Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 17.185
Filtrar
1.
Nat Commun ; 11(1): 3847, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737299

RESUMO

Reporter systems are routinely used in plant genetic engineering and functional genomics research. Most such plant reporter systems cause accumulation of foreign proteins. Here, we demonstrate a protein-independent reporter system, 3WJ-4 × Bro, based on a fluorescent RNA aptamer. Via transient expression assays in both Escherichia coli and Nicotiana benthamiana, we show that 3WJ-4 × Bro is suitable for transgene identification and as an mRNA reporter for expression pattern analysis. Following stable transformation in Arabidopsis thaliana, 3WJ-4 × Bro co-segregates and co-expresses with target transcripts and is stably inherited through multiple generations. Further, 3WJ-4 × Bro can be used to visualize virus-mediated RNA delivery in plants. This study demonstrates a protein-independent reporter system that can be used for transgene identification and in vivo dynamic analysis of mRNA.


Assuntos
Aptâmeros de Nucleotídeos/genética , Arabidopsis/genética , Brassica/genética , Engenharia Genética/métodos , RNA Mensageiro/genética , Tabaco/genética , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Arabidopsis/metabolismo , Compostos de Benzil/química , Brassica/metabolismo , Fluorescência , Corantes Fluorescentes/química , Regulação da Expressão Gênica , Genes Reporter , Imidazolinas/química , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , RNA Mensageiro/metabolismo , Tabaco/metabolismo , Transformação Genética
2.
Zhongguo Zhong Yao Za Zhi ; 45(13): 3112-3119, 2020 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-32726019

RESUMO

The WRKY family genes, which play an important role in plant morphogenesis and stress response, were selected based on the data of the full-length transcriptome of Asarum heterotropoides. Using AtWRKY33, which regulates the synthesis of the camalexin in the model plant Arabidopsis to compare homologous genes in A. heterotropoides, primers were designed to amplify the open reading frame(ORF) fragment of AhWRKY33 gene by RT-PCR using total RNA of A. heterotropoides leaves as template. Real-time PCR results showed that there was a significant difference between the aerial part and the underground part of A. heterotropoides, the toxic aristolochic acid content is highly expressed in the leaves higher than the root. After verification, the WRKY33 gene of A. heterotropoides is ORF long 1 686 bp, encoding 561 amino acids.AhWRKY33 had two conserved WRKYGQK domains. According to the classical classification, it belongs to group Ⅰ WRKY transcription factor. A. heterotropoides WRKY33 had some homology with amino acids of other species. The study successfully constructed the plant eukaryotic expression vector PHG-AhWRKY33 and transformed Arabidopsis thaliana, the transgenic Arabidopsis was obtained by PCR detection and hygromycin resistant plate screening. It found that the germination of transgenic Arabidopsis seeds was accelerated and the stress resistance was increased. It laid a foundation for further analysis of WRKY transcription factor in the growth and development of A. heterotropoides and the synthesis of secondary metabolites.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Asarum , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Folhas de Planta , Proteínas de Plantas/genética , Fatores de Transcrição , Transformação Genética
3.
PLoS One ; 15(6): e0233911, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479550

RESUMO

Promoters are very important for transcriptional regulation and gene expression, and have become invaluable tools for genetic engineering. Owing to the characteristics of obligate biotrophs, molecular research into obligate biotrophic fungi is seriously lagging behind, and very few of their endogenous promoters have been developed. In this study, a WY7 fragment was predicted in the genome of Oidium heveae Steinmann using PromoterScan. Its promoter function was verified with transient transformations (Agrobacterium tumefaciens-mediated transformation, ATMT) in Nicotiana tabacum cv. Xanthi nc. The analysis of the transcription range showed that WY7 could regulate GUS expression in both monocots (Zea mays Linn and Oryza sativa L. spp. Japonica cv. Nipponbare) and dicots (N. tabacum and Hylocereus undulates Britt). The results of the quantitative detection showed that the GUS transient expression levels when regulated by WY7 was more than 11.7 times that of the CaMV 35S promoter in dicots (N. tabacum) and 5.13 times that of the ACT1 promoter in monocots (O. sativa). GUS staining was not detected in the T1 generation of the WY7-GUS transgenic N. tabacum. This showed that WY7 is an inducible promoter. The cis elements of WY7 were predicted using PlantCARE, and further experiments indicated that WY7 was a low temperature- and salt-inducible promoter. Soluble proteins produced by WY7-hpa1Xoo transgenic tobacco elicited hypersensitive responses (HR) in N. tabacum leaves. N. tabacum transformed with pBI121-WY7-hpa1Xoo exhibited enhanced resistance to the tobacco mosaic virus (TMV). The WY7 promoter has a lot of potential as a tool for plant genetic engineering. Further in-depth studies will help to better understand the transcriptional regulation mechanisms of O. heveae.


Assuntos
Fungos/genética , Regulação Fúngica da Expressão Gênica , Engenharia Genética/métodos , Doenças das Plantas/prevenção & controle , Regiões Promotoras Genéticas , Fungos/patogenicidade , Genoma Fúngico , Hevea/genética , Hevea/microbiologia , Interações Hospedeiro-Patógeno/genética , Magnoliopsida/genética , Magnoliopsida/microbiologia , Oryza/genética , Oryza/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Tabaco/genética , Tabaco/microbiologia , Transformação Genética , Zea mays/genética , Zea mays/microbiologia
4.
Nat Commun ; 11(1): 2965, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528082

RESUMO

Trajectories of cellular ontogeny are tightly controlled and often involve feedback-regulated molecular antagonism. For example, sieve element differentiation along developing protophloem cell files of Arabidopsis roots requires two antagonistic regulators of auxin efflux. Paradoxically, loss-of-function in either regulator triggers similar, seemingly stochastic differentiation failures of individual sieve element precursors. Here we show that these patterning defects are distinct and non-random. They can be explained by auxin-dependent bistability that emerges from competition for auxin between neighboring cells. This bistability depends on the presence of an auxin influx facilitator, and can be triggered by either flux enhancement or repression. Our results uncover a hitherto overlooked aspect of auxin uptake, and highlight the contributions of local auxin influx, efflux and biosynthesis to protophloem formation. Moreover, the combined experimental-modeling approach suggests that without auxin efflux homeostasis, auxin influx interferes with coordinated differentiation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Transformação Genética/genética
5.
J Genet ; 992020.
Artigo em Inglês | MEDLINE | ID: mdl-32529984

RESUMO

CmFT homologous gene in muskmelon was obtained by homologous cloning, introducing CmFT gene by Agrobacterium mediated transformation. The results of subcellular localization showed that CmFT protein was expressed in cytoplasm and nucleus. qRTPCR results showed that the expression levels of AtLFY, AtFT, AtCO, AtFLC, AtSOC1 and AtAP1 were upregulated in the 35S::MeFT Arabidopsis line. The CmFT gene was introduced into wild-type Arabidopsis by Agrobacterium-mediated transformation, and the growth status of T2 transgenic Arabidopsis thaliana and wild-type A. thaliana was observed. The results showed that wild-type Arabidopsis began to bolt on the 25th day after sowing, we can initially confirm that the FT gene of melon can promote the early flowering of melon in the growth and development of melon.


Assuntos
Agrobacterium/genética , Arabidopsis/genética , Clonagem Molecular , Cucumis melo/genética , Genes de Plantas , Fenótipo , Plantas Geneticamente Modificadas , Transformação Genética
6.
PLoS One ; 15(5): e0232770, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32369513

RESUMO

Cereal cyst nematodes cause serious yield losses of wheat in Hunaghuai winter wheat growing region in China. Beauveria bassiana 08F04 isolated from the surface of cysts is a promising biological control agent for cereal cyst nematodes. As the colonization capacity is a crucial criteria to assess biocontrol effectiveness for a microbial agent candidate, we aimed to label B. bassiana 08F04 for efficient monitoring of colonization in the soil. The binary pCAM-gfp plasmid containing sgfp and hph was integrated into B. bassiana 08F04 using the Agrobacterium tumefaciens-mediated transformation. The transformation caused a significant change in mycelial and conidial yields, and in extracellular chitinase activity in some transformants. The cultural filtrates of some transformants also decreased acetylcholinesterase activity and the survival of Heterodera filipjevi second-stage juveniles relative to the wild-type strain. One transformant (G10) had a growth rate and biocontrol efficacy similar to the wild-type strain, so it was used for a pilot study of B. bassiana colonization conducted over 13 weeks. Real-time PCR results and CFU counts revealed that the population of G10 increased quickly over the first 3 weeks, then decreased slowly over the following 4 weeks before stabilizing. In addition, the application of wild-type B. bassiana 08F04 and transformant G10 significantly reduced the number of H. filipjevi females in roots by 64.4% and 60.2%, respectively. The results of this study have practical applications for ecological, biological and functional studies of B. bassiana 08F04 and for bionematicide registration.


Assuntos
Beauveria/fisiologia , Controle Biológico de Vetores , Doenças das Plantas/parasitologia , Triticum/parasitologia , Tylenchida/fisiologia , Agrobacterium tumefaciens/genética , Animais , Beauveria/genética , Feminino , Raízes de Plantas/parasitologia , Microbiologia do Solo , Transformação Genética
7.
Sheng Wu Gong Cheng Xue Bao ; 36(4): 643-651, 2020 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-32347059

RESUMO

Cucumber (Cucumis sativus) is an important vegetable crop in the world. Agrobacterium-mediated transgenic technology is an important way to study plant gene functions and improve varieties. In order to further accelerate the transgenic research and breeding process of cucumber, we described the progress and problems of Agrobacterium tumefaciens-mediated transgenic cucumber, from the influencing factors of cucumber regeneration ability, genetic transformation conditions and various additives in the process. We prospected for improving the genetic transformation efficiency and safety selection markers of cucumber, and hoped to provide reference for the research of cucumber resistance breeding and quality improvement.


Assuntos
Agrobacterium tumefaciens , Cucumis sativus , Plantas Geneticamente Modificadas , Transformação Genética , Agrobacterium tumefaciens/metabolismo , Cruzamento , Cucumis sativus/genética , Cucumis sativus/microbiologia , Plantas Geneticamente Modificadas/microbiologia , Pesquisa
8.
Sheng Wu Gong Cheng Xue Bao ; 36(4): 700-706, 2020 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-32347064

RESUMO

The responsibility of root is absorbing water and nutrients, it is an important plant tissue, but easily to be affected by biotic and abiotic stresses, affecting crop growth and yield. The design of a synthetic root-specific promoter provides candidate promoters for the functional analysis and efficient expression of stress-related genes in crop roots. In this study, a synthetic root-specific module (pro-SRS) was designed using tandem four-copies of root specific cis-acting elements (OSE1ROOTNODULE, OSE2ROOTNODULE, SP8BFIBSP8AIB, and ROOTMOTIFAPOX1), and fused with minimal promoter from the CaMV 35S promoter to synthesize an artificially synthetic SRSP promoter. The SRSP promoter was cloned in pCAMBIA2300.1 by replacing CaMV 35S promoter so as to drive GUS expression. The constructs with SRSP promoter were transformed in tobacco by Agrobacterium-mediated method. SRSP promoter conferred root-specific expression in transgenic tobacco plants through Real-time PCR (RT-PCR) analysis and GUS histochemical staining analysis. It is indicated that the repeated arrangement of cis-acting elements can realize the expected function of the promoter. This study laid a theoretical foundation for the rational design of tissue-specific promoters.


Assuntos
Regulação da Expressão Gênica de Plantas , Raízes de Plantas , Regiões Promotoras Genéticas , Tabaco , Agrobacterium/genética , Clonagem Molecular , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Estresse Fisiológico , Tabaco/genética , Tabaco/crescimento & desenvolvimento , Transformação Genética
9.
Nat Methods ; 17(5): 481-494, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32251396

RESUMO

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.


Assuntos
DNA/administração & dosagem , Eucariotos/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Biologia Marinha , Modelos Biológicos , Transformação Genética , Biodiversidade , Ecossistema , Meio Ambiente , Eucariotos/classificação , Especificidade da Espécie
10.
Plant Mol Biol ; 103(4-5): 443-456, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32270430

RESUMO

KEY MESSAGE: The simultaneous expression of AmRosea1 and AmDelila transcription factors from snapdragon can activate the anthocyanin pathway in orange carrots, leading to the synthesis and accumulation of anthocyanins in the taproots. Anthocyanins are phenolic compounds produced in various parts of plants. They are used as natural food dyes and are reported as beneficial antioxidants for humans. Black carrot is an important source for anthocyanins; however, the reason for the lack of anthocyanin production in the orange carrot is unknown. Anthocyanins are synthesized by a specific branch of the phenylpropanoid pathway that has previously been reported to be activated by a triad of R2R3-MYB, basic helix-loop helix (bHLH) and WD40 transcription factors (TFs). In the current study, orange carrots were turned purple by simultaneous expression of R2R3-MYB and bHLH TFs, i.e. AmRosea1 and AmDelila from snapdragon (Antirrhinum majus). Simultaneous transgenic expression of the TFs under a constitutive promoter in the orange carrot cultivar 'Danvers 126' lead to consistent upregulation of anthocyanin-related biosynthetic genes and significant accumulation of anthocyanins in leaves, stems and taproots. Highest overall content of soluble anthocyanins in the taproot among the transformants amounted to 44.38 mg g-1 dry weight. The anthocyanin profile of the transformants were significantly different from the profile in the reference black carrot 'Deep Purple'. The main anthocyanins present in the transformed taproots were cyanidin 3-xylosyl(sinapoylglucosyl)galactoside, whereas the main anthocyanin present in Deep Purple was cyanidin 3-xylosyl(feruloylglucosyl)galactoside. This study confirms the presence of the necessary biosynthetic genes in orange carrots for production of anthocyanins and demonstrates the absence of suitable R2R3-MYB and bHLH TFs for stimulating anthocyanin biosynthesis in the orange carrot.


Assuntos
Antocianinas/biossíntese , Antocianinas/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Daucus carota/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Vias Biossintéticas/genética , Cor , Daucus carota/genética , Genes de Plantas/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Pigmentos Biológicos/biossíntese , Pigmentos Biológicos/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Fatores de Transcrição , Transformação Genética
11.
PLoS One ; 15(3): e0230126, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32226034

RESUMO

The generation of genetically modified mouse models derived from gene targeting (GT) in mouse embryonic stem (ES) cells (mESCs) has greatly advanced both basic and clinical research. Our previous finding that gene targeting at the Myh9 exon2 site in mESCs has a pronounced high homologous recombination (HR) efficiency (>90%) has facilitated the generation of a series of nonmuscle myosin II (NM II) related mouse models. Furthermore, the Myh9 gene locus has been well demonstrated to be a new safe harbor for site-specific insertion of other exogenous genes. In the current study, we intend to investigate the molecular biology underlying for this high HR efficiency from other aspects. Our results confirmed some previously characterized properties and revealed some unreported observations: 1) The comparison and analysis of the targeting events occurring at the Myh9 and several widely used loci for targeting transgenesis, including ColA1, HPRT, ROSA26, and the sequences utilized for generating these targeting constructs, indicated that a total length about 6 kb with approximate 50% GC-content of the 5' and 3' homologous arms, may facilitate a better performance in terms of GT efficiency. 2) Despite increasing the length of the homologous arms, shifting the targeting site from the Myh9 exon2, to intron2, or exon3 led to a gradually reduced GT frequency (91.7, 71.8 and 50.0%, respectively). This finding provides the first evidence that the HR frequency may also be associated with the targeting site even in the same locus. Meanwhile, the decreased trend of the GT efficiency at these targeting sites was consistent with the reduced percentage of simple sequence repeat (SSR) and short interspersed nuclear elements (SINEs) in the sequences for generating the targeting constructs, suggesting the potential effects of these DNA elements on GT efficiency; 3) Our series of targeting experiments and analyses with truncated 5' and 3' arms at the Myh9 exon2 site demonstrated that GT efficiency positively correlates with the total length of the homologous arms (R = 0.7256, p<0.01), confirmed that a 2:1 ratio of the length, a 50% GC-content and the higher amount of SINEs for the 5' and 3' arms may benefit for appreciable GT frequency. Though more investigations are required, the Myh9 gene locus appears to be an ideal location for identifying HR-related cis and trans factors, which in turn provide mechanistic insights and also facilitate the practical application of gene editing.


Assuntos
Marcação de Genes , Recombinação Homóloga/genética , Cadeias Pesadas de Miosina/genética , Animais , Edição de Genes/métodos , Marcação de Genes/métodos , Técnicas de Genotipagem , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas , Transformação Genética
12.
J Vis Exp ; (157)2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32225142

RESUMO

Tartary buckwheat (TB) [Fagopyrum tataricum (L.) Gaertn] possesses various biological and pharmacological activities because it contains abundant secondary metabolites such as flavonoids, especially rutin. Agrobacterium rhizogenes have been gradually used worldwide to induce hairy roots in medicinal plants to investigate gene functions and increase the yield of secondary metabolites. In this study, we have described a detailed method to generate A. rhizogenes-mediated hairy roots in TB. Cotyledons and hypocotyledonary axis at 7-10 days were selected as explants and infected with A. rhizogenes carrying a binary vector, which induced adventitious hairy roots that appeared after 1 week. The generated hairy root transformation was identified based on morphology, resistance selection (kanamycin), and reporter gene expression (green fluorescent protein). Subsequently, the transformed hairy roots were self-propagated as required. Meanwhile, a myeloblastosis (MYB) transcription factor, FtMYB116, was transformed into the TB genome using the A. rhizogenes-mediated hairy roots to verify the role of FtMYB116 in synthesizing flavonoids. The results showed that the expression of flavonoid-related genes and the yield of flavonoid compounds (rutin and quercetin) were significantly (p < 0.01) promoted by FtMYB116, indicating that A. rhizogenes-mediated hairy roots can be used as an effective alternative tool to investigate gene functions and the production of secondary metabolites. The detailed step-by-step protocol described in this study for generating hairy roots can be adopted for any genetic transformation or other medicinal plants after adjustment.


Assuntos
Agrobacterium/metabolismo , Fagopyrum/genética , Fagopyrum/microbiologia , Raízes de Plantas/microbiologia , Transformação Genética , Regulação da Expressão Gênica de Plantas , Genes Reporter , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Luz , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Rutina/biossíntese , Rutina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
13.
J Vis Exp ; (157)2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32225152

RESUMO

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to agriculture. However, the Ralstonia model is considerably underexplored in comparison to other models involving bacterial plant pathogens, such as Pseudomonas syringae in Arabidopsis. Research targeted to understanding the interaction between Ralstonia and crop plants is essential to develop sustainable solutions to fight against bacterial wilt disease but is currently hindered by the lack of straightforward experimental assays to characterize the different components of the interaction in native host plants. In this scenario, we have developed a method to perform genetic analysis of Ralstonia infection of tomato, a natural host of Ralstonia. This method is based on Agrobacterium rhizogenes-mediated transformation of tomato roots, followed by Ralstonia soil-drenching inoculation of the resulting plants, containing transformed roots expressing the construct of interest. The versatility of the root transformation assay allows performing either gene overexpression or gene silencing mediated by RNAi. As a proof of concept, we used this method to show that RNAi-mediated silencing of SlCESA6 in tomato roots conferred resistance to Ralstonia. Here, we describe this method in detail, enabling genetic approaches to understand bacterial wilt disease in a relatively short time and with small requirements of equipment and plant growth space.


Assuntos
Lycopersicon esculentum/genética , Lycopersicon esculentum/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Raízes de Plantas/microbiologia , Ralstonia solanacearum/fisiologia , Transformação Genética , Agrobacterium/metabolismo , Antibacterianos/farmacologia , Arabidopsis/microbiologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Lycopersicon esculentum/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Ralstonia solanacearum/efeitos dos fármacos , Ralstonia solanacearum/crescimento & desenvolvimento , Reprodutibilidade dos Testes , Solo , Transformação Genética/efeitos dos fármacos
14.
J Biosci Bioeng ; 130(1): 89-97, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32192841

RESUMO

Rational metabolic-flow switching, which we proposed recently, is an effective strategy to produce an exogenous high-value natural product using transformed plant cells; the proof of this concept was demonstrated using bamboo (Phyllostachys nigra; Pn) cells as a model system. Pn cells were transformed to express 4-hydroxycinnamoyl-CoA hydratase/lyase of Pseudomonas putida KT2440 (PpHCHL), which catalyzes the formation of 4-hydroxybenzaldehyde and vanillin from p-coumaroyl-CoA and feruloyl-CoA, respectively. The PpHCHL-transformed cells accumulated glucose conjugates of 4-hydroxybenzoic acid and vanillic acid, indicating that the PpHCHL products (aldehydes) were further metabolized by inherent enzymes in the Pn cells. The production titers of 4-hydroxybenzoic acid glucose ester, vanillic acid glucose ester, and 4-hydroxybenzoic acid glucoside reached 1.7, 0.17, and 0.14 g/L at the maximum, respectively. These results proved the versatility of Pn cells for producing vanillin-related compounds based on rational metabolic-flow switching.


Assuntos
Proteínas de Bactérias/genética , Bambusa/metabolismo , Glucose/metabolismo , Hidroliases/genética , Parabenos/metabolismo , Pseudomonas putida/enzimologia , Ácido Vanílico/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bambusa/genética , Benzaldeídos/metabolismo , Catálise , Expressão Gênica , Hidroliases/química , Hidroliases/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Transformação Genética
15.
J Vis Exp ; (156)2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32116304

RESUMO

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.


Assuntos
Agrobacterium tumefaciens/genética , Genes de Plantas , Endogamia , Morfogênese/genética , Transformação Genética , Zea mays/embriologia , Zea mays/genética , DNA Bacteriano/genética , Plantas Geneticamente Modificadas , Plasmídeos/genética , Polinização , Zea mays/crescimento & desenvolvimento
16.
PLoS One ; 15(3): e0229909, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32134988

RESUMO

Stable transformation of common bean (Phaseolus vulgaris L.) has been successful, to date, only using biolistic-mediated transformation and shoot regeneration from meristem-containing embryo axes. In this study, using precultured embryo axes, and optimal co-cultivation conditions resulted in a successful transformation of the common bean cultivar Olathe using Agrobacterium tumefaciens strain EHA105. Plant regeneration through somatic embryogenesis was attained through the preculture of embryo axes for 12 weeks using induced competent cells for A. tumefaciens-mediated gene delivery. Using A. tumefaciens at a low optical density (OD) of 0.1 at a wavelength of 600 nm for infection and 4-day co-cultivation, compared to OD600 of 0.5, increased the survival rate of the inoculated explants from 23% to 45%. Selection using 0.5 mg L-1 glufosinate (GS) was effective to identify transformed cells when the bialaphos resistance (bar) gene under the constitutive 35S promoter was used as a selectable marker. After an 18-week selection period, 1.5% -2.5% inoculated explants, in three experiments with a total of 600 explants, produced GS-resistant plants through somatic embryogenesis. The expression of bar was confirmed in first- and second-generation seedlings of the two lines through reverse polymerase chain reaction. Presence of the bar gene was verified through genome sequencing of two selected transgenic lines. The induction of regenerable, competent cells is key for the successful transformation, and the protocols described may be useful for future transformation of additional Phaseolus germplasm.


Assuntos
Agrobacterium tumefaciens/genética , Phaseolus/genética , Melhoramento Vegetal/métodos , Plantas Geneticamente Modificadas/genética , Transformação Genética , Agrobacterium tumefaciens/efeitos dos fármacos , Aminobutiratos/farmacologia , DNA de Plantas/genética , Farmacorresistência Bacteriana/genética , Vetores Genéticos , Herbicidas/farmacologia , Compostos Organofosforados/farmacologia , Fenótipo , RNA de Plantas/genética
17.
PLoS One ; 15(3): e0230362, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32176712

RESUMO

Fungi in the genus Cercospora cause crop losses world-wide on many crop species. The wide host range and success of these pathogens has been attributed to the production of a photoactivated toxin, cercosporin. We engineered tobacco for resistance to Cercospora nicotianae utilizing two strategies: 1) transformation with cercosporin autoresistance genes isolated from the fungus, and 2) transformation with constructs to silence the production of cercosporin during disease development. Three C. nicotianae cercosporin autoresistance genes were tested: ATR1 and CFP, encoding an ABC and an MFS transporter, respectively, and 71cR, which encodes a hypothetical protein. Resistance to the pathogen was identified in transgenic lines expressing ATR1 and 71cR, but not in lines transformed with CFP. Silencing of the CTB1 polyketide synthase and to a lesser extent the CTB8 pathway regulator in the cercosporin biosynthetic pathway also led to the recovery of resistant lines. All lines tested expressed the transgenes, and a direct correlation between the level of transgene expression and disease resistance was not identified in any line. Resistance was also not correlated with the degree of silencing in the CTB1 and CTB8 silenced lines. We conclude that expression of fungal cercosporin autoresistance genes as well as silencing of the cercosporin pathway are both effective strategies for engineering resistance to Cercospora diseases where cercosporin plays a critical role.


Assuntos
Ascomicetos/genética , Resistência à Doença/genética , Farmacorresistência Fúngica/genética , Inativação Gênica , Genes Fúngicos , Engenharia Genética , Perileno/análogos & derivados , Tabaco/microbiologia , Ascomicetos/efeitos dos fármacos , Resistência à Doença/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Perileno/farmacologia , Plantas Geneticamente Modificadas , Transformação Genética , Transgenes
18.
Lett Appl Microbiol ; 70(5): 388-393, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32077122

RESUMO

Four Agrobacterium tumefaciens strains AGL-1, C58C1, EHA105 and LBA4404 were tested for the effects of strain types on the transformation efficiency in Mortierella alpina. Results showed that AGL-1, C58C1 and EHA105 transformed M. alpina successfully. Among them, A. tumefaciens EHA105 was first proven successful transformation of M. alpina. AGL-1 and EHA105 had the highest transformation efficiency among the four strains, while LBA4404 failed to transform M. alpina. The reason leading to the transformation efficiency difference among the four strains was explored by determining transcription levels of the virulence (vir) gene in the induction medium. Results showed that the expressions of virD1, virD2, virD4 and virE1 genes were obviously induced by acetosyringone in all the strains, and their transcriptional levels as well as virA's of AGL-1, C58C1 and EHA105 were higher than that of LBA4404, suggesting high transcriptional levels of vir genes were important for successful transformation. The study selected A. tumefaciens with high transformation efficiency of M. alpina, and would accelerate the genetic management of M. alpina. SIGNIFICANCE AND IMPACT OF THE STUDY: Oleaginous filamentous fungus Mortierella alpina is a commercial strain for the production of arachidonic acid. Genetic manipulation of M. alpina requires highly efficient transformation method. In this study, we explore the effect of Agrobacterium tumefaciens strain types on the transformation efficiency of M. alpina and select A. tumefaciens with the highest transformation efficiency, which accelerates the genetic manipulation of M. alpina. Besides, high transcriptional levels of virulence genes in A. tumefaciens were proven to play an important role for successful transformation.


Assuntos
Agrobacterium tumefaciens/genética , Mortierella/genética , Transformação Genética , Eletroporação , Microbiologia Industrial , Microrganismos Geneticamente Modificados , Plasmídeos , Virulência , Fatores de Virulência/genética
19.
Nat Commun ; 11(1): 680, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015344

RESUMO

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most destructive diseases that pose a great threat to wheat production. Wheat landraces represent a rich source of powdery mildew resistance. Here, we report the map-based cloning of powdery mildew resistance gene Pm24 from Chinese wheat landrace Hulutou. It encodes a tandem kinase protein (TKP) with putative kinase-pseudokinase domains, designated WHEAT TANDEM KINASE 3 (WTK3). The resistance function of Pm24 was validated by transgenic assay, independent mutants, and allelic association analyses. Haplotype analysis revealed that a rare 6-bp natural deletion of lysine-glycine codons, endemic to wheat landraces of Shaanxi Province, China, in the kinase I domain (Kin I) of WTK3 is critical for the resistance function. Transgenic assay of WTK3 chimeric variants revealed that only the specific two amino acid deletion, rather than any of the single or more amino acid deletions, in the Kin I of WTK3 is responsible for gaining the resistance function of WTK3 against the Bgt fungus.


Assuntos
Resistência à Doença/genética , Mutação com Ganho de Função , Genes de Plantas/genética , Doenças das Plantas/microbiologia , Triticum/genética , Ascomicetos/patogenicidade , China , Peróxido de Hidrogênio/metabolismo , Mutagênese , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Domínios Proteicos , Proteínas Quinases/genética , Transformação Genética
20.
J Vis Exp ; (155)2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32065159

RESUMO

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in elucidating the mechanisms that C. trachomatis use to create and maintain a privileged intracellular niche has been limited due to a lack of genetic tools. Fortunately, there have recently been several new advances in genetic manipulation techniques. Among these is the development of fluorescence-reported allelic exchange mutagenesis (FRAEM). This method allows targeted gene deletion coupled with insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Reliance on this strategy can be complicated when targeting genes within polycistronic operons due to the potential of polar effects on downstream genes. Floxed cassette allelic exchange mutagenesis (FLAEM), the protocol for which is described here, was developed to alleviate cassette-induced polar effects. FLAEM utilizes Cre-loxP genome editing to remove the selection cassette after targeted deletion by allelic exchange. The resulting strains contain markerless gene deletions of one or more coding sequences. This technique facilitates direct assessment of gene function and expands the repertoire of tools for genetic manipulation in C. trachomatis.


Assuntos
Alelos , Chlamydia trachomatis/genética , Deleção de Genes , Mutagênese Insercional/genética , Mutagênese/genética , Sequência de Bases , DNA Bacteriano/genética , Genoma Bacteriano , Proteínas de Fluorescência Verde/genética , Integrases/metabolismo , Transformação Genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA