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A transgenic perspective on plant functional genomics 总被引:17,自引:0,他引:17
Pereira A 《Transgenic research》2000,9(4-5):245-260
Transgenic crops are very much in the news due to the increasing public debate on their acceptance. In the scientific community
though, transgenic plants are proving to be powerful tools to study various aspects of plant sciences. The emerging scientific
revolution sparked by genomics based technologies is producing enormous amounts of DNA sequence information that, together
with plant transformation methodology, is opening up new experimental opportunities for functional genomics analysis. An overview
is provided here on the use of transgenic technology for the functional analysis of plant genes in model plants and a link
made to their utilization in transgenic crops. In transgenic plants, insertional mutagenesis using heterologous maize transposons
or Agrobacterium mediated T-DNA insertions, have been valuable tools for the identification and isolation of genes that display a mutant phenotype.
To discover functions of genes that do not display phenotypes when mutated, insertion sequences have been engineered to monitor
or change the expression pattern of adjacent genes. These gene detector insertions can detect adjacent promoters, enhancers
or gene exons and precisely reflect the expression pattern of the tagged gene. Activation tag insertions can mis-express the
adjacent gene and confer dominant phenotypes that help bridge the phenotype gap. Employment of various forms of gene silencing
technology broadens the scope of recovering knockout phenotypes for genes with redundant function. All these transgenic strategies
describing gene-phenotype relationships can be addressed by high throughput reverse genetics methods that will help provide
functions to the genes discovered by genome sequencing. The gene functions discovered by insertional mutagenesis and silencing
strategies along with expression pattern analysis will provide an integrated functional genomics perspective and offer unique
applications in transgenic crops.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
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Jeanice Hwang Archana Hari Raymond Cheng Jeffrey G. Gardner Daniel Lobo 《Biotechnology and bioengineering》2020,117(12):3876-3890
Understanding the complex growth and metabolic dynamics in microorganisms requires advanced kinetic models containing both metabolic reactions and enzymatic regulation to predict phenotypic behaviors under different conditions and perturbations. Most current kinetic models lack gene expression dynamics and are separately calibrated to distinct media, which consequently makes them unable to account for genetic perturbations or multiple substrates. This challenge limits our ability to gain a comprehensive understanding of microbial processes towards advanced metabolic optimizations that are desired for many biotechnology applications. Here, we present an integrated computational and experimental approach for the development and optimization of mechanistic kinetic models for microbial growth and metabolic and enzymatic dynamics. Our approach integrates growth dynamics, gene expression, protein secretion, and gene-deletion phenotypes. We applied this methodology to build a dynamic model of the growth kinetics in batch culture of the bacterium Cellvibrio japonicus grown using either cellobiose or glucose media. The model parameters were inferred from an experimental data set using an evolutionary computation method. The resulting model was able to explain the growth dynamics of C. japonicus using either cellobiose or glucose media and was also able to accurately predict the metabolite concentrations in the wild-type strain as well as in β-glucosidase gene deletion mutant strains. We validated the model by correctly predicting the non-diauxic growth and metabolite consumptions of the wild-type strain in a mixed medium containing both cellobiose and glucose, made further predictions of mutant strains growth phenotypes when using cellobiose and glucose media, and demonstrated the utility of the model for designing industrially-useful strains. Importantly, the model is able to explain the role of the different β-glucosidases and their behavior under genetic perturbations. This integrated approach can be extended to other metabolic pathways to produce mechanistic models for the comprehensive understanding of enzymatic functions in multiple substrates. 相似文献
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Identification of genes that affect the product accumulation phenotype of recombinant strains is an important problem in industrial strain construction and a central tenet of metabolic engineering. We have used systematic (model-based) and combinatorial (transposon-based) methods to identify gene knockout targets that increase lycopene biosynthesis in strains of Escherichia coli. We show that these two search strategies yield two distinct gene sets, which affect product synthesis either through an increase in precursor availability or through (largely unknown) kinetic or regulatory mechanisms, respectively. Exhaustive exploration of all possible combinations of the above gene sets yielded a unique set of 64 knockout strains spanning the metabolic landscape of systematic and combinatorial gene knockout targets. This included a global maximum strain exhibiting an 8.5-fold product increase over recombinant K12 wild type and a twofold increase over the engineered parental strain. These results were further validated in controlled culture conditions. 相似文献
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Systematic and combinatorial genetic approaches for the identification of gene knockout and overexpression targets have been
effectively employed in the improvement of cellular phenotypes. Previously, we demonstrated how two of these tools, metabolic
modeling and transposon mutagenesis, can be combined to identify strains of interest spanning the metabolic landscape of recombinant
lycopene production in Escherichia coli. However, it is unknown how to best select multiple-gene knockout targets. Hence, this study seeks to understand how the
overall order of gene selection, or search trajectory, biases the exploration and topology of the metabolic landscape. In
particular, transposon mutagenesis and selection were employed in the background of eight different knockout genotypes. Collectively,
800,000 mutants were analyzed in hopes of exhaustively identifying all advantageous gene knockout targets. Several interesting
observations, including clusters of gene functions, recurrence, and divergent genotypes, demonstrate the complexity of mapping
only one genotype to one phenotype. One particularly interesting mutant, the ΔhnrΔyliE genotype, exhibited a drastically improved lycopene production capacity in basic minimal medium in comparison to the best
strains identified in previous studies. 相似文献
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Dominant negative mutator mutations in the mutS gene of Escherichia coli. 总被引:10,自引:1,他引:9
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The MutS protein of Escherichia coli is part of the dam-directed MutHLS mismatch repair pathway which rectifies replication errors and which prevents recombination between related sequences. In order to more fully understand the role of MutS in these processes, dominant negative mutS mutations on a multicopy plasmid were isolated by screening transformed wild-type cells for a mutator phenotype, using a Lac+ papillation assay. Thirty-eight hydroxylamine- and 22 N-methyl-N'-nitro-N-nitrosoguanidine-induced dominant mutations were isolated. Nine of these mutations altered the P-loop motif of the ATP-binding site, resulting in four amino acid substitutions. With one exception, the remaining sequenced mutations all caused substitution of amino acids conserved during evolution. The dominant mutations in the P-loop consensus caused severely reduced repair of heteroduplex DNA in vivo in a mutS mutant host strain. In a wild-type strain, the level of repair was decreased by the dominant mutations to between 12 to 90% of the control value, which is consistent with interference of wild-type MutS function by the mutant proteins. Increasing the wild-type mutS gene dosage resulted in a reversal of the mutator phenotype in about 60% of the mutant strains, indicating that the mutant and wild-type proteins compete. In addition, 20 mutant isolates showed phenotypic reversal by increasing the gene copies of either mutL or mutH. There was a direct correlation between the levels of recombination and mutagenesis in the mutant strains, suggesting that these phenotypes are due to the same function of MutS. 相似文献
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The whole MATA cassette from Yarrowia lipolytica, a dimorphic fungus, was replaced by the URA3 gene through a double homologous recombination. This MAT-less strain lost its mate capacity with A or B Y. lipolytica strains. Introduction of polymerase chain reaction-synthesized idiomorph MATB in a null strain of A locus by double homologous recombination gave rise to a "transsexual" B strain. Mating capacity of this engineered mutant was assayed using Y. lipolytica strains of either A or B mating type. Mating took place only with an A strain, demonstrating the MATB idiomorph functionality in a MATA phenotype. Our data suggest that specific downstream genes are responsible for the final A or B phenotypes present in all Y. lipolytica cells, independent of their MAT idiomorph phenotype. 相似文献
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Genetic variables that influence phenotype 总被引:3,自引:0,他引:3
Linder CC 《ILAR journal / National Research Council, Institute of Laboratory Animal Resources》2006,47(2):132-140
Characterization of genetically engineered mice requires consideration of the gene of interest and the genetic background on which the mutation is maintained. A fundamental prerequisite to deciphering the genetic factors that influence the phenotype of a mutant mouse is an understanding of genetic nomenclature. Mutations and transgenes are often maintained on segregating or mixed backgrounds of often-unspecified origin. Minimizing the importance of strain and substrain differences, especially among 129 strains, can lead to poor experimental design or faulty interpretations of data. Genetic factors that influence phenotype can be categorized as traits that are unique to the background strain, unique to the gene of interest, or an interaction of both the background strain and the gene of interest. The commonly used inbred strains are generally well characterized and understood; however, specific genetic alterations combined with genes unique to the background inbred strain may lead to unexpected results. Genetic background effects can be analyzed and controlled for by using specific targeting and breeding strategies. Selection of appropriate experimental controls is critical. Ideally, mutations or transgenes should be characterized on more than one genetic background and in hybrids of the two progenitor strains. This approach may lead to the identification of novel genetic modifiers of the "gene of interest." Conditional mutagenesis technologies increase the options for controlling genetic background effects in addition to permitting the study of developmental and temporal changes in gene and protein expression and thus phenotype. 相似文献
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Min基因突变小鼠模型在肠道肿瘤研究中的应用 总被引:3,自引:0,他引:3
迄今为止, 肠道肿瘤相关的基因突变小鼠或敲除小鼠大约有30多种, Min(multiple intestinal neoplasia)小鼠是具有肠道多发性腺瘤特征的Apc基因突变小鼠, 被认为是当前较为理想的家族性腺瘤性息肉病(FAP)的研究模型。APC基因是Wnt途径中重要的抑癌基因, 该途径不仅是动物胚胎发育过程中关键的信号转导途径, 也对结直肠肿瘤的发生发展起到不同寻常的作用。对Min小鼠模型历史、分子遗传学与表型特征、肠道肿瘤与Wnt途径异常、抑癌基因甲基化、TGF-b途径和多药耐药基因等内容进行了介绍, 并分析了该小鼠模型在抗结直肠肿瘤药物研究中的应用和意义。 相似文献
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Joseph H. Nadeau Jiri Forejt Toyoyuki Takada Toshihiko Shiroishi 《Mammalian genome》2012,23(9-10):693-705
Laboratory mice are valuable in biomedical research in part because of the extraordinary diversity of genetic resources that are available for studies of complex genetic traits and as models for human biology and disease. Chromosome substitution strains (CSSs) are important in this resource portfolio because of their demonstrated use for gene discovery, genetic and epigenetic studies, functional characterizations, and systems analysis. CSSs are made by replacing a single chromosome in a host strain with the corresponding chromosome from a donor strain. A complete CSS panel involves a total of 22 engineered inbred strains, one for each of the 19 autosomes, one each for the X and Y chromosomes, and one for mitochondria. A genome survey simply involves comparing each phenotype for each of the CSSs with the phenotypes of the host strain. The CSS panels that are available for laboratory mice have been used to dissect a remarkable variety of phenotypes and to characterize an impressive array of disease models. These surveys have revealed considerable phenotypic diversity even among closely related progenitor strains, evidence for strong epistasis and for heritable epigenetic changes. Perhaps most importantly, and presumably because of their unique genetic constitution, CSSs, and congenic strains derived from them, the genetic variants underlying quantitative trait loci (QTLs) are readily identified and functionally characterized. Together these studies show that CSSs are important resource for laboratory mice. 相似文献
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T Doetschman 《Laboratory animal science》1999,49(2):137-143
BACKGROUND AND PURPOSE: In mice, genetic engineering involves two general approaches-addition of an exogenous gene, resulting in transgenic mice, and use of knockout mice, which have a targeted mutation of an endogenous gene. The advantages of these approaches is that questions can be asked about the function of a particular gene in a living mammalian organism, taking into account interactions among cells, tissues, and organs under normal, disease, injury, and stress situations. METHODS: Review of the literature concentrating principally on knockout mice and questions of unexpected phenotypes, lack of phenotype, redundancy, and effect of genetic background on phenotype will be discussed. CONCLUSION: There is little gene redundancy in mammals; knockout phenotypes exist even if none are immediately apparent; and investigating phenotypes in colonies of mixed genetic background may reveal not only more phenotypes, but also may lead to better understanding of the molecular or cellular mechanism underlying the phenotype and to discovery of modifier gene(s). 相似文献
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Hao-Man Li Eric V. Virtudazo Akio Toh-e Masashi Yamaguchi Susumu Kawamoto Kiminori Shimizu 《Mycoscience》2010,51(4):272-280
The efficiency of gene targeting by integration through homologous recombination (homologous integration, HI) in the human
pathogen Cryptococcus neoformans remains unsatisfactory. In order to achieve a much more efficient gene targeting system in C. neoformans, a new double knockout strain in genes involved in the non-homologous end joining (NHEJ) pathway was constructed. HI frequency
was elevated by as much as approximately fivefold in the single or double knockout strains in NHEJ genes, and the frequency
depended on the gene targeted. None of the NHEJ gene knockouts showed significant differences in regular growth, sensitivity
to DNA-damaging drugs or UV, and virulence compared to the wild-type control, suggesting that the NHEJ pathway does not play
a significant role in these biological stresses in C. neoformans. It was also suggested that the genes analyzed in this study are components of a single NHEJ pathway, as the mutants (including
the double mutant) displayed the same phenotypes. 相似文献
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Siniashina LN Siniashina LS Semin EG Amelina IP Karataev GI 《Molekuliarnaia genetika, mikrobiologiia i virusologiia》2010,(3):31-36
The recombinant modified (attenuated) bacteria A. pertussis were constructed. These bacteria contained knockout mutation of the dnt gene and produced nontoxic pertussis toxin derivative. The immunological properties of the mutant bacteria B. pertussis strain KS were studied. The recombinant bacteria B. pertussis strain KS were found to be devoid of dermonecrotic toxin activity, conserved the structure of the mutant dnt gene in condition of cultivation on selective growth media, and long-term survival in laboratory animal organism. Intranasal immunization of mice with living bacteria B. pertussis, attenuated strain KS provided protection of animals from virulent strains of the pertussis. The efficiency of the protection was comparable with protection efficiency provided by standard corpuscular pertussis vaccine OSO-3. 相似文献
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Adrienne Zaprasis Tamara Hoffmann Lorena Stannek Katrin Gunka Fabian M. Commichau Erhard Bremer 《Journal of bacteriology》2014,196(3):515-526
PutP and OpuE serve as proline transporters when this imino acid is used by Bacillus subtilis as a nutrient or as an osmostress protectant, respectively. The simultaneous inactivation of the PutP and OpuE systems still allows the utilization of proline as a nutrient. This growth phenotype pointed to the presence of a third proline transport system in B. subtilis. We took advantage of the sensitivity of a putP opuE double mutant to the toxic proline analog 3,4-dehydro-dl-proline (DHP) to identify this additional proline uptake system. DHP-resistant mutants were selected and found to be defective in the use of proline as a nutrient. Whole-genome resequencing of one of these strains provided the lead that the inactivation of the γ-aminobutyrate (GABA) transporter GabP was responsible for these phenotypes. DNA sequencing of the gabP gene in 14 additionally analyzed DHP-resistant strains confirmed this finding. Consistently, each of the DHP-resistant mutants was defective not only in the use of proline as a nutrient but also in the use of GABA as a nitrogen source. The same phenotype resulted from the targeted deletion of the gabP gene in a putP opuE mutant strain. Hence, the GabP carrier not only serves as an uptake system for GABA but also functions as the third proline transporter of B. subtilis. Uptake studies with radiolabeled GABA and proline confirmed this conclusion and provided information on the kinetic parameters of the GabP carrier for both of these substrates. 相似文献
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【目的】考察茎瘤固氮根瘤菌ORS571中c-di-GMP合成酶AZC-2412的编码基因缺失的突变表型,初步探究其功能机理。【方法】本实验构建基于cre-loxp重组酶系统的根瘤菌基因敲除系统,以及采用三亲接合技术构建突变株。测定野生型和突变株的生长速率、趋化能力、胞外多糖产量、生物膜形成等表型。【结果】突变株与野生型生长速率几乎相同。与野生型相比突变株由于细胞内c-di-GMP水平降低,胞外多糖、生物膜产量等均有所下降。【结论】实验表明,环二鸟苷酸合成酶AZC-2412缺失,使得c-di-GMP水平降低,对胞外多糖生成、细菌的运动能力、生物膜的形成、细胞絮凝、与植物的互作等均有调控作用。 相似文献
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The low prevalence rate of orphan diseases (OD) requires special combined efforts to improve diagnosis, prevention, and discovery of novel therapeutic strategies. To identify and investigate relationships based on shared genes or shared functional features, we have conducted a bioinformatic-based global analysis of all orphan diseases with known disease-causing mutant genes. Starting with a bipartite network of known OD and OD-causing mutant genes and using the human protein interactome, we first construct and topologically analyze three networks: the orphan disease network, the orphan disease-causing mutant gene network, and the orphan disease-causing mutant gene interactome. Our results demonstrate that in contrast to the common disease-causing mutant genes that are predominantly nonessential, a majority of orphan disease-causing mutant genes are essential. In confirmation of this finding, we found that OD-causing mutant genes are topologically important in the protein interactome and are ubiquitously expressed. Additionally, functional enrichment analysis of those genes in which mutations cause ODs shows that a majority result in premature death or are lethal in the orthologous mouse gene knockout models. To address the limitations of traditional gene-based disease networks, we also construct and analyze OD networks on the basis of shared enriched features (biological processes, cellular components, pathways, phenotypes, and literature citations). Analyzing these functionally-linked OD networks, we identified several additional OD-OD relations that are both phenotypically similar and phenotypically diverse. Surprisingly, we observed that the wiring of the gene-based and other feature-based OD networks are largely different; this suggests that the relationship between ODs cannot be fully captured by the gene-based network alone. 相似文献
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Davisson MT Bronson RT Tadenev AL Motley WW Krishnaswamy A Seburn KL Burgess RW 《PloS one》2011,6(12):e29538
Mutations in the gene encoding the immunoglobulin-superfamily member cell adhesion molecule contactin1 (CNTN1) cause lethal congenital myopathy in human patients and neurodevelopmental phenotypes in knockout mice. Whether the mutant mice provide an accurate model of the human disease is unclear; resolving this will require additional functional tests of the neuromuscular system and examination of Cntn1 mutations on different genetic backgrounds that may influence the phenotype. Toward these ends, we have analyzed a new, spontaneous mutation in the mouse Cntn1 gene that arose in a BALB/c genetic background. The overt phenotype is very similar to the knockout of Cntn1, with affected animals having reduced body weight, a failure to thrive, locomotor abnormalities, and a lifespan of 2-3 weeks. Mice homozygous for the new allele have CNTN1 protein undetectable by western blotting, suggesting that it is a null or very severe hypomorph. In an analysis of neuromuscular function, neuromuscular junctions had normal morphology, consistent with previous studies in knockout mice, and the muscles were able to generate appropriate force when normalized for their reduced size in late stage animals. Therefore, the Cntn1 mutant mice do not show evidence for a myopathy, but instead the phenotype is likely to be caused by dysfunction in the nervous system. Given the similarity of CNTN1 to other Ig-superfamily proteins such as DSCAMs, we also characterized the expression and localization of Cntn1 in the retinas of mutant mice for developmental defects. Despite widespread expression, no anomalies in retinal anatomy were detected histologically or using a battery of cell-type specific antibodies. We therefore conclude that the phenotype of the Cntn1 mice arises from dysfunction in the brain, spinal cord or peripheral nervous system, and is similar in either a BALB/c or B6;129;Black Swiss background, raising a possible discordance between the mouse and human phenotypes resulting from Cntn1 mutations. 相似文献