico/genética , Transgenes
19.
Dev Growth Differ ; 61(4): 276-282, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30968390

RESUMO

Skin development is tightly temporally coordinated with its sensory innervation, which consists of the peripheral branches of the dorsal root ganglion (DRG) axons. Various studies suggest that the skin produces a long-range attractant for the sensory axons. However, the exact identity of the guidance cue(s) remains unclear. To reveal the detailed molecular mechanism that controls DRG axon guidance and targeting, manipulation of specific skin layers at specific time points are required. To test a variety of attractants that can be expressed in specific skin layers at specific timepoints, we combined in utero electroporation with the Tol2 transposon system to induce long-term transgene expression in the developing mouse skin, including in the highly proliferative epidermal stem cells (basal layer) and their descendants (spinous and granular layer cells). The plasmid solution was injected as close to the hindpaw plantar surface as possible. Immediately, electric pulses were passed through the embryo to transduce the plasmid DNA into hindpaw skin cells. Balancing outcome measurements including: embryo survival, transfection efficiency, and the efficiency of transgene integration into host cells, we found that IUE was best performed on E13.5, and using an electroporation voltage of 34V. After immunostaining embryonic and early postnatal skin tissue sections for keratinocyte and sensory axon markers, we observe the growth of axons into skin epidermal layers including areas expressing EGFP. Therefore, this method is useful for studying the interaction between axon growth and epidermal cell division/differentiation.


Assuntos
Epiderme/inervação , Epiderme/metabolismo , Neurônios/metabolismo , Pele/inervação , Pele/metabolismo , Transgenes/genética , Animais , Axônios/metabolismo , Células Epidérmicas/citologia , Células Epidérmicas/metabolismo , Epiderme/embriologia , Epiderme/crescimento & desenvolvimento , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Gravidez , Pele/embriologia , Pele/crescimento & desenvolvimento
20.
Mitochondrion ; 46: 15-21, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30980913

RESUMO

Mitochondrial genomes (mtDNA) depend on the nuclear genome with which they have evolved to provide essential replication functions and have been known to replicate as xenotransplants only in the cells of closely related species. We now report that complete mouse mitochondrial genomes can be stably transplanted into the mitochondrial network in yeast devoid of their own mtDNA. Our analyses of these xenomitochondrial yeast cells show that they are accurately replicating intact mouse mtDNA genomes without rearrangement and that these mtDNA genomes have the same overall topology as the mtDNA present in the mouse mitochondrial network (i.e., circular monomers). Moreover, non-mtDNA replication and selection sequences required for maintaining the mitochondrial genomes in bacterial hosts are dispensable in these yeast mitochondria and could be efficiently and seamlessly removed by targeted homologous recombination within the mitochondria. These findings demonstrate that the yeast mtDNA replication system is capable of accurately replicating intact mammalian mtDNA genomes without sequence loss or rearrangement and that yeast mitochondria are a highly versatile host system for engineering complete mammalian mitochondrial genomes.


Assuntos
Replicação do DNA , DNA Mitocondrial/metabolismo , Genoma Mitocondrial , Camundongos/genética , Organismos Geneticamente Modificados , Transgenes , Leveduras/genética , Animais , DNA Mitocondrial/genética , Instabilidade Genômica
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