RESUMO
Plants have evolved the ability to distinguish between symbiotic and pathogenic microbial signals. However, potentially cooperative plant-microbe interactions often abort due to incompatible signaling. The Nodulation Specificity 1 (NS1) locus in the legume Medicago truncatula blocks tissue invasion and root nodule induction by many strains of the nitrogen-fixing symbiont Sinorhizobium meliloti. Controlling this strain-specific nodulation blockade are two genes at the NS1 locus, designated NS1a and NS1b, which encode malectin-like leucine-rich repeat receptor kinases. Expression of NS1a and NS1b is induced upon inoculation by both compatible and incompatible Sinorhizobium strains and is dependent on host perception of bacterial nodulation (Nod) factors. Both presence/absence and sequence polymorphisms of the paired receptors contribute to the evolution and functional diversification of the NS1 locus. A bacterial gene, designated rns1, is required for activation of NS1-mediated nodulation restriction. rns1 encodes a type I-secreted protein and is present in approximately 50% of the nearly 250 sequenced S. meliloti strains but not found in over 60 sequenced strains from the closely related species Sinorhizobium medicae. S. meliloti strains lacking functional rns1 are able to evade NS1-mediated nodulation blockade.
Assuntos
Medicago truncatula , Sinorhizobium meliloti , Sinorhizobium meliloti/genética , Medicago truncatula/genética , Medicago truncatula/microbiologia , Simbiose/genética , Genes Bacterianos , Especificidade da Espécie , Fixação de NitrogênioRESUMO
KEY MESSAGE: Isoflavones are not involved in rhizobial signaling in red clover, but likely play a role in defense in the rhizosphere. Red clover (Trifolium pratense) is a high-quality forage legume, well suited for grazing and hay production in the temperate regions of the world. Like many legumes, red clover produces a number of phenylpropanoid compounds including anthocyanidins, flavan-3-ols, flavanols, flavanones, flavones, and isoflavones. The study of isoflavone biosynthesis and accumulation in legumes has come into the forefront of biomedical and agricultural research due to potential for medicinal, antimicrobial, and environmental implications. CRISPR/Cas9 was used to knock out the function of a key enzyme in the biosynthesis of isoflavones, isoflavone synthase (IFS1). A hemizygous plant carrying a 9-bp deletion in the IFS1 gene was recovered and was intercrossed to obtain homozygous mutant plants. Levels of the isoflavones formononetin, biochanin A and genistein were significantly reduced in the mutant plants. Wild-type and mutant plants were inoculated with rhizobia to test the effect of the mutation on nodulation, but no significant differences were observed, suggesting that these isoflavones do not play important roles in nodulation. Gene expression profiling revealed an increase in expression of the upstream genes producing the precursors for IFS1, namely, phenylalanine ammonium lyase and chalcone synthase, but there were no significant differences in IFS1 gene expression or in the downstream genes in the production of specific isoflavones. Higher expression in genes involved in ethylene response was observed in the mutant plants. This response is normally associated with biotic stress, suggesting that the plants may have been responding to cues in the surrounding rhizosphere due to lower levels of isoflavones.
Assuntos
Isoflavonas/metabolismo , Oxigenases/genética , Proteínas de Plantas/genética , Trifolium/genética , Trifolium/metabolismo , Sistemas CRISPR-Cas , Deleção de Genes , Regulação da Expressão Gênica de Plantas , Genisteína/metabolismo , Isoflavonas/genética , Oxigenases/metabolismo , Proteínas de Plantas/metabolismo , Nodulação/genética , Plantas Geneticamente Modificadas , Rhizobium/fisiologia , RizosferaRESUMO
The legume-rhizobial symbiosis results in the formation of root nodules that provide an ecological niche for nitrogen-fixing bacteria. However, plant-bacteria genotypic interactions can lead to wide variation in nitrogen fixation efficiency, and it is not uncommon that a bacterial strain forms functional (Fix+) nodules on one plant genotype but nonfunctional (Fix-) nodules on another. Host genetic control of this specificity is unknown. We herein report the cloning of the Medicago truncatula NFS1 gene that regulates the fixation-level incompatibility with the microsymbiont Sinorhizobium meliloti Rm41. We show that NFS1 encodes a nodule-specific cysteine-rich (NCR) peptide. In contrast to the known role of NCR peptides as effectors of endosymbionts' differentiation to nitrogen-fixing bacteroids, we demonstrate that specific NCRs control discrimination against incompatible microsymbionts. NFS1 provokes bacterial cell death and early nodule senescence in an allele-specific and rhizobial strain-specific manner, and its function is dependent on host genetic background.
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Medicago truncatula , Fixação de Nitrogênio/fisiologia , Proteínas de Plantas , Rizoma , Nódulos Radiculares de Plantas , Sinorhizobium meliloti/metabolismo , Simbiose/fisiologia , Transaminases , Medicago truncatula/genética , Medicago truncatula/metabolismo , Medicago truncatula/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Rizoma/genética , Rizoma/metabolismo , Rizoma/microbiologia , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , Nódulos Radiculares de Plantas/microbiologia , Transaminases/genética , Transaminases/metabolismoRESUMO
Legumes engage in root nodule symbioses with nitrogen-fixing soil bacteria known as rhizobia. In nodule cells, bacteria are enclosed in membrane-bound vesicles called symbiosomes and differentiate into bacteroids that are capable of converting atmospheric nitrogen into ammonia. Bacteroid differentiation and prolonged intracellular survival are essential for development of functional nodules. However, in the Medicago truncatula-Sinorhizobium meliloti symbiosis, incompatibility between symbiotic partners frequently occurs, leading to the formation of infected nodules defective in nitrogen fixation (Fix-). Here, we report the identification and cloning of the M. truncatula NFS2 gene that regulates this type of specificity pertaining to S. meliloti strain Rm41. We demonstrate that NFS2 encodes a nodule-specific cysteine-rich (NCR) peptide that acts to promote bacterial lysis after differentiation. The negative role of NFS2 in symbiosis is contingent on host genetic background and can be counteracted by other genes encoded by the host. This work extends the paradigm of NCR function to include the negative regulation of symbiotic persistence in host-strain interactions. Our data suggest that NCR peptides are host determinants of symbiotic specificity in M. truncatula and possibly in closely related legumes that form indeterminate nodules in which bacterial symbionts undergo terminal differentiation.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Bactérias/metabolismo , Medicago truncatula , Fixação de Nitrogênio/fisiologia , Proteínas de Plantas/metabolismo , Microbiologia do Solo , Simbiose/fisiologia , Medicago truncatula/metabolismo , Medicago truncatula/microbiologiaRESUMO
Medicago truncatula shows a high level of specificity when interacting with its symbiotic partner Sinorhizobium meliloti. This specificity is mainly manifested at the nitrogen-fixing stage of nodule development, such that a particular bacterial strain forms nitrogen-fixing nodules (Nod+/Fix+) on one plant genotype but ineffective nodules (Nod+/Fix-) on another. Recent studies have just begun to reveal the underlying molecular mechanisms that control this specificity. The S. meliloti strain A145 induces the formation of Fix+ nodules on the accession DZA315.16 but Fix- nodules on Jemalong A17. A previous study reported that the formation of Fix- nodules on Jemalong A17 by S. meliloti A145 was conditioned by a single recessive allele named Mtsym6. Here we demonstrate that the specificity associated with S. meliloti A145 is controlled by multiple genes in M. truncatula, including NFS1 and NFS2 that encode nodule-specific cysteine-rich (NCR) peptides. The two NCR peptides acted dominantly to block rather than promote nitrogen fixation by S. meliloti A145. These two NCR peptides are the same ones that negatively regulate nitrogen-fixing symbiosis associated with S. meliloti Rm41.
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Medicago truncatula/fisiologia , Fixação de Nitrogênio/fisiologia , Peptídeos/metabolismo , Proteínas de Plantas/metabolismo , Nódulos Radiculares de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Peptídeos/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Nódulos Radiculares de Plantas/químicaRESUMO
Rj4 is a dominant gene in soybeans (Glycine max) that restricts nodulation by many strains of Bradyrhizobium elkanii. The soybean-B. elkanii symbiosis has a low nitrogen-fixation efficiency, but B. elkanii strains are highly competitive for nodulation; thus, cultivars harboring an Rj4 allele are considered favorable. Cloning the Rj4 gene is the first step in understanding the molecular basis of Rj4-mediated nodulation restriction and facilitates the development of molecular tools for genetic improvement of nitrogen fixation in soybeans. We finely mapped the Rj4 locus within a small genomic region on soybean chromosome 1, and validated one of the candidate genes as Rj4 using both complementation tests and CRISPR/Cas9-based gene knockout experiments. We demonstrated that Rj4 encodes a thaumatin-like protein, for which a corresponding allele is not present in the surveyed rj4 genotypes, including the reference genome Williams 82. Our conclusion disagrees with the previous report that Rj4 is the Glyma.01G165800 gene (previously annotated as Glyma01g37060). Instead, we provide convincing evidence that Rj4 is Glyma.01g165800-D, a duplicated and unique version of Glyma.01g165800, that has evolved the ability to control symbiotic specificity.
Assuntos
Glycine max/genética , Glycine max/microbiologia , Proteínas de Plantas/genética , Nodulação/genética , Sequência de Aminoácidos , Bradyrhizobium/fisiologia , Sistemas CRISPR-Cas , Mapeamento Cromossômico , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , Nodulação/fisiologia , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas , Polimorfismo GenéticoRESUMO
The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some-including the infamous ergot alkaloids-have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.
Assuntos
Alcaloides , Claviceps , Epichloe , Alcaloides de Claviceps , Seleção Genética , Alcaloides/química , Alcaloides/classificação , Alcaloides/genética , Alcaloides/metabolismo , Claviceps/genética , Claviceps/metabolismo , Claviceps/patogenicidade , Epichloe/genética , Epichloe/metabolismo , Epichloe/patogenicidade , Alcaloides de Claviceps/genética , Alcaloides de Claviceps/metabolismo , Regulação Fúngica da Expressão Gênica , Hypocreales/genética , Hypocreales/metabolismo , Neotyphodium , Poaceae/genética , Poaceae/metabolismo , Poaceae/parasitologia , Simbiose/genéticaRESUMO
BACKGROUND: Leguminous plants are able to form a root nodule symbiosis with nitrogen-fixing soil bacteria called rhizobia. This symbiotic association shows a high level of specificity. Beyond the specificity for the legume family, individual legume species/genotypes can only interact with certain restricted group of bacterial species or strains. Specificity in this system is regulated by complex signal exchange between the two symbiotic partners and thus multiple genetic mechanisms could be involved in the recognition process. Knowledge of the molecular mechanisms controlling symbiotic specificity could enable genetic improvement of legume nitrogen fixation, and may also reveal the possible mechanisms that restrict root nodule symbiosis in non-legumes. RESULTS: We screened a core collection of Medicago truncatula genotypes with several strains of Sinorhizobium meliloti and identified a naturally occurring dominant gene that restricts nodulation by S. meliloti Rm41. We named this gene as Mt-NS1 (for M.truncatulanodulation specificity 1). We have mapped the Mt-NS1 locus within a small genomic region on M. truncatula chromosome 8. The data reported here will facilitate positional cloning of the Mt-NS1 gene. CONCLUSIONS: Evolution of symbiosis specificity involves both rhizobial and host genes. From the bacterial side, specificity determinants include Nod factors, surface polysaccharides, and secreted proteins. However, we know relatively less from the host side. We recently demonstrated that a component of this specificity in soybeans is defined by plant NBS-LRR resistance (R) genes that recognize effector proteins delivered by the type III secretion system (T3SS) of the rhizobial symbionts. However, the lack of a T3SS in many sequenced S. meliloti strains raises the question of how the specificity is regulated in the Medicago-Sinorhizobium system beyond Nod-factor perception. Thus, cloning and characterization of Mt-NS1 will add a new dimension to our knowledge about the genetic control of nodulation specificity in the legume-rhizobial symbiosis.
Assuntos
Genes Dominantes , Genes de Plantas , Medicago truncatula/genética , Medicago truncatula/microbiologia , Nodulação/genética , Sinorhizobium meliloti/fisiologia , Mapeamento Cromossômico , Loci Gênicos , Variação Genética , Mutação INDEL/genética , Medicago truncatula/crescimento & desenvolvimento , Microscopia de Fluorescência , Mutação/genética , Fenótipo , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Polissacarídeos/metabolismo , Simbiose/genéticaRESUMO
Host range specificity is a prominent feature of the legume-rhizobial symbiosis. Sinorhizobium meliloti and Sinorhizobium medicae are two closely related species that engage in root nodule symbiosis with legume plants of the Medicago genus, but certain Medicago species exhibit selectivity in their interactions with the two rhizobial species. We have identified a Medicago receptor-like kinase, which can discriminate between the two bacterial species, acting as a genetic barrier against infection by most S. medicae strains. Activation of this receptor-mediated nodulation restriction requires a bacterial gene that encodes a glycine-rich octapeptide repeat protein with distinct variants capable of distinguishing S. medicae from S. meliloti. This study sheds light on the coevolution of host plants and rhizobia, shaping symbiotic selectivity in their respective ecological niches.
Assuntos
Simbiose , Especificidade da Espécie , Medicago/microbiologia , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Nódulos Radiculares de Plantas/microbiologia , Nódulos Radiculares de Plantas/metabolismo , Proteínas Quinases/metabolismo , Proteínas Quinases/genéticaRESUMO
Low-salt stress germination is an effective way to improve the nutritional composition of food crops. A novel soluble dietary fiber (MS-SDF) was isolated from low-salt stress mung bean sprouts that were exposed to low-salt stress using anion exchange and gel permeation techniques. Structural analysis revealed that MS-SDF was a homogeneous heteropolysaccharide with an average molecular weight of 164.997 KDa. It featured a loose structure and contained the characteristic functional groups typical of polysaccharides. MS-SDF was composed of arabinose, galactose, glucose, and mannose with a molar ratio of 3.95:3.86:82.69:9.02. The structure was mainly composed of â6)-α-D-Glcp-(1â, â5)-α-L-Araf-(1â, and â3,6)-α-D-Glcp-(1â as the main chain. Branched at O-3 position with single ß-D-Manp-(1â as major the side chain. Furthermore, in vitro hypoglycemic assays indicate that MS-SDF exhibits α-glucosidase inhibitory activity, significantly enhancing glucose uptake, glycogen synthesis, and pyruvate kinase activity in insulin-resistant HepG2 cells. Overall, MS-SDF could be used as a promising source of functional hypoglycemic foods.
RESUMO
As a biodegradable material, magnesium alloy has a modulus similar to that of bone, and given the biological activity of its degradation products, it has the potential to be a bone grafting material. Oxidation heat treatment is a very effective passivation method that may reduce the rate of magnesium alloy degradation. Oxidation heat treatment increases the rare earth oxide content of the scaffold as well as the corrosion resistance of the scaffold. The overall cytotoxicity of the as-printed scaffolds (APSs) and oxidation heat-treated scaffolds (OHSs) showed that OHSs accelerated cell proliferation. In the apoptosis experiment, the OHS group had a cell survival rate between that of the control group and of the as-printed group. In the osteogenic induction experiment, the alkaline phosphatase activity and the quantity of mineralized nodules were greater in the APS and OHS groups than in the control group. Marker proteins for bone growth were expressed at higher levels in the APS and OHS groups than in the control group. Therefore, oxidation heat-treated 3D printing scaffolds with good biocompatibility and osteogenic properties have great potential to be made into advanced biomaterials that can be used to fix bone defects.
RESUMO
The effects of pore size in additively manufactured biodegradable porous magnesium on the mechanical properties and biodegradation of the scaffolds as well as new bone formation have rarely been reported. In this work, we found that high temperature oxidation improves the corrosion resistance of magnesium scaffold. And the effects of pore size on the mechanical characteristics and biodegradation of scaffolds, as well as new bone formation, were investigated using magnesium scaffolds with three different pore sizes, namely, 500, 800, and 1400 µm (P500, P800, and P1400). We discovered that the mechanical characteristics of the P500 group were much better than those of the other two groups. In vitro and in vivo investigations showed that WE43 magnesium alloy scaffolds supported the survival of mesenchymal stem cells and did not cause any local toxicity. Due to their larger specific surface area, the scaffolds in the P500 group released more magnesium ions within reasonable range and improved the osteogenic differentiation of bone mesenchymal stem cells compared with the other two scaffolds. In a rabbit femoral condyle defect model, the P500 group demonstrated unique performance in promoting new bone formation, indicating its great potential for use in bone defect regeneration therapy.
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Laser powder bed fusion (L-PBF) of magnesium (Mg) alloy porous scaffolds is expected to solve the dual challenges from customized structures and biodegradable functions required for repairing bone defects. However, one of the key technical difficulties lies in the poor L-PBF process performance of Mg, contributed by the high susceptibility to oxidation, vaporization, thermal expansion, and powder attachment etc. This work investigated the influence of L-PBF energy input and scanning strategy on the formation quality of porous scaffolds by using WE43 powder, and characterized the microstructure, mechanical properties, biocompatibility, biodegradation and osteogenic effect of the as-built WE43 porous scaffolds. With the customized energy input and scanning strategy, the relative density of struts reached over 99.5%, and the geometrical error between the designed and the fabricated porosity declined to below 10%. Massive secondary phases including intermetallic precipitates and oxides were observed. The compressive strength (4.37-23.49 MPa) and elastic modulus (154.40-873.02 MPa) were comparable to those of cancellous bone. Good biocompatibility was observed by in vitro cell viability and in vivo implantation. The biodegradation of as-built porous scaffolds promoted the osteogenic effect, but the structural integrity devastated after 12 h by the immersion tests in Hank's solution and after 4 weeks by the implantation in rabbits' femur, indicating an excessively rapid degradation rate.
RESUMO
Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that certain of the Transwell cell migration assay data shown in Fig. 5A were strikingly similar to data appearing in different form in other articles by different authors. Owing to the fact that the contentious data in the above article had already been published elsewhere, or were already under consideration for publication, prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive any reply. The Editor apologizes to the readership for any inconvenience caused. [the original article was published in Molecular Medicine Reports 14: 2651-2656, 2016; DOI: 10.3892/mmr.2016.5534].
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The influence of tin foil and Ni coatings on microstructures, mechanical properties, and the interfacial reaction mechanism was investigated during laser welding/brazing of Al/Cu lap joints. In the presence of a Zn-based filler, tin foil as well as Ni coating strengthened the Al/Cu joints. The tin foil only slightly influenced the joint strength. It considerably improved the spreading/wetting ability of the weld filler; however, it weakened the bonding between the seam and the Al base metal. The Ni coating considerably strengthened the Al/Cu lap joints; the highest tensile strength was 171 MPa, which was higher by 15.5% than that of a joint without any interlayer. Microstructure analysis revealed that composite layers of Ni3Zn14-(τ2 Zn-Ni-Al ternary phase)-(α-Zn solid solution)-Al3Ni formed at the fusion zone (FZ)/Cu interface. Based on the inferences about the microstructures at the interfaces, thermodynamic results were calculated to analyze the interfacial reaction mechanism. The diffusion of Cu was limited by the Ni coating and the mutual attraction between the Al and Ni atoms. The microstructure comprised Zn, Ni, and Al, and they replaced the brittle Cu-Zn intermetallic compounds, successfully strengthening the bonding of the FZ/Cu interface.
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Laser powder bed fusion (LPBF) of Zn-based metals exhibits prominent advantages to produce customized biodegradable implants. However, massive evaporation occurs during laser melting of Zn so that it becomes a critical issue to modulate laser energy input and gas shielding conditions to eliminate the negative effect of evaporation fume during the LPBF process. In this research, two numerical models were established to simulate the interaction between the scanning laser and Zn metal as well as the interaction between the shielding gas flow and the evaporation fume, respectively. The first model predicted the evaporation rate under different laser energy input by taking the effect of evaporation on the conservation of energy, momentum, and mass into consideration. With the evaporation rate as the input, the second model predicted the elimination effect of evaporation fume under different conditions of shielding gas flow by taking the effect of the gas circulation system including geometrical design and flow rate. In the case involving an adequate laser energy input and an optimized shielding gas flow, the evaporation fume was efficiently removed from the processing chamber during the LPBF process. Furthermore, the influence of evaporation on surface quality densification was discussed by comparing LPBF of pure Zn and a Titanium alloy. The established numerical analysis not only helps to find the adequate laser energy input and the optimized shielding gas flow for the LPBF of Zn based metal, but is also beneficial to understand the influence of evaporation on the LPBF process.
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Legumes are the second most important family of crop plants. One defining feature of legumes is their unique ability to establish a nitrogen-fixing root nodule symbiosis with soil bacteria known as rhizobia. Since domestication from their wild relatives, crop legumes have been under intensive breeding to improve yield and other agronomic traits but with little attention paid to the belowground symbiosis traits. Theoretical models predict that domestication and breeding processes, coupled with high-input agricultural practices, might have reduced the capacity of crop legumes to achieve their full potential of nitrogen fixation symbiosis. Testing this prediction requires characterizing symbiosis traits in wild and breeding populations under both natural and cultivated environments using genetic, genomic, and ecological approaches. However, very few experimental studies have been dedicated to this area of research. Here, we review how legumes regulate their interactions with soil rhizobia and how domestication, breeding and agricultural practices might have affected nodulation capacity, nitrogen fixation efficiency, and the composition and function of rhizobial community. We also provide a perspective on how to improve legume-rhizobial symbiosis in sustainable agricultural systems.
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Chemical gene synthesis is a powerful tool for basic biological research and biotechnology applications. During the last 30 years, major advances have been made in the chemical synthesis of DNA sequences ranging from fragments of <1 kb to multi-gene sequences of >30 kb. There is a need for simple, reproducible, less error-prone and cost-effective methods that guarantee successful synthesis of the desired genes and are amenable to automation. Many polymerase chain reaction (PCR)-based and non-polymerase-cycling-assembly (PCA)-based strategies have been developed for chemical gene synthesis. The PCR-based method has been the subject of several recent reviews. Here, we provide an overview of the progress in non-PCA-based chemical gene synthesis using different strategies and methods, including enzymatic gene synthesis, annealing and ligation reaction, simultaneous synthesis of two genes via a hybrid gene, shotgun ligation and co-ligation, insertion gene synthesis, gene synthesis via one strand of DNA, template-directed ligation, ligase chain reaction, microarray-mediated gene synthesis, Blue Heron solid support technology and Sloning building block technology. The fundamental principle underlying each strategy, an example where applicable, and the advantages and disadvantages are discussed. The emphasis is on discussion of the most recent technologies and their potential applications, particularly for microarray-based genomics research.
Assuntos
Biotecnologia/métodos , Biotecnologia/tendências , DNA/síntese química , Previsões , Genes , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/tendências , Reação em Cadeia da PolimeraseRESUMO
Legumes are able to form a symbiotic relationship with nitrogen-fixing soil bacteria called rhizobia. The result of this symbiosis is to form nodules on the plant root, within which the bacteria can convert atmospheric nitrogen into ammonia that can be used by the plant. Establishment of a successful symbiosis requires the two symbiotic partners to be compatible with each other throughout the process of symbiotic development. However, incompatibility frequently occurs, such that a bacterial strain is unable to nodulate a particular host plant or forms nodules that are incapable of fixing nitrogen. Genetic and molecular mechanisms that regulate symbiotic specificity are diverse, involving a wide range of host and bacterial genes/signals with various modes of action. In this review, we will provide an update on our current knowledge of how the recognition specificity has evolved in the context of symbiosis signaling and plant immunity.
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To achieve a thermostable beta-glucuronidase (GUS) and identify key mutation sites, we applied in vitro directed evolution strategy through DNA shuffling and obtained a highly thermostable mutant GUS gene, gus-tr, after four rounds of DNA shuffling and screening. This variant had mutations in 15 nucleic acid sites, resulting in changes in 12 amino acids (AAs). Using gus-tr as the template, we further performed site-directed mutagenesis to reverse the individual mutation to the wild-type protein. We found that six sites (Q493R, T509A, M532T, N550S, G559S and N566S) present in GUS-TR3337, were the key AAs needed to confer its high thermostability. Of these, Q493R and T509A were not reported previously as important residues for thermostability of GUS. Furthermore, all of these six mutations must be present concurrently to confer the high thermostability. We expressed the gus-tr3337 gene and purified the GUS-TR3337 protein that contained the six AA mutations. Compared with the wild-type protein which lost its activity completely after 10 min at 70 degrees C, the mutant GUS-TR3337 protein retained 75% of its activity when heated at 80 degrees C for 10 min. The GUS-TR3337 exhibited high activity even heated at 100 degrees C for 30 min on nitrocellulose filter. The comparison of molecular models of the mutated and wild-type enzyme revealed the relation of protein function and these structural modifications.