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1.
以拟南芥野生型和类受体蛋白激酶基因CRK45的T-DNA插入突变体crk45为材料,采用差异基因表达筛选技术检测ABA处理后野生型和crk45中基因表达的差异。结果显示:(1)crk45突变体中有1个基因的表达比野生型高约4倍。(2)NCBI数据库检索表明,该基因编码的蛋白具有EF手型结构,蛋白序列全长为130个氨基酸,是典型的Ca2+结合蛋白,故命名为CRK45抑制的钙离子结合蛋白(CICBP)。(3)Northern blotting分析结果显示,ABA处理后crk45突变体中CICBP的表达明显升高,证明CICBP基因的确受ABA诱导,且其表达受CRK45的抑制。(4)外源75mmol/L的Ca2+处理后,crk45突变体的萌发率(30.8%)显著高于野生型(17.16%),说明在Ca2+介导下CRK45的功能是抑制种子萌发。(5)qRT-PCR检测显示,野生型中CRK45的表达受Ca2+诱导明显升高,而crk45突变体中的表达一直保持很低,说明crk45突变体是一个基因敲除突变体;Ca2+处理后crk45突变体中CICBP基因表达上调,而野生型中CICBP的表达反而降低,说明Ca2+处理下CRK45抑制CICBP基因的表达。研究表明,ABA或Ca2+处理后,CRK45通过负调控CICBP基因的表达,从而抑制拟南芥种子萌发。  相似文献   

2.
The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex (S. rolfsii) and inter-cellular growth in the medulla (S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry.  相似文献   

3.
【背景】环境中高毒性As3+的微生物氧化在砷的生物地球化学循环中起重要作用,具有潜在的应用价值。【目的】Bacillus sp.ZJS3菌株是本实验室前期分离鉴定的一株As3+耐受菌株,而且对多种重金属具有耐受性,期望进一步明确该菌株在As3+胁迫下菌体形态变化及应对砷胁迫的遗传基础,为As3+耐受细菌的研究提供基础数据。【方法】使用单分子实时测序(single-molecule real-time sequencing,SMRT)及Illumina测序技术对Bacillus sp.ZJS3菌株进行全基因组测序,对其基因进行功能注释和生物信息学分析,并结合绝对定量PCR技术对砷抗性及砷代谢相关基因进行分析。【结果】Bacillus sp.ZJS3菌株基因组大小为5.82 Mb,GC含量为35.9%,包含染色体1个、质粒3个、CDS数量为5 981个、tRNA 104个、sRNA 136个、rRNA 42个、串联重复序列173个、基因岛13个、转运蛋白1 023个、跨膜蛋白1 717个和双组分调控基因160个。NR、Swiss-Prot、Pfam、COG、GO和KEGG数据库分别可注释Bacillus sp.ZJS3菌株基因组中97.66%、69.30%、78.52%、65.49%、67.65%和43.87%的基因。绝对定量PCR结果表明,arsC基因在砷处理条件下显著高于对照组,而arsB基因在砷处理条件下显著低于对照组。【结论】Bacillus sp.ZJS3菌株在As3+胁迫下可能导致细胞分裂无法正常进行,进而影响细胞形态。基因组中aqpZarsAarsBarsC等基因的存在表明该菌株具有As3+外排和还原As5+的能力,phoUpstBACS的存在表明菌株可以吸收As5+,但菌株受到外界环境As3+胁迫时arsB表达水平降低。  相似文献   

4.
A halophilic, thermotolerant Bacillus strain (B3-15), isolated from water of a shallow, marine hot spring at Vulcano Island (Eolian Islands, Italy), produced an exocellular polysaccharide at 165 mg l–1. It grew on kerosene as sole carbon source and was resistant to Cd2+, Zn2+, As2+ and Hg2+. From 16S rDNA analysis, strain B3-15 was related to B. licheniformis. The exopolysaccharide was a tetrasaccharide repeating unit essentially constituted by sugars having a manno-pyranosidic configuration.  相似文献   

5.
【目的】研究地毯草黄单胞菌双组分系统VgrS-VgrR与致病性的关系,为木薯细菌性病害的高效防控提供分子生物学证据。【方法】采用同源重组方法构建vgrS和vgrR的插入失活突变体,用可移动的cosmid载体p HM1构建互补菌株。检测突变体的致病性、细菌游动性、胞外酶、胞外多糖的变化,观察细菌对H_2O_2和金属离子胁迫的反应。【结果】相比野生型菌株,vgrS和vgrR突变体接种寄主植物木薯后致病力显著降低,突变体的游动性减少、蛋白酶活性减弱、H_2O_2耐受性降低,在高浓度金属离子Fe2+、Fe3+、Cu2+、Ni2+、Zn2+、Co2+的胁迫条件下菌体生长显著减弱。然而,vgrS和vgrR突变体的胞外多糖含量显著升高,分别是野生型的2.14和1.89倍。【结论】阐明了VgrS-VgrR系统在细菌致病过程中发挥的重要作用,为鉴定VgrS-VgrR调控机制提供线索,为药物筛选提供靶向目标。  相似文献   

6.
7.
A part of the gene encoding cbb 3-type cytochrome oxidase CcoN subunit was cloned from Azotobacter vinelandii and a mutant strain of this bacterium with disrupted ccoN gene was constructed. In contrast to the wild type strain, this one is unable to oxidize cytochromes c 4 and c 5. Thus, the A. vinelandii respiratory chain is shown to contain cbb 3-type cytochrome c oxidase. It is also shown that the activity of this enzyme is not necessary for diazotrophic growth of A. vinelandii at high oxygen concentrations.  相似文献   

8.
利用构建的烟曲霉金属还原酶基因(AFUA-1G00350,Fre B2)缺失突变株,对烟曲霉金属还原酶基因Fre B2功能进行初步研究,为揭示该基因与烟曲霉的致病关系提供依据。比较野生株和基因缺失突变株在AMM和无铁AMM液体培养基中生长时高铁还原酶的活性,绘制不同时间野生株和基因缺失突变株在AMM和无铁AMM液体培养基中生长时高铁还原酶活性曲线。利用Real-Time PCR方法分析Sre A、Sid A、Fet C、Ftr A和Fre B这些与铁的吸收相关基因的mRNA的表达量变化。测定野生株和基因缺失突变株对氧化压力的敏感性及胞内活性氧物质含量。不论在AMM液体培养基中还是在无铁AMM液体培养基中培养时,突变株高铁还原酶的活性都明显高于野生株高铁还原酶活性。与野生株相比培养60 h时,突变株Sre A、Sid A、Fet C、Ftr A和Fre B这些与铁的吸收相关基因的表达量出现明显上调。氧化压力敏感性实验显示,基因缺失突变株对H2O2的敏感性显著增强,同时胞内活性氧物质含量明显增多。金属还原酶基因Fre B2在烟曲霉铁吸收及氧化压力应答过程中发挥作用;烟曲霉与铁吸收相关基因之间存在功能互补效应。  相似文献   

9.
The mechanism of Zn resistance in multiple metal-resistant Pseudomonas putida strain S4 is based on inducible efflux. An ATPase in the strain S4 mediated active extrusion of Zn2+, which occurred during the exponential phase of growth. The ATPase activity was inhibited by micromolar concentrations (50 M) of vanadate, suggesting the involvement of a P-type ATPase. The effluxed Zn2+ were not ejected out of the cell but stored in the outer membrane and periplasm, which provided the required binding sites. The strain S4, thus, employs a dual strategy of efflux and binding to bring about a proper management of essential ions like Zn.  相似文献   

10.
The lack of eye pigment in the Aedes aegypti WE (white eye) colony was confirmed to be due to a mutation in the kynurenine hydroxylase gene, which catalyzes one of the steps in the metabolic synthesis of ommochrome eye pigments. Partial restoration of eye color (orange to red phenotype) in pupae and adults occurred in both sexes when first or second instar larvae were reared in water containing 3-hydroxykynurenine, the metabolic product of the enzyme kynurenine hydroxylase. No eye color restoration was observed when larvae were reared in water containing kynurenine sulfate, the precursor of 3-hydroxykynurenine in the ommochrome synthesis pathway. In addition, a plasmid clone containing the wild type Drosophila melanogaster gene encoding kynurenine hydroxylase, cinnabar (cn), was also able to complement the kynurenine hydroxylase mutation when it was injected into embryos of the A. aegypti WE strain. The ability to complement this A. aegypti mutant with the transiently expressed D. melanogaster cinnabar gene supports the value of this gene as a transformation reporter for use with A. aegypti WE and possibly other Diptera with null mutations in the kynurenine hydroxylase gene.  相似文献   

11.
This study provides first hand comparative account of growth and antioxidative defense system of the wild type, Cu2+ and temperature treated wild type and acclimated strains of Anabaena doliolum Bharadwaja against Cu2+ and high temperature. The acclimated strains showed perceptible growth at 250 μM Cu2+ and 47°C temperatures, respectively. In contrast to this the wild type strain on exposure to 50 μM Cu2+ and 47°C temperature depicted almost complete inhibition of growth. However, the peroxide content was significantly higher in the acclimated strains than the wild type. Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) showed maximum activity at high temperature followed by Cu2+ acclimated and minimum in the wild type strains. The ascorbate (ASC) and glutathione (GSH) contents were increased by 2.3 and 43.3, and 15.5 and 36.5-fold in Cu2+ and 47°C acclimated strains, respectively. However, when the wild type strain was subjected to Cu2+ and temperature all antioxidative enzymes except SOD showed inhibition of their activity. In case of wild type the GSH content was inhibited by 0.39-fold at 50 μM Cu2+ but the ASC content registered increase by 2 and 2.7-fold on subjecting to Cu2+ and temperature, respectively. Thus increased activity of enzymatic antioxidants as well as accumulation of ascorbate and glutathione in both the acclimated strains suggests that enzymatic and non-enzymatic antioxidants help in the acclimation of A. doliolum Bharadwaja against Cu2+ and high temperature. However, inhibition of antioxidative defense system of wild type under Cu2+ and heat stress appears to be the reason for its non survival. In view of the appreciable increase in the level of antioxidants as well as greater inhibition of specific growth rate in temperature than Cu2+ acclimated strains, temperature (47°C) is proposed to be is more deleterious to the organism than copper (250 μM).  相似文献   

12.
Streptomyces coeruleorubidus strain SIPI-1482 is an important industrial microbial strain which produces daunorubicin, the precursor for semi-synthesis of first-line anti-tumor antibiotics doxorubicin and epirubicin. dnmV, the C4 ketoreductase gene in the biosynthetic pathway of TDP-l-daunosamine was successfully disrupted by homologue recombination. The SIPI-1482 dnmV-blocked mutant lost the ability to produce daunorubicin and aggregate the intermediate ε-rhodomycinone. By introducing dnmV, the daunorubicin biosynthetic pathway in S. coeruleorubidus was reconstituted. Further more, aveBIV from S. avermitilis, as well as oleU from S. antibiotics, and novS from S. niveus were introduced into the dnmV-blocked mutant. The SIPI-1482 dnmV::aveBIV mutant could produce 4′-epidaunorubicin instead of daunorubicin, but dnmV::oleU and dnmV::novS mutant could not. Our study showed that the genetically engineered strain had a different fermentation condition and extraction protocol compared with the wild type daunorubicin producer. These results suggest that metabolic engineering is a powerful tool to produce novel hybrid antibiotics and a good alternative to chemical synthesis.  相似文献   

13.
ARR5-gene expression was studied in the course of natural leaf senescence and detached leaf senescence in the dark using Arabidopsis thaliana plants transformed with the P ARR5 -GUS gene construct. GUS-activity was measured as a marker of ARR5-gene expression. Chlorophyll and total protein amounts were also estimated to evaluate leaf senescence. Natural leaf senescence was accompanied by the progressive decline in the GUS-activity in leaves of the 2nd and 3rd nodes studied, and this shift of GUS-activity was more pronounced than the loss of chlorophyll content. The ability of the ARR5-gene promoter to respond to cytokinin was not eliminated during natural leaf senescence, as was demonstrated by a cytokinin-induced increase in GUS activity in leaves after their detachment and incubation on benzyladenine (BA, 5 × 10−6 M) in the dark. Leaf senescence in the dark was associated with the further decrease in the GUS-activity. The ARR5-gene promoter response to cytokinin was enhanced with the increase of the age of plants, taken as a source of leaves for cytokinin treatments. Hence, although the expression of the ARR5 gene reduces during natural and dark/detached leaf senescence, the ARR5-gene sensitivity to cytokinin was maintained in both cases and even increased with the leaf age. This data suggest that the ARR5 gene, which belongs to the type-A negative regulators of plant response to cytokinin, could be a feedback regulator able to prevent retardation by cytokinin of leaf senescence when it is important for plant life. Growth regulators either reduced ARR5 gene response to cytokinin during senescence of mature detached leaves in the dark (SA, meJA, ABA, SP) or increased it (IAA), thus modifying the resulting rate of its expression.  相似文献   

14.
Summary Using genetic hybridization analysis we identified seven polymorphic genes for the fermentation of melibiose in different Mel+ strains of Saccharomyces cerevisiae. Four laboratory strains (1453-3A, 303-49, N2, C.B.11) contained only the MEL1 gene and a wild strain (VKM Y-1830) had only the MEL2 gene. Another wild strain (CBS 4411) contained five genes: MEL3, MEL4, MEL5, MEL6 and MEL7. MEL3-MEL7 were isolated and identified by backcrosses with Mel parents (X2180-1A, S288C). A cloned MEL1 gene was used as a probe to investigate the physical structure and chromosomal location of the MEL gene family and to check the segregation of MEL genes from CBS 4411 in six complete tetrads. Restriction and Southern hybridization analyses showed that all seven genes are physically very similar. By electrokaryotyping we found that all seven genes are located on different chromosomes MEL1 on chromosome II as shown previously by Vollrath et al. (1988), MEL2 on VII, MEL3 on XVI, MEL4 on XI, MEL5 on IV, MEL6 on XIII, and MEL7 on VI. Molecular analysis of the segregation of MEL genes from strain CBS 4411 gave results identical to those from the genetic analyses. The homology in the physical structure of this MEL gene family suggests that the MEL loci have evolved by transposition of an ancestral gene to specific locations within the genome.  相似文献   

15.
This study demonstrates a pH-dependent inhibition of Mg2+- and Ca2+- ATPase activities ofNostoc linckia andChlorella vulgaris exposed to AlCl3, AlF3, NaF and AlCl3 + NaF together. AlF3 and the combination of AlCl3 + NaF were more inhibitory to both the enzymes as compared with AlCl3 and NaF. Toxicity of the test compounds increased with increasing acidity. Interaction of AlCl3 + NaF was additive onN. linckia andC. vulgaris, respectively, at pH 7.5 and 6.8, and synergistic at pH 6.0 and 4.5. In the presence of 60 and 100 m PO4 3- an increased NaF concentration (in the AlCl3 + NaF combination) was required to produce the same degree of inhibition in ATP synthesis and ATPase activity. Toxicity of fluoroaluminate was reduced in the presence of EDTA and citrate. Except for beryllium to some extent, combinations of cadmium, cobalt, iron, manganese, tin and zinc with fluoride were not as effective as aluminium in inhibiting the ATPase activity. The presence of a 100 kDa protein band in SDS-PAGE of both control as well as AlCl3 + NaF-treated samples suggested that AlF4 inhibits the ATPase activity by acting as a functional barrier without affecting the structure of the enzyme.  相似文献   

16.
In this study, rpoS gene was identified from Edwardsiella tarda EIB202 and its functional role was analyzed by using an in-frame deletion mutant ∆rpoS and the complemental strain rpoS +. Compared with the wild type and rpoS +, ∆rpoS was impaired in terms of the ability to survive under oxidative stress and nutrient starvation, as well as the resistance to 50% serum of Scophthalmus maximus in 3 h, demonstrating essential roles of RpoS in stress adaptation. The rpoS mutant also displayed markedly increased chondroitinase activity and biofilm formation. Real-time polymerase chain reaction revealed that the expression level of quorum sensing autoinducer synthetase genes luxS and edwI was increased by 3.7- and 2.5-fold in the rpoS mutant strain. Those results suggested that rpoS might be involved in the negative or positive regulation of chondroitinase and biofilm formation, or quorum sensing networks in E. tarda, respectively. Although there were no obvious differences between the wild-type and the rpoS mutant in adherence of epithelioma papulosum cyprini (EPC) cell and in the lethality on fish model, rpoS deletion leads to the drastically reduced capacity for E. tarda to internalize in EPC cells, indicating that RpoS was, while not the main, the factor required for the virulence network of E. tarda.  相似文献   

17.
Two moderately halophilic low G + C Gram-positive bacteria were isolated from a sample of salted skate (Class Chondrychthyes, Genus Raja). Phylogenetic analysis of the 16S rRNA gene sequence of strains RH1T and RH4 showed that these organisms represented a novel species of the genus Salinicoccus. The new isolates formed pink–red colonies and flocculated in liquid media, with optimum growth in media containing 4% NaCl and pH of about 8.0. These organisms are aerobic but reduce nitrate to nitrite under anaerobic conditions. Acid is produced from several carbohydrates. Oxidase and catalase were detected. Menaquinone 6 was the major respiratory quinone. The major fatty acids of strains RH1T and RH4 were 15:0 anteiso and 15:0 iso. The G + C contents of DNA were 46.2 and 46.0 mol%, respectively. The peptidoglycan was of A3alpha L-Lys-Gly5–6 type. On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we suggest that strain RH1T (=LMG 22840 = CIP 108576) represents a new species of the genus Salinicoccus, for which we propose the name Salinicoccus salsiraiae.  相似文献   

18.
A new alkaliphilic and moderately halophilic, strictly anaerobic, fermentative bacterium (strain IMP-300T) was isolated from a groundwater sample in the zone of the former soda lake Texcoco in Mexico. Strain IMP-300T was Gram-positive, non-sporulated, motile and rod-shaped. It grew within a pH range from 7.5 to 10.5, and an optimum at 9.5. The organism was obligately dependent on the presence of sodium salts. Growth showed an optimum at 35°C with absence of growth above 45°C. It fermented peptone and a few amino acids, preferentially arginine and ornithine, with production of acetate, propionate, and ammonium. Its fatty acid pattern was mainly composed of straight chain saturated, unsaturated, and cyclopropane fatty acids. The G + C content of genomic DNA was 40.0 mol%. Analysis of the 16S rRNA gene sequence indicated that the new isolate belongs to the genus Tindallia, in the low G + C Gram-positive phylum. Phylogenetically, strain IMP-300T has Tindallia californiensis, as closest relative with a 97.5% similarity level between their 16S rDNA gene sequences, but the DNA–DNA re-association value between the two DNAs was only 42.2%. On the basis of differences in genotypic, phenotypic, and phylogenetic characteristics, strain IMP-300T is proposed as a new species of the genus Tindallia, T. texcoconensis sp. nov. (type strain IMP-300T = DSM 18041T = JCM 13990T).  相似文献   

19.
TheSaccharomyces cerevisiae PMR1 gene encodes a Ca2+-ATPase localized in the Golgi. We have investigated the effects ofPMR1 disruption inS. cerevisiae on the glycosylation and secretion of three heterologous glycoproteins, human α1-antitrypsin (α1-AT), human antithrombin III (ATHIII), andAspergillus niger glucose oxidase (GOD). Thepmr1 null mutant strain secreted larger amounts of ATHIII and GOD proteins per a unit cell mass than the wild type strain. Despite a lower growth rate of thepmr1 mutant, two-fold higher level of human ATHIII was detected in the culture supernatant from thepmr1 mutant compared to that of the wild-type strain. Thepmr1 mutant strain secreted α1-AT and the GOD proteins mostly as core-glycosylated forms, in contrast to the hyperglycosylated proteins secreted in the wild-type strain. Furthermore, the core-glycosylated forms secreted in thepmr1 mutant migrated slightly faster on SDS-PAGE than those secreted in themnn9 deletion mutant and the wild type strains. Analysis of the recombinant GOD with anti-α1,3-mannose antibody revealed that GOD secreted in thepmr1 mutant did not have terminal α1,3-linked mannoses unlike those secreted in themnn9 mutant and the wild type strains. The present results indicate that thepmr1 mutant, with the super-secretion phenotype, is useful as a host system to produce recombinant glycoproteins lacking high-mannose outer chains.  相似文献   

20.
A bacterial strain, designated Iso4T, was isolated from the East Sea of Korea and was subjected to a poly-phasic taxonomy study including phenotypic and chemotaxonomic characteristics as well as 16S rRNA gene sequence analysis. Cells of the strain were Gram-negative, motile, non-budding, non-stalked, and strictly aerobic. Strain Iso4T grew optimally at 20°C in the presence of 1∼2% (w/v) NaCl and at pH 6.9∼7.6. The major respiratory quinone was Q-10 and the major cellular fatty acids were C18:1 ω7c (53.5%), C17:1 ω5c (11.7%), C17:1 ω6c (8.1%), C16:0 (7.8%), C17:0 (4.8%), C15:0 (2.9%), and C16:1 ω5c (2.2%). The DNA G+C content of strain Iso4T was 56.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Iso4T formed a monophyletic clade in the family Hyphomonadaceae, supported by high bootstrap value and was most closely related to the genus Hyphomonas (92∼94%), a member of marine bacteria in the family. The phenotypic, genotypic, and chemotaxonomic evidences also suggest strain Iso4T represents a novel genus and species in the family Hyphomonadaceae, for which the name Henriciella gen. nov., sp. nov. is proposed. The type strain is Iso4T (=KCTC 12513T =DSM 19595T =JCM 15116T).  相似文献   

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