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杜氏盐藻DCA1启动子内GT重复序列在盐诱导调控中的作用 总被引:1,自引:0,他引:1
为了研究杜氏盐藻双拷贝碳酸酐酶(DCA1)启动子中高度重复的GT序列在盐诱导表达时的调控作用,设计不同的引物,通过PCR法获得6条不同长度的DCA1启动子片段,分别与gus报告基因融合后构建6个表达载体;电击法转化杜氏盐藻细胞。组织化学染色和荧光定量法检测GUS在不同盐浓度下的瞬时表达。结果显示,DCA1启动子内高度重复的GT序列无论与其上游、下游或上下游片段同时结合均能驱动gus基因的表达,并且其表达受氯化钠浓度调控,其中和上下游均结合时活性最强;无GT重复序列的融合片段及GT 重复的下游片段也能驱动gus基因的表达,但其表达不受氯化钠浓度调控;而GT重复的上游片段不能驱动gus基因的表达。结果提示:盐藻DCA1启动子中高度重复的GT序列在盐诱导调控中起重要作用,可能为一种新型的盐诱导元件。 相似文献
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桃PpMADS1基因启动子的克隆及功能分析 总被引:3,自引:0,他引:3
PpMADS1基因属于一类MADS box 基因,在植物的花发育调控中起着重要的作用。通过Genome Walking的方法从桃基因组中分离了长度为1 814bp的PpMADS1基因启动子片段,序列分析表明,在此启动子上不仅含有TATA box 和CAAT box基本元件,而且含有大量的与光调节有关的调控元件,如GT-1,Sp1和as-2-box,另外存在两个CArG-box元件、一个G-box元件和一个TGA-element,说明该启动子可能受光周期和激素的调控。将该启动子通过5′端缺失,分区段与GUS报告基因连接构建表达载体,并转化拟南芥。GUS组织化学染色分析结果表明,在-197到-454bp有促使GUS在花原基中表达的花原基特异性元件,在-454到-678bp之间存在促使GUS在萼片和花瓣表达的特异性元件,在-678到-978bp存在负调控作用元件,阻遏了GUS基因在花药中的表达。 相似文献
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锌指蛋白作为植物体内一类重要的转录因子,对植物生长发育、基因调控以及响应外界环境变化方面发挥重要作用。Os BBX6基因属于水稻锌指蛋白B-Box基因家族成员,启动子元件分析发现其含有高温应答元件(HSE)、干旱应答元件(MBS)及非生物胁迫响应元件(TC-rich repeats)等逆境相关元件。组织特异性定量表达分析表明,Os BBX6在叶片中表达最高,根其次,茎和幼穗中表达最低。胁迫处理后的荧光定量PCR发现其受低温诱导上调,受高温、干旱、盐胁迫等抑制表达,表明其正向响应低温胁迫,负向响应高温、干旱、盐胁迫等。另外,本研究还克隆了OsBBX6基因,并对其进行了系统进化、蛋白跨膜、蛋白亚细胞定位及OsBBX6基因共表达等分析,为进一步研究其生物学功能奠定基础。 相似文献
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A substrate-dependent biological containment system for Pseudomonas putida based on the Escherichia coli gef gene. 总被引:4,自引:4,他引:0 下载免费PDF全文
A model substrate-dependent suicide system to biologically contain Pseudomonas putida KT2440 is reported. The system consists of two elements. One element carries a fusion between a synthetic lac promoter (PA1-04/03) and the gef gene, which encodes a killing function. This element is contained within a transposaseless mini-Tn5 transposon so that it can be integrated at random locations on the Pseudomonas chromosome. The second element, harbored by plasmid pCC102, is designed to control the first and bears a fusion between the promoter of the P. putida TOL plasmid-encoded meta-cleavage pathway operon (Pm) and the lacI gene, encoding the Lac repressor, plus xylS2, coding for a positive regulator of Pm. In liquid culture under optimal growth conditions and in sterile and nonsterile soil microcosms, P. putida KT2440 (pWWO) bearing the containment system behaves as designed. In the presence of a XylS effector, such as m-methylbenzoate, the LacI protein is synthesized, preventing the expression of the killing function. In the absence of effectors, expression of the PA1-04/03::gef cassette is no longer prevented and a high rate of cell killing is observed. Fluctuation test analyses revealed that mutants resistant to cell killing arise at a frequency of around 10(-5) to 10(-6) per cell per generation. Mutations are linked to the killing element rather than to the regulatory one. In bacteria bearing two copies of the killing cassette, the rate of appearance of mutants resistant to killing decreased to as low as 10(-8) per cell per generation. 相似文献
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Construction and behavior of biologically contained bacteria for environmental applications in bioremediation. 总被引:4,自引:3,他引:1 下载免费PDF全文
The survival of microorganisms can be predicted through the use of active biological containment systems. We have constructed contained Pseudomonas putida strains that degrade alkylbenzoates. The modified strain carries a fusion of the Plac promoter to the gef gene, which encodes a killing protein. Expression from Plac is controlled through a regulatory cascade, so that Plac is switched on or off by the absence or presence of alkylbenzoates, respectively. Similar uncontained strains were also constructed and tested as a control. Contained and uncontained strains were genetically stable, and their survival and functionality in soil microcosms were as expected. Both contained and uncontained strains survived well in soils supplemented with alkylaromatics, whereas survival of the contained strain in soil microcosms without methylbenzoates was markedly reduced, in contrast to the control strain, which survived in these soils in the absence of alkylbenzoates. The TOL plasmid was transferred in soils between Pseudomonas strains but was not able to mobilize the elements of the containment system. 相似文献
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A conditional suicide system in Escherichia coli based on the intracellular degradation of DNA. 总被引:5,自引:1,他引:4 下载免费PDF全文
The potential risks associated with the intentional or unintentional release of genetically engineered microorganisms led to the construction of biological containment systems by which bacteria are killed in a controlled suicide process. In previously published suicide systems, cell killing was caused by proteins destroying the cell membrane or cell wall. Here a conditional cell killing system based on the intracellular degradation of cellular DNA is presented. The nuclease gene used was that of the extracellular nuclease of Serratia marcescens. The nuclease gene was deleted for the leader-coding sequence, and the truncated gene was put under the control of the lambda pL promoter. Following thermoinduction of the nuclease gene cassette in Escherichia coli, cell survival dropped to 2 x 10(-5), and more than 80% of the radioactively labeled DNA was converted to acid-soluble material within 2.5 h in the absence of cell lysis. The majority (84%) of clones which survived thermoinduced killing turned out to be as sensitive to a second thermoinduction as the original strain. The other clones showed somewhat slower killing kinetics or slightly higher final levels of survivors. The suicide system described combines the regulated killing of cells with the destruction of intracellular DNA otherwise potentially available for horizontal gene transfer processes. 相似文献
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Development of efficient suicide mechanisms for biological containment of bacteria. 总被引:8,自引:7,他引:1 下载免费PDF全文
To optimize plasmid containment, we have systematically investigated the factors that limit the killing efficiency of a suicide system based on the relF gene from Escherichia coli controlled by inducible lac promoters and placed on plasmids. In induction experiments with this suicide system, killing efficiency was unaffected by temperature and growth medium; there was no requirement for great promoter strength or high plasmid copy number. We could demonstrate that the factors limiting killing were the mutation rate of the suicide function and the reduced growth rate caused by a basal level of expression of the suicide gene during normal growth, which can give a selective growth advantage to cells with mutated suicide functions. The capacity of the plasmid-carried killing system to contain the plasmid was tested in transformation, transduction, and conjugational mobilization. The rate of plasmid transfer detected in these experiments seemed too high to provide adequate biological containment. As expected from the induction experiments, plasmids that escaped containment in these transfer experiments turned out to be mutated in the suicide function. With lac-induced suicide as a test, the efficiency of the system was improved by tightening the repression of the suicide gene, thereby preventing selection of cells mutated in the killing function. Reduction of the mutational inactivation rate of the suicide system by duplication of the suicide function augmented the efficiency of the suicide dramatically. These results permit the construction of extremely efficient biological containment systems. 相似文献
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To optimize plasmid containment, we have systematically investigated the factors that limit the killing efficiency of a suicide system based on the relF gene from Escherichia coli controlled by inducible lac promoters and placed on plasmids. In induction experiments with this suicide system, killing efficiency was unaffected by temperature and growth medium; there was no requirement for great promoter strength or high plasmid copy number. We could demonstrate that the factors limiting killing were the mutation rate of the suicide function and the reduced growth rate caused by a basal level of expression of the suicide gene during normal growth, which can give a selective growth advantage to cells with mutated suicide functions. The capacity of the plasmid-carried killing system to contain the plasmid was tested in transformation, transduction, and conjugational mobilization. The rate of plasmid transfer detected in these experiments seemed too high to provide adequate biological containment. As expected from the induction experiments, plasmids that escaped containment in these transfer experiments turned out to be mutated in the suicide function. With lac-induced suicide as a test, the efficiency of the system was improved by tightening the repression of the suicide gene, thereby preventing selection of cells mutated in the killing function. Reduction of the mutational inactivation rate of the suicide system by duplication of the suicide function augmented the efficiency of the suicide dramatically. These results permit the construction of extremely efficient biological containment systems. 相似文献
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The genetic determinant of the lethal antibiotic colicin E3 was cloned under the control of a tightly regulated promoter in the absence of the gene for its cognate inhibitor. Combination of this killing cassette with a stringent regulatory element provided a substrate-dependent conditional suicide system that was exploited for the biological containment of a Pseudomonas putida strain. The lethality of a single gene copy and the distinct and universal cellular target of the antibiotic suggest colicin E3 as an ideal candidate for combination with other lethal functions to design highly efficient containment systems for microorganisms. 相似文献
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Ronald M. Atlas 《Biodegradation》1992,3(2-3):137-146
Plans to introduce genetically engineered microorganisms into the environment has led to concerns over safety and has raised questions about how to detect and to contain such microorganisms. Specific gene sequences, such as lacZ, have been inserted into genetically engineered microorganisms to permit their phenotypic detection. Molecular methods have been developed based upon recovery of DNA from environmental samples and gene probe hybridization to specific diagnostic gene sequences for the specific detection of genetically engineered microorganisms. DNA amplification using the polymerase chain reaction has been applied to enhance detection sensitivity so that single gene targets can be detected. Detection of messenger RNA has permitted the monitoring of gene expression in the environment. The use of reporter genes, such as the lux gene for bioluminescence, likewise has permitted the observation of gene expression. Conditional lethal constructs have been developed as models for containment of genetically engineered microorganisms. Suicide vectors, based upon the hok gene have been developed as model containment systems. 相似文献
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Conditional-Suicide Containment System for Bacteria Which Mineralize Aromatics 总被引:11,自引:8,他引:3 下载免费PDF全文
A model conditional-suicide system to control genetically engineered microorganisms able to degrade substituted benzoates is reported. The system is based on two elements. One element consists of a fusion between the promoter of the Pseudomonas putida TOL plasmid-encoded meta-cleavage pathway operon (Pm) and the lacI gene encoding Lac repressor plus xylS, coding for the positive regulator of Pm. The other element carries a fusion between the Ptac promoter and the gef gene, which encodes a killing function. In the presence of XylS effectors, LacI protein is synthesized, preventing the expression of the killing function. In the absence of effectors, expression of the Ptac::gef cassette is no longer prevented and a high rate of cell killing is observed. The substitution of XylS for XylSthr45, a mutant regulator with altered effector specificity and increased affinity for benzoates, allows the control of populations able to degrade a wider range of benzoates at micromolar substrate concentrations. Given the wide effector specificity of the key regulators, the wild-type and mutant XylS proteins, the system should allow the control of populations able to metabolize benzoate; methyl-, dimethyl-, chloro-, dichloro-, ethyl-, and methoxybenzoates; salicylate; and methyl- and chlorosalicylates. A small population of genetically engineered microorganisms became Gef resistant; however, the mechanism of such survival remains unknown. 相似文献
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A stochastic killing system for biological containment of Escherichia coli. 总被引:1,自引:0,他引:1 下载免费PDF全文
Bacteria with a stochastic conditional lethal containment system have been constructed. The invertible switch promoter located upstream of the fimA gene from Escherichia coli was inserted as expression cassette in front of the lethal gef gene deleted of its own natural promoter. The resulting fusion was placed on a plasmid and transformed to E. coli. The phenotype connected with the presence of such a plasmid was to reduce the population growth rate with increasing significance as the cell growth rate was reduced. In very fast growing cells, there was no measurable effect on growth rate. When a culture of E. coli harboring the plasmid comprising the containment system is left as stationary cells in suspension without nutrients, viability drops exponentially over a period of several days, in contrast to the control cells, which maintain viability nearly unaffected during the same period of time. Similar results were obtained with a strain in which the killing cassette was inserted in the chromosome. In competition with noncontained cells during growth, the contained cells are always outcompeted. Stochastic killing obtained by the fim-gef fusion is at present relevant only as a containment approach for E. coli, but the model may be mimicked in other organisms by using species-specific stochastic expression systems. 相似文献
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Active biological containment (ABC) systems have been designed to control at will the survival or death of a bacterial population. These systems are based on the use of a killing gene, e.g., a porin-inducing protein such as the one encoded by the Escherichia coli gef gene, and a regulatory circuit that controls expression of the killing gene in response to the presence or absence of environmental signals. An ABC system for recombinant microorganisms that degrade a model pollutant was designed on the basis of the Pseudomonas putida TOL plasmid meta-cleavage regulatory circuit. The system consists of a fusion of the Pm promoter to lacI, whose expression is controlled by XylS with 3-methylbenzoate, and a fusion of a synthetic P(lac) promoter to gef. In the presence of the model pollutant, bacterial cells survived and degraded the target compound, whereas in the absence of the aromatic carboxylic acid cell death was induced. The system had two main drawbacks: (i) the slow death of the bacterial cells in soil versus the fast killing rate in liquid cultures in laboratory assays, and (ii) the appearance of mutants, at a rate of about 10(-8) per cell and generation, that did not die after the pollutant had been exhausted. We reinforced the ABC system by including it in a Deltaasd P. putida background. A P. putida Deltaasd mutant is viable only in complex medium supplemented with diaminopimelic acid, methionine, lysine, and threonine. We constructed a P. putida Deltaasd strain, called MCR7, with a Pm::asd fusion in the host chromosome. This strain was viable in the presence of 3-methylbenzoate because synthesis of the essential metabolites was achieved through XylS-dependent induction. In the P. putida MCR7 strain, an ABC system (Pm::lacI, xylS, P(lac)::gef) was incorporated into the host chromosome to yield strain MCR8. The number of MCR8 mutants that escaped killing was below our detection limit (<10(-9) mutants per cell and generation). The MCR8 strain survived and colonized rhizosphere soil with 3-methylbenzoate at a level similar to that of the wild-type strain. However, it disappeared in less than 20 to 25 days in soils without the pollutant, whereas an asd(+), biologically contained counterpart such as P. putida CMC4 was still detectable in soils after 100 days. 相似文献