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1.
The risk that the plant pathogen Phytophthora melonis develops resistance to carboxylic acid amide (CAA) fungicides was determined by measuring baseline sensitivities of field isolates, generating resistant mutants, and measuring the fitness of the resistant mutants. The baseline sensitivities of 80 isolates to flumorph, dimethomorph and iprovalicarb were described by unimodal curves, with mean EC(50) values of 0.986 (±0.245), 0.284 (±0.060) and 0.327 (±0.068) μg/ml, respectively. Seven isolates with different genetic background (as indicated by RAPD markers) were selected to generate CAA-resistance. Fifty-five resistant mutants were obtained from three out of seven isolates by spontaneous selection and UV-mutagenesis with frequencies of 1×10(-7) and 1×10(-6), respectively. CAA-resistance was stable for all mutants. The resistance factors of these mutants ranged from 7 to 601. The compound fitness index (CFI = mycelial growth × zoospore production × pathogenicity) was often lower for the CAA-resistant isolates than for wild-type isolates, suggesting that the risk of P. melonis developing resistance to CAA fungicides is low to moderate. Among the CAA-resistant isolates, a negative correlation between EC(50) values was found for iprovalicarb vs. flumorph and for iprovalicarb vs. dimethomorph. Comparison of the full-length cellulose synthase 3 (CesA3) between wild-type and CAA-resistant isolates revealed only one point mutation at codon position 1109: a valine residue (codon GTG in wild-type isolates) was converted to leucine (codon CTG in resistant mutants). This represents a novel point mutation with respect to mutations in CesA3 conferring resistance to CAA fungicides. Based on this mutation, an efficient allelic-specific PCR (AS-PCR) method was developed for rapid detection of CAA-resistance in P. melonis populations. 相似文献
2.
Rio A. Stamler Omar Holguin Barry Dungan Tanner Schaub Soumaila Sanogo Natalie Goldberg Jennifer J. Randall 《PloS one》2015,10(5)
Induced resistance in plants is a systemic response to certain microorganisms or chemicals that enhances basal defense responses during subsequent plant infection by pathogens. Inoculation of chile pepper with zoospores of non-host Phytophthora nicotianae or the chemical elicitor beta-aminobutyric acid (BABA) significantly inhibited foliar blight caused by Phytophthora capsici. Tissue extract analyses by GC/MS identified conserved change in certain metabolite concentrations following P. nicotianae or BABA treatment. Induced chile pepper plants had reduced concentrations of sucrose and TCA cycle intermediates and increased concentrations of specific hexose-phosphates, hexose-disaccharides and amino acids. Galactose, which increased significantly in induced chile pepper plants, was shown to inhibit growth of P. capsici in a plate assay. 相似文献
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G. Wei Xu Julia I. Toth Sara R. da Silva Stacey-Lynn Paiva Julie L. Lukkarila Rose Hurren Neil Maclean Mahadeo A. Sukhai Rabindra N. Bhattacharjee Carolyn A. Goard Patrick T. Gunning Sirano Dhe-Paganon Matthew D. Petroski Aaron D. Schimmer 《PloS one》2014,9(4)
The NEDD8-activating enzyme (NAE) initiates neddylation, the cascade of post-translational NEDD8 conjugation onto target proteins. MLN4924, a selective NAE inhibitor, has displayed preclinical anti-tumor activity in vitro and in vivo, and promising clinical activity has been reported in patients with refractory hematologic malignancies. Here, we sought to understand the mechanisms of resistance to MLN4924. K562 and U937 leukemia cells were exposed over a 6 month period to MLN4924 and populations of resistant cells (R-K562MLN, R-U937MLN) were selected. R-K562MLN and R-U937MLN cells contain I310N and Y352H mutations in the NAE catalytic subunit UBA3, respectively. Biochemical analyses indicate that these mutations increase the enzyme’s affinity for ATP while decreasing its affinity for NEDD8. These mutations effectively contribute to decreased MLN4924 potency in vitro while providing for sufficient NAE function for leukemia cell survival. Finally, R-K562MLN cells showed cross-resistance to other NAE-selective inhibitors, but remained sensitive to a pan-E1 (activating enzyme) inhibitor. Thus, our work provides insight into mechanisms of MLN4924 resistance to facilitate the development of more effective second-generation NAE inhibitors. 相似文献
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为科学评价中国灌木辣椒种质,选取有代表性的8份辣椒材料,开展了中国灌木辣椒农艺性状鉴定和疫病抗性分析。结果表明:中国灌木辣椒长势强,株高均在1.0 m以上,叶片阔大,花瓣白绿色;果实直立向上,单果质量在0.51~2.04 g之间,平均为1.26 g;果实辣椒素与二氢辣椒素含量之和在565.00~1821.00 mg/kg,平均为1328.33mg/kg,是一年生辣椒B9431的407倍;对疫霉菌抗性水平表现为中抗至高抗,其中,海南野生灌木辣椒H108表现高抗。基于表型数据的主成分分析将中国灌木辣椒与一年生辣椒及美洲灌木辣椒有效区分开来。本研究结果为中国灌木辣椒优异基因的发掘和有效利用提供了理论参考。 相似文献
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Inoculation of the stems of three Capsicum annuum L. cultivars showing different degrees of sensitivity to the fungal pathogen Phytophthora capsici , resulted in a hypersensitive reaction being expressed along the stems. One of the peppers (cv. Smith-5) showed resistance by total inhibition of fungal growth. Capsidiol, a phytoalexin, which accumulates in the area of necrosis appears to be involved in this resistance. Capsidiol accumulation was analyzed by gas chromatography and was correlated with the restricted growth of P. capsici , in vivo and in vitro, confirming the former's fungistatic and fungitoxic properties. The capacity to inhibit pathogenic growth was evident only when capsidiol production exceeded 1 204 μg ml-1 , a level reached in the resistant variety after 6 days of incubation. Experiments on induced resistance showed that a second inoculation of the stems of the three cultivars also resulted in necrosis and in an accumulation of capsidiol, although to a lesser extent than in the first inoculation. The greater accumulation of capsidiol in the stems of cv. Smith-5 is in accordance with the resistance shown by this cultivar to P. capsici , and confirms the implication of capsidiol in the disease resistance of this cultivar to fungal pathogens. Capsidiol has a fungistatic character at a mean concentration of 3.75 mM, and is fungitoxic at levels above 5 mM. This level must be exceeded and all the growing hyphae must be affected for capsidiol to qualify from being fungistatic to being fungitoxic. 相似文献
9.
Peroxidase isoenzymes in the defense response of Capsicum annuum to Phytophthora capsici 总被引:1,自引:0,他引:1
M. Dolores Alcázar Catalina Egea Antonio Espín M. Emilia Candela 《Physiologia plantarum》1995,94(4):736-742
Quantitative and qualitative changes in isoperoxidase patterns from stems of three cultivars of pepper ( Capsicum annuum L.). one susceptible, one intermediate and one resistant, were found upon inoculation with Phytophthora capsici using a decapitation method. The peroxidase activity was determined in the intercellular fluid as well as in the cytosolic fraction of the necrotic, healthy and intermediate zones of stems of the three cultivars, 6 days after inoculation. In the intercellular fluid, peroxidase activity of the susceptible cv. Yolo Wonder increased somewhat from 4.7 (healthy zone) to 12.9 (intermediate zone) μmol mg−1 protein min−1 , whereas in the intermediate cv. Americano, the peroxidase activity decreased from 123 (healthy zone) to 78 (intermediate zone) μmol mg−1 protein min−1 . The most dramatic increase (5.7 to 662 μmol mg−1 protein min−1 ) in intercellular peroxidase activity was found in the resistant cv. Smith-5. This, in conjunction with the appearance of an additional acidic isoperoxidase (pI 4.4) specific for the cv. Smith-5, could be the reason for the resistance of this cultivar against the fungus attack. The release of peroxidase into the intercellular space as a defense reaction was confirmed by histochemical analysis, showing that peroxidase activity occurred in the intercellular spaces of those stems of the resistant cultivar that had not yet been invaded by the fungus, but was detected neither in the other cultivars nor in the intercellular spaces of such stems of the intermediate and susceptible cultivars that contained growing mycelium of P. capsici. The lack of staining in the intercellular spaces of the susceptible cultivars could be attributed to their low content in peroxidase. 相似文献
10.
ByungKook Hwang Jae Young Yoon W.-D. Ibenthal R. Heitefuss 《Journal of Phytopathology》1991,132(2):129-138
Patterns of soluble proteins and isoenzymes of esterase and superoxide dismutase were investigated in healthy and infected stems of two pepper cultivars resistant and susceptible to Phytophthora capsici. By the use of two-dimensional SDS-polyacrylamide gel electrophoresis, it was possible to compare precisely the cultivars Hanbyul and Kingkun susceptible or resistant to P. capsici with respect to their protein patterns. The two-dimensional electrophoresis identified three proteins (25—27 kD) from the healthy stem extracts of Kingkun, which were absent in Hanbyul. Some particular proteins appeared in pepper stems of both cultivars at later developmental stage of plants, suggesting their role in the expression of age-related resistance. Some proteins which were not detectable in the healthy stem extracts also existed in large amounts in the diseased ones. By contrast, other proteins present in the healthy stems disappeared from the diseased stems. Some esterase isoenzymes appeared in the two cultivars only at late developmental stage. Other esterase isoenzymes were produced only in the diseased stems. There were no differences in the patterns of superoxide dismutases between the cultivars and also between developmental stages. Large activities of several superoxide dismutases were detected in the diseased stems. 相似文献
11.
Functional analysis of the cellulose synthase genes CesA1, CesA2, and CesA3 in Arabidopsis 总被引:1,自引:0,他引:1
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Polysaccharide analyses of mutants link several of the glycosyltransferases encoded by the 10 CesA genes of Arabidopsis to cellulose synthesis. Features of those mutant phenotypes point to particular genes depositing cellulose predominantly in either primary or secondary walls. We used transformation with antisense constructs to investigate the functions of CesA2 (AthA) and CesA3 (AthB), genes for which reduced synthesis mutants are not yet available. Plants expressing antisense CesA1 (RSW1) provided a comparison with a gene whose mutant phenotype (Rsw1(-)) points mainly to a primary wall role. The antisense phenotypes of CesA1 and CesA3 were closely similar and correlated with reduced expression of the target gene. Reductions in cell length rather than cell number underlay the shorter bolts and stamen filaments. Surprisingly, seedling roots were unaffected in both CesA1 and CesA3 antisense plants. In keeping with the mild phenotype compared with Rsw1(-), reductions in total cellulose levels in antisense CesA1 and CesA3 plants were at the borderline of significance. We conclude that CesA3, like CesA1, is required for deposition of primary wall cellulose. To test whether there were important functional differences between the two, we overexpressed CesA3 in rsw1 but were unable to complement that mutant's defect in CesA1. The function of CesA2 was less obvious, but, consistent with a role in primary wall deposition, the rate of stem elongation was reduced in antisense plants growing rapidly at 31 degrees C. 相似文献
12.
Eggplant (Solanum melongena L.) is an important solanaceous crop with high phenotypic diversity and moderate genotypic diversity. Ninety-nine genotypes of eggplant germplasm (species (S. melongena, S. incanum, S. linnaeanum and S. gilo), landraces and heirloom cultivars) from 32 countries and five continents were evaluated for genetic diversity, population structure, fruit shape, and disease resistance to Phytophthora fruit rot. Fruits from each line were measured for fruit shape and evaluated for resistance to two Phytophthora capsici isolates seven days post inoculation. Only one accession (PI 413784) was completely resistant to both isolates evaluated. Partial resistance to Phytophthora fruit rot was found in accessions from all four eggplant species evaluated in this study. Genetic diversity and population structure were assessed using 22 polymorphic simple sequence repeats (SSRs). The polymorphism information content (PIC) for the population was moderate (0.49) in the population. Genetic analyses using the program STRUCTURE indicated the existence of four genetic clusters within the eggplant collection. Population structure was detected when eggplant lines were grouped by species, continent of origin, country of origin, fruit shape and disease resistance. 相似文献
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The relationship between age-related resistance of peper plants to Phytophthora capsici and contents of carbohydrates, amino acids, phenolics and mineral nutrients in pepper stems was studied using two pepper cultivars, Hanbyul (susceptible) and Kingkun (resistant). With increasing age of pepper plants, the two cultivars, which differ in their susceptibility to Phytophthora blight, became gradually resistant to the disease. The cultivar Kingkun distinctly showed the age-related resistance to Phytophthora blight at the second branch stage. The weight of dry matter in healthy stems of pepper plants at the second branch stage was twice that at the six leaf stage. The resistant cultivar Kingkun contained lower levels of fructose, glucose and sucrose in stems than the susceptible cultivar Hanbyul at the different developmental stages. No consistent differences between the developmental stages of the plants were recognized with regard to their glucose content. However, the contents of fructose and sucrose in the cultivar Hanbyul greatly increased at the second branch stage. The levels of inositol reduced in both pepper cultivars during plant development. In view of the fact that there were only slight changes in the amount of total amino acids, it seems unlikely that there is a relationship between the amino acid metabolism and the retardation of Phytophthora infection during plant development. The amounts of total phenolic compounds in pepper stems were relatively low at the later growth stages of the plants and also in the resistant cultivar Kingkun. The contents of macroelemental nutrients such as nitrogen, phosphorus, potassium, calcium and magnesium were drastically reduced in pepper stems at the later plant growth stage. No significant differences between the cultivars or the plant growth stages were found in the silicon and microelemental nutrients such as sodium, iron, zinc and manganese. These results suggest that the expression of age-related resistance of pepper plants may be due to the morphological and nutritional changes in tissues of pepper stems during ageing, i.e. the pronounced increase in weight of dry matter, the significant decrease in amounts of mineral nutrients such as nitrogen, phosphorus, potassium, calcium and magnesium, and the tow contents of fructose, glucose and sucrose in the stem tissues. 相似文献
14.
Deborah A. Ribardo Lacey K. Bingham-Ramos David R. Hendrixson 《Journal of bacteriology》2010,192(7):1890-1901
Campylobacter jejuni is a leading cause of gastroenteritis in humans and a commensal bacterium of the intestinal tracts of many wild and agriculturally significant animals. We identified and characterized a locus, which we annotated as rdxAB, encoding two nitroreductases. RdxA was found to be responsible for sensitivity to metronidazole (Mtz), a common therapeutic agent for another epsilonproteobacterium, Helicobacter pylori. Multiple, independently derived mutations in rdxA but not rdxB resulted in resistance to Mtz (Mtzr), suggesting that, unlike the case in H. pylori, Mtzr might not be a polygenic trait. Similarly, Mtzr C. jejuni was isolated after both in vitro and in vivo growth in the absence of selection that contained frameshift, point, insertion, or deletion mutations within rdxA, possibly revealing genetic variability of this trait in C. jejuni due to spontaneous DNA replication errors occurring during normal growth of the bacterium. Similar to previous findings with H. pylori RdxA, biochemical analysis of C. jejuni RdxA showed strong oxidase activity, with reduction of Mtz occurring only under anaerobic conditions. RdxB showed similar characteristics but at levels lower than those for RdxA. Genetic analysis confirmed that rdxA and rdxB are cotranscribed and induced during in vivo growth in the chick intestinal tract, but an absence of these genes did not strongly impair C. jejuni for commensal colonization. Further studies indicate that rdxA is a convenient locus for complementation of mutants in cis. Our work contributes to the growing knowledge of determinants contributing to susceptibility to Mtz (Mtzs) and supports previous observations of the fundamental differences in the activities of nitroreductases from epsilonproteobacteria.Nitroreductases form a large family of enzymes whose physiological roles have been implicated or proposed to function in diverse processes, such as the generation of nitrogen sources for metabolism, degradation of potentially toxic nitro compounds, vitamin and bioluminescence production, redox balancing, and oxidative stress responses (20, 31, 32, 35, 41, 43, 58). These enzymes can been subdivided into two main categories based on characteristics of their reductive processes, including the mechanism of electron transfer and sensitivity to oxygen. Type I (O2-insensitive) nitroreductases catalyze a sequential two- or three-step reduction of the nitro group on heterocyclic compounds via paired-electron transfer to produce either hydroxylamine or amino derivatives. Type II (O2-sensitive) nitroreductases catalyze a single-electron reduction of heterocyclic nitro compounds that is reversible in the presence of oxygen (40). Nitroreductases are common in bacteria, with a given bacterial species often containing multiple paralogs that presumably reduce different substrates. The genes encoding nitroreductases have received intense study due to their unusual nature in degrading or transforming xenobiotic chemicals. Consequently, these enzymes have become attractive candidates for bioremediation processes, and some are utilized in cancer chemotherapies (27, 49). However, the nitroreductases are also a puzzling class of enzymes, because the natural substrates for most remain unknown and these proteins likely did not evolve to exclusively manipulate xenobiotic compounds.Metronidazole (Mtz) has been used in multidrug therapy for Helicobacter pylori infections due to production of factors that convert this 5-nitroimidazole product to a toxic form (1, 45, 53). Therapeutic failure with Mtz has been predominantly associated with mutations occurring in one of two genes of H. pylori encoding the nitroreductases RdxA and FrxA. A previous biochemical study characterized the Mtz reductase activity of RdxA (37). Even though RdxA was capable of reducing other nitro compounds under aerobic conditions, the enzyme was unable to reduce Mtz. However, under anaerobic conditions, RdxA was shown to catalyze the reduction of Mtz, and its specific activity for this reaction was 60-fold greater than that of the NfsB nitroreductase of Escherichia coli under similar conditions. In addition, this work revealed that RdxA exhibited a potent NADPH oxidase activity not appreciated in other nitroreductases. Not only did this study demonstrate a direct reduction of Mtz by a nitroreductase, but results from this work implied that RdxA of H. pylori possessed novel biochemical properties relative to other nitroreductases.Like H. pylori, Campylobacter jejuni is a Gram-negative bacterium belonging to the epsilonproteobacteria class. C. jejuni is a common commensal bacterium of the intestinal tracts of wild and agriculturally significant animals, especially poultry. In contrast, C. jejuni causes acute diarrhea in humans, ranging from a mild enteritis to a bloody diarrheal syndrome, and is one of the most prevalent causes of food-borne gastritis (4, 5, 34, 38). Additionally, postinfectious sequelae can develop in a small percentage of patients following a C. jejuni infection. One major complication is Guillan-Barré syndrome, a temporary and partial paralysis of the peripheral nervous system (21).Many individuals with C. jejuni enteritis resolve the infection without therapeutic treatment. If antibiotics are administered, fluoroquinolones or macrolides, such as ciprofloxacin or erythromycin, are common drugs of choice, with therapeutic use of Mtz for C. jejuni infections being unconventional. However, Mtz-resistant (Mtzr) C. jejuni isolates have been recovered from humans and animals. In agriculture, 19 to 92% of C. jejuni isolates from avian species (including chickens and turkeys) and 6 to 20% of isolates from lambs, sheep, and cows were Mtzr (13, 47). One study also demonstrated that 62% of C. jejuni clinical isolates from humans were Mtzr (47). These data are curious, since these C. jejuni isolates would have likely developed Mtzr during infections in the absence of selection.Because susceptibility to Mtz (Mtzs) was also found in isolates associated with each host in studies described above and since C. jejuni is closely related to H. pylori, we hypothesized that C. jejuni may produce a nitroreductase to reduce Mtz to its toxic form, leading to Mtzs. In this report, we identify and characterize the gene required for Mtzs in C. jejuni. Mutations in this gene, encoding a putative nitroreductase, but not in a downstream paralog were linked to the development of Mtzr, indicating that Mtzr in C. jejuni appears to be linked to mutation of only one nitroreductase. Supporting our findings, we provide evidence that a proportion of Mtzr isolates of C. jejuni are due to spontaneous errors during DNA replication in the absence of Mtz exposure, resulting in a variety of mutations. Biochemical analysis of these C. jejuni nitroreductases demonstrated that these proteins had potent NADPH oxidase activity and could reduce Mtz under anaerobic conditions. These results, along with previous biochemical analysis of H. pylori RdxA (37), demonstrate that the nitroreductases of epsilonproteobacteria have unique characteristics in comparison to other bacterial counterparts. 相似文献
15.
Identification of rad27 Mutations That Confer Differential Defects in Mutation Avoidance, Repeat Tract Instability, and Flap Cleavage
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Yali Xie Yuan Liu Juan Lucas Argueso Leigh A. Henricksen Hui-I Kao Robert A. Bambara Eric Alani 《Molecular and cellular biology》2001,21(15):4889-4899
In eukaryotes, the nuclease activity of Rad27p (Fen1p) is thought to play a critical role in lagging-strand DNA replication by removing ribonucleotides present at the 5' ends of Okazaki fragments. Genetic analysis of Saccharomyces cerevisiae also has identified a role for Rad27p in mutation avoidance. rad27Delta mutants display both a repeat tract instability phenotype and a high rate of forward mutations to canavanine resistance that result primarily from duplications of DNA sequences that are flanked by direct repeats. These observations suggested that Rad27p activities in DNA replication and repair could be altered by mutagenesis and specifically assayed. To test this idea, we analyzed two rad27 alleles, rad27-G67S and rad27-G240D, that were identified in a screen for mutants that displayed repeat tract instability and mutator phenotypes. In chromosome stability assays, rad27-G67S strains displayed a higher frequency of repeat tract instabilities relative to CAN1 duplication events; in contrast, the rad27-G240D strains displayed the opposite phenotype. In biochemical assays, rad27-G67Sp displayed a weak exonuclease activity but significant single- and double-flap endonuclease activities. In contrast, rad27-G240Dp displayed a significant double-flap endonuclease activity but was devoid of exonuclease activity and showed only a weak single-flap endonuclease activity. Based on these observations, we hypothesize that the rad27-G67S mutant phenotypes resulted largely from specific defects in nuclease function that are important for degrading bubble intermediates, which can lead to DNA slippage events. The rad27-G240D mutant phenotypes were more difficult to reconcile to a specific biochemical defect, suggesting a structural role for Rad27p in DNA replication and repair. Since the mutants provide the means to relate nuclease functions in vitro to genetic characteristics in vivo, they are valuable tools for further analyses of the diverse biological roles of Rad27p. 相似文献
16.
Aziz Zaanan Koichi Okamoto Hisato Kawakami Khashayarsha Khazaie Shengbing Huang Frank A. Sinicrope 《The Journal of biological chemistry》2015,290(39):23838-23849
In colorectal cancers with oncogenic GTPase Kras (KRAS) mutations, inhibition of downstream MEK/ERK signaling has shown limited efficacy, in part because of failure to induce a robust apoptotic response. We studied the mechanism of apoptosis resistance in mutant KRAS cells and sought to enhance the efficacy of a KRAS-specific MEK/ERK inhibitor, GDC-0623. GDC-0623 was shown to potently up-regulate BIM expression to a greater extent versus other MEK inhibitors in isogenic KRAS HCT116 and mutant KRAS SW620 colon cancer cells. ERK silencing enhanced BIM up-regulation by GDC-0623 that was due to its loss of phosphorylation at Ser69, confirmed by a BIM-EL phosphorylation-defective mutant (S69G) that increased protein stability and blocked BIM induction. Despite BIM and BIK induction, the isogenic KRAS mutant versus wild-type cells remained resistant to GDC-0623-induced apoptosis, in part because of up-regulation of BCL-XL. KRAS knockdown by a doxycycline-inducible shRNA attenuated BCL-XL expression. BCL-XL knockdown sensitized KRAS mutant cells to GDC-0623-mediated apoptosis, as did the BH3 mimetic ABT-263. GDC-0623 plus ABT-263 induced a synergistic apoptosis by a mechanism that includes release of BIM from its sequestration by BCL-XL. Furthermore, mutant KRAS activated p-STAT3 (Tyr705) in the absence of IL-6 secretion, and STAT3 knockdown reduced BCL-XL mRNA and protein expression. These data suggest that BCL-XL up-regulation by STAT3 contributes to mutant KRAS-mediated apoptosis resistance. Such resistance can be overcome by potent BIM induction and concurrent BCL-XL antagonism to enable a synergistic apoptotic response. 相似文献
17.
Isolation and Characterization of Point Mutations in Mismatch Repair Genes That Destabilize Microsatellites in Yeast
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Elaine Ayres Sia Margaret Dominska Lela Stefanovic Thomas D. Petes 《Molecular and cellular biology》2001,21(23):8157-8167
The stability of simple repetitive DNA sequences (microsatellites) is a sensitive indicator of the ability of a cell to repair DNA mismatches. In a genetic screen for yeast mutants with elevated microsatellite instability, we identified strains containing point mutations in the yeast mismatch repair genes, MSH2, MSH3, MLH1, and PMS1. Some of these mutations conferred phenotypes significantly different from those of null mutations in these genes. One semidominant MSH2 mutation was identified. Finally we showed that strains heterozygous for null mutations of mismatch repair genes in diploid strains in yeast confer subtle defects in the repair of small DNA loops. 相似文献
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We have isolated and characterized extragenic suppressors of mutations in two different target genes that affect DNA replication in Salmonella typhimurium. Both the target and the suppressor genes are functional homologues of known replication genes of E. coli that were identified in intergeneric complementation tests. Our results point to interactions in vivo involving the dnaB and dnaC proteins in one case and the dnaQ and dnaE proteins in the other case. The suppressor mutations, which were isolated as derivatives of lambda-Salmonella in vitro recombinants, were detected by an adaptation of the red plaque complementation assay. This method was applicable even when the locus of suppressor mutations was not chosen in advance. 相似文献
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The site-specific recombinase IntI1 found in class 1 integrons catalyzes the excision and integration of mobile gene cassettes, especially antibiotic resistance gene cassettes, with a site-specific recombination system. The integron integrase belongs to the tyrosine recombinase (phage integrase) family. The members of this family, exemplified by the lambda integrase, do not share extensive amino acid identities, but three invariant residues are found within two regions, designated box I and box II. Two conserved residues are arginines, one located in box I and one in box II, while the other conserved residue is a tyrosine located at the C terminus of box II. We have analyzed the properties of IntI1 variants carrying point mutations at the three conserved residues of the family in in vivo recombination and in vitro substrate binding. We have made four proteins with mutations of the conserved box I arginine (R146) and three mutants with changes of the box II arginine (R280); of these, MBP-IntI1(R146K) and MBP-IntI1(R280K) bind to the attI1 site in vitro, but only MBP-IntI1(R280K) is able to excise cassettes in vivo. However, the efficiency of recombination and DNA binding for MBP-IntI1(R280K) is lower than that obtained with the wild-type MBP-IntI1. We have also made two proteins with mutations of the tyrosine residue (Y312), and both mutant proteins are similar to the wild-type fusion protein in their DNA-binding capacity but are unable to catalyze in vivo recombination.Integrons are DNA elements that capture genes, especially antibiotic resistance genes, by a site-specific recombination system (32). The recombination system consists of a DNA integrase (Int) and two types of recombination sites, attI and attC (59-base element). The integrase gene (int) is located in the 5′ conserved segment of the integron structure (Fig. (Fig.1)1) and is a member of the tyrosine recombinase family (1, 4, 13, 23, 24). Three types of integrases, sharing around 50% identity among themselves, have been identified; they define integron classes 1, 2, and 3 (30). The 5′ conserved segment found in class 1 integrons also contains a promoter region responsible for the expression of inserted cassettes (11, 21) and the recombination site attI1 (31). The 3′ conserved segment of the class 1 integrons includes an ethidium bromide resistance determinant (qacEΔ1), a sulfonamide resistance gene (sulI), an open reading frame (ORF5) of unknown function, and further sequences that differ from one integron to another (5, 6, 28). The 3′ conserved segment of class 2 integrons includes transposition genes (20) while that of class 3 integrons has not yet been studied (2). The variable region, located between the two conserved segments, usually contains antibiotic resistance genes; In0 contains no inserted genes while In21 possesses eight cassettes with ten genes (or ORFs) in this region (5, 16). These genes are part of mobile cassettes which include a recombination site, attC, that differs from one gene to another (18, 33). Incoming genes must be associated with an attC to be recognized by the integron integrase and are preferentially inserted at the recombination site attI1 (11). Cassettes are excised as circular intermediates and integrated at core sites by the action of the integrase (8–10). The core site, defined as GTTRRRY, makes up the 3′ end of attI1 and attC, with the crossover taking place between the G and the first T (19). Antibiotic selection pressure can reveal cassette rearrangements in which a given resistance is nearest the promoter and thus most strongly expressed (10). Open in a separate windowFIG. 1General structure of class 1 integrons. Cassettes are inserted in the integron variable region by a site-specific recombination mechanism. The attI1 site is shown by a black circle, core sites are represented by ovals, the attC site is indicated by a black rectangle, and promoters are denoted by P. intIl, integrase gene; qacEΔ1, antiseptic resistance gene; sulI, sulfonamide resistance gene; orf5, gene of unknown function.Site-specific recombination, unlike homologous recombination, is characterized by relatively short, specific DNA sequences and requires only limited homology of the recombining partners (12). Site-specific recombination is an entirely conservative process since all DNA strands that are broken (two per exchange site) are rejoined in a process that involves neither ATP nor DNA synthesis. Homology alignments of site-specific recombinases assign them to two families: the resolvase family, named after the TnpR proteins encoded by the transposons γδ and Tn3, and the integrase family. The integrase family includes over 140 members to date, but they are highly diversified proteins (13, 23). Members of this family, which include the well-studied λ integrase, recombine DNA duplexes by executing two consecutive strand breakage and rejoining steps and a topoisomerization of the reactants. The first pair of exchanges form a four-way Holliday junction and the second pair resolve the junction to complete the recombination. The integrase nucleophile is a conserved tyrosine that becomes associated with a phosphate group on DNA. The cleavage sites on each DNA duplex are separated by 6 to 8 bp with a 5′ stagger, and the tyrosine joins to the 3′ phosphate (17).The initial definition of the integrase family was based on comparisons of seven sequences, and three invariant residues were identified: an HXXR cluster and a Y residue (4). Alignment of 28 sequences identified a fourth invariant position, occupied by an arginine residue (1). These four conserved residues are found in two boxes located in the second half of the protein. A recent analysis has shown that the conserved histidine is present in 136 of the 147 members (93%); this residue is then not conserved in all members of the family (13). Another recent analysis has identified three patches of residues located around box I, which seem to be important in the secondary structure of these proteins (23). In this study, we analyzed the properties of several mutants of the conserved residues R146, R280, and Y312 of the integron integrase IntI1 in in vivo recombination and in vitro substrate binding.
Open in a separate windowaAkr, Apr, and Cmr, resistance to amikacin, ampicillin, and chloramphenicol. Open in a separate windowFIG. 3Electrophoresis of PCR products obtained with the pACYC184 primer pair and 100 ng of DNA preparations from overexpressed cultures on a 1% agarose gel. Lane 1, 1-kb DNA ladder (Gibco BRL); lane 2, DNA preparation of pLQ428-pLQ377 (R146E); lane 3, pLQ428-pLQ378 (R146I); lane 4, pLQ428-pLQ376 (R146K); lane 5, pLQ428-pLQ379 (R146V); lane 6, pLQ428-pLQ390 (R280E); lane 7, pLQ428-pLQ388 (R280G); lane 8, pLQ428-pLQ391 (R280K); lane 9, pLQ428-pLQ394 (Y312F); lane 10, pLQ428-pLQ393 (Y312S); lane 11, pLQ428-pLQ369 (wild type); lane 12, pLQ428-pMAL-c2 (MBP).The 2,499-bp PCR product was not obtained in the reaction containing the wild-type MBP-IntI1-expressing clone pLQ369 (Fig. (Fig.3,3, lane 11), indicating that there were no remaining full-length pLQ428 molecules. This shows that the wild-type fusion protein is very efficient in site-specific recombination and that all pLQ428 clones have undergone an excision of one or both cassettes. In this PCR, we observed two major bands of 1,341 and 889 bp. The 1,341-bp PCR product was digested with restriction enzymes to show that it represents a pLQ428 clone which has lost the aacA1-ORFG cassette (data not shown). The 889-bp band was also digested with restriction enzymes to show that it represents a pLQ428 clone which has lost both aacA1-ORFG and ORFH cassettes (data not shown). These two PCR products are also observed in the reaction containing the mutant clone pLQ391, which expresses the MBP-IntI1(R280K) fusion protein. This mutant protein is, however, less efficient than the wild-type protein, as seen by the intensity of the PCR products (Fig. (Fig.3,3, lane 8). We were not able to detect a PCR product of 2,047 bp, corresponding to the excision of the ORFH cassette alone; this is not surprising since this event has been shown in another study to be rare (16). It is possible to observe another band in pLQ428-pLQ391 (R280K) and pLQ428-pLQ369 (wild type) PCRs (Fig. (Fig.3,3, lanes 8 and 11); this PCR product is 1,100 bp long and probably represents a recombination event at a secondary site. Restriction enzyme digestions were done on this product, but we were unable to identify its origin. This product results from an event mediated by the integron integrase since it is seen only in reactions containing active proteins. An 1,800-bp PCR band is also present in the negative control and in all PCRs containing a mutant clone. This product appears to be nonspecific, and the fact that it is not seen in the PCR containing the pLQ428-pLQ369 (wild-type) clones probably results from the PCR being more favorable to smaller PCR products.
Open in a separate windowaLess efficient than the wild-type protein. bND, not determined. We have also made proteins with mutations at position 280; these were MBP-IntI1(R280E), MBP-IntI1(R280G), and MBP-IntI1(R280K). We found that the MBP-IntI1(R280K) mutant protein binds the attI1 site and excises integron cassettes with a lower efficiency than the wild-type MBP-IntI1, while MBP-IntI1(R280E) and MBP-IntI1(R280G) have completely lost both phenotypes. The Flp(R308K) mutant protein has been shown to bind DNA as well as the wild-type protein, but it recombines DNA with a lower efficiency than wild-type Flp (27). Another mutant protein of Flp [Flp(R308G)] has also been shown to bind DNA as well as the wild-type protein, but it was unable to catalyze in vivo or in vitro recombination (27). These results show that Flp(R308K) and MBP-IntI1(R280K) act similarly but that the other Flp mutant [Flp(R308G)] can bind DNA while the MBP-IntI1 mutant [MBP-IntI1(R280G)] cannot (Table (Table2).2). We also think that the positive charge of this residue is important for the binding of the recombinase to DNA, but Arg-280 does not seem to be implicated in the positioning of the tyrosine residue, since the MBP-IntI1(R280K) mutant protein can perform recombination.We found that MBP-IntI1(Y312S) and MBP-IntI1(Y312F) mutant proteins bind the attI1 site with the same efficiency as the wild-type protein but are not able to catalyze in vivo recombination. As expected, these results are the same as those obtained with the lambda integrase [λ(Y342F)], the XerC and XerD recombinases [XerC(Y275F) and XerD(Y279F)], and the Flp recombinases [Flp(Y343S) and Flp(Y343F)] (Table (Table2)2) (3, 22, 26, 29). The loss of the catalytic activity of the MBP-IntI1(Y312F) mutant protein is not surprising, since the hydroxyl group of the tyrosine, which is responsible for the nucleophilic attack of the DNA at the recombination site, is not present on the phenylalanine residue. The phenotype of MBP-IntI1(Y312S) indicates that the conformation of the tyrosine residue is important for the good activity of the recombinase, because even if the serine residue has a hydroxyl group, it is not able to catalyze recombination. These results indicate that the integron integrase IntI1 uses the hydroxyl group of the conserved tyrosine (Y312) to catalyze site-specific recombination, like other members of the family. However, in vitro recombination using this mutant protein needs to be done to confirm this.These results of point mutations show that mutations of the conserved arginines by nonpositively charged residues abolish substrate recognition, unlike the corresponding mutants of other members of the family. However, further mutational analysis, such as of residues around and in patch III, would be interesting, since only integron integrases contain more residues in this region than other members of the family (23). In vitro recombination assays with purified mutant proteins also need to be done in order to study thoroughly the mechanism of site-specific recombination in integrons. 相似文献
Construction of plasmids overexpressing mutant MBP-IntI1 fusion proteins.
The plasmids encoding various mutants of MBP-IntI1 were constructed by PCR using pLQ369 (50 ng) as a template (15). Two primer pairs, designed with the OLIGO software package (version 4.1; National Biosciences, Plymouth, Minn.), were used to construct each set of mutants. The R146 mutants were constructed with an XcmI-BamHI primer pair [IntI1(R146)-XcmI, 5′-TTCACCAGCTTCTGTATGGAACGGGCATG(A/G)(A/T)AATCAG-3′; IntI1(R146)-BamHI, 5′-CCGGATCCCTACCTCTCACT-3′], the R280 mutants were constructed with an NruI-XmnI primer pair [IntI1(R280)-NruI, 5′-AGCCGTCGCGAACGAGTGC(C/T)(C/T)GAGGG-3′; IntI1(R280)-XmnI, 5′-ACCCCTAATGAAGTGGTTCGTATCC-3′], and the Y312 mutants were constructed with a AatII-ScaI primer pair [IntI1(Y312)-AatII, 5′-ATTCCGACGTCTCTACTACGATGATTT(C/T)CACGC-3′; pLQ369-ScaI, 5′-ATGCTTTTCTGTGACTGGTG-3′] (restriction sites within primer sequences are underlined). PCR conditions were 10 min at 94°C, three cycles consisting of 45 s at 94°C, 45 s at 47°C, and 90 s at 72°C, 30 cycles consisting of 45 s at 94°C, 45 s at 60°C (50°C for Y312 mutants), and 90 s at 72°C, and a final elongation step of 10 min at 72°C. The XcmI, NruI, and AatII primers were degenerate in one or two positions, so that a single primer could give all mutants. Mutant PCR fragments were digested and cloned directly into pLQ369 digested with the same enzymes, except for the R146 mutant fragments that were subcloned into pLQ364 at first. New mutant PCR fragments were then amplified on these subclones, using IntI1(R146)-BamHI and IntI1(R280)-XmnI primers. These mutant PCR fragments were cleaved with BamHI and XmnI, and the resulting fragments were cloned into pLQ369. This avoids the necessity of partial digestion of pLQ369 with XcmI. Mutant clones were digested with restriction endonucleases and sequenced to determine the mutation.In vivo recombination.
Mutant MBP-IntI1 clones were introduced into Escherichia coli TB1 {F′ araΔ(lac-proAB) rpsL (Strr) [φ80dlacΔ(lacZ)M15] hsdR(rK−mK−)} containing pLQ428 by transformation (Fig. (Fig.22 and Table Table1).1). E. coli TB1 cells containing pLQ428 and one of the MBP-IntI1 mutants were grown at 37°C for 3 h in Luria-Bertani medium. Excision of the aacA1-ORFG and/or ORFH cassettes was induced by the overexpression of the malE-intI1 gene by using 0.3 mM isopropyl-β-d-thiogalactopyranoside (IPTG; Sigma Chemical Co.) and by incubation at 37°C for another 3 h. Cell culture was done in the presence of 50 μg of ampicillin per ml, 15 μg of amikacin per ml, and 50 μg of chloramphenicol per ml. Plasmid DNA was then prepared from 5-ml cultures with the Perfect Prep DNA extraction kit (Mandel Corporation). In order to determine the capacity of mutant MBP-IntI1 proteins to excise aacA1-ORFG and/or ORFH cassettes of In21, we used PCR primers pACYC184-5′ (5′-TGTAGCACCTGAAGTCAGCC-3′) and pACYC184-3′ (5′-ATACCCACGCCGAAACAAG-3′) (Fig. (Fig.2,2, primers 1 and 2) to detect the reduction of pLQ428 length. PCR conditions were 10 min at 94°C, 30 cycles consisting of 1 min at 94°C, 1 min at 60°C, and 5 min at 72°C, and a final elongation step of 10 min at 72°C. A major PCR fragment can be seen in each lane containing a DNA preparation from a mutant clone (Fig. (Fig.3,3, lanes 2 to 9). This band is 2,499 bp long and, as determined by restriction enzyme digestions, represents the pLQ428 clone without any cassette excision (data not shown). This band is also observed in the negative control, which is the pMAL-c2 vector without any gene fused to malE (Fig. (Fig.3,3, lane 12). Open in a separate windowFIG. 2Representation of plasmids used in this study. The positions of the three invariant residues of the integrase family are indicated, along with restriction sites used to construct mutant proteins. Core sites are represented by black circles, and attCs are shown by white boxes. The numbered arrows represent the PCR primers used to detect excision events, pACYC184-5′ (1) and pACYC184-3′ (2). bla, gene encoding β-lactamase; cat, gene encoding chloramphenicol acetyltransferase; intIl, gene encoding the integron integrase (IntI1); malE, gene encoding the maltose binding protein (MBP); ori, origin of replication; Ptac, tac promoter; Ptet, tetracycline promoter. Only relevant restriction sites are indicated.TABLE 1
Plasmids used in this studyPlasmid | Characteristic(s)a | Reference or source |
---|---|---|
pLQ363 | 2,190-bp EcoRI-HincII fragment of pLQ161 cloned in pLQ402 (Apr) | 16 |
pLQ364 | 1,027-bp NcoI-BamHI PCR fragment amplified on pLQ860 and cloned in pET-3d (Apr) | This study |
pLQ369 | 1,019-bp NdeI-BamHI PCR fragment modified to create a blunt-end 5′-ATG and cloned in pMAL-c2 cut with XmnI-BamHI (Apr) | 15 |
pLQ376 | pLQ369 MBP-IntI1(R146K) (Apr) | This study |
pLQ377 | pLQ369 MBP-IntI1(R146E) (Apr) | This study |
pLQ378 | pLQ369 MBP-IntI1(R146I) (Apr) | This study |
pLQ379 | pLQ369 MBP-IntI1(R146V) (Apr) | This study |
pLQ388 | pLQ369 MBP-IntI1(R280G) (Apr) | This study |
pLQ390 | pLQ369 MBP-IntI1(R280E) (Apr) | This study |
pLQ391 | pLQ369 MBP-IntI1(R280K) (Apr) | This study |
pLQ393 | pLQ369 MBP-IntI1(Y312S) (Apr) | This study |
pLQ394 | pLQ369 MBP-IntI1(Y312F) (Apr) | This study |
pLQ428 | 2,133-bp EcoRI (filled in)-BglII fragment of pLQ363 cloned in pACYC184 cut with EcoRV-BamHI (Akr Cmr) | This study |
pLQ860 | 2,900-bp BamHI fragment of pVS1 cloned in pTZ19R (Apr Sulr) | 5 |
In vitro substrate binding.
The experiments described above demonstrate that only one of our mutants of IntI1 protein is able to catalyze in vivo recombination. Can all mutant proteins recognize and bind to the IntI1 recombination site in a manner similar to the wild-type protein? To investigate this question, we used purified fusion proteins and a gel retardation assay with the complete attI1 site (5′ site) of the integron. MBP-IntI1 fusion proteins were purified as suggested by New England Biolabs. The concentration of the purified fusion protein was determined by using the Bradford protein assay (Bio-Rad). The protein solution was then made 20% in glycerol and stored at −80°C. The purity of MBP-IntI1 was evaluated as >90% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (data not shown). Binding reactions were done with labeled 5′-site DNA fragments (20,000 cpm, 0.25 pmol), incubated with different concentrations of MBP-IntI1 in a 10-μl volume containing 10 mM HEPES (K+, pH 8.0), 60 mM KCl, 4 mM MgCl2, 100 μM EDTA (pH 8.0), 100 μg of bovine serum albumin per ml, 250 μM dithiothreitol, 100 ng of poly(dI-dC), and 10% glycerol. Reaction mixtures were incubated at room temperature for 15 min prior to electrophoresis through 4 or 5% prerun, nondenaturing polyacrylamide gels buffered with 0.5× Tris-borate-EDTA. Dried gels were subjected to autoradiography. The wild-type fusion protein and native IntI1 were shown to lead to the same four distinct complexes (I, II, III, and IV) with this DNA substrate (Fig. (Fig.4)4) (15). These complexes represent the binding of four IntI1 molecules to four different sites in the attI1 site (15). Figure Figure44 shows results obtained with nine mutants of the MBP-IntI1 fusion protein. We observed that MBP-IntI1(R146E), MBP-IntI1(R146I), and MBP-IntI1(R146V) lost their ability to bind to the attI1 site, as no complexes are seen in the gel retardation experiment (Fig. (Fig.4A).4A). However, MBP-IntI1(R146K) formed four IntI1-DNA complexes with the 5′ site DNA fragment. The band pattern and the intensity observed with this mutant protein are similar to those observed with the wild-type protein, suggesting that MBP-IntI1(R146K) and MBP-IntI1 bind DNA with similar affinities. Open in a separate windowFIG. 4Binding of mutant MBP-IntI1 fusion proteins purified from E. coli TB1 to the 5′-site DNA fragment containing the complete attI1 site of the In2 integron (from nucleotide −96 to nucleotide +71, relative to the G residue of the core site as position 0). (A) MBP-IntI1(R146) mutants; (B) MBP-IntI1(R280) mutants; (C) MBP-IntI1(Y312) mutants. A purified labeled fragment was incubated with different concentrations of mutant fusion proteins. Free DNA (F) and protein-DNA complexes (I, II, III, and IV) were separated on 4 or 5% polyacrylamide gels and are indicated by arrows. Lanes 1, free DNA; lanes 2 through 7, purified fusion protein at 250, 375, 500, 12.5, 37.5, and 62.5 nM, respectively. The wild-type (WT) lanes in panel C were from a separate gel.Competition with a specific fragment with a 30-fold excess of unlabeled DNA competed away all four complexes, while a 100-fold excess of a nonspecific unlabeled DNA fragment did not compete away any complexes, indicating their specificity (data not shown) (15). We observed that MBP-IntI1(R280G) and MBP-IntI1(R280E) lost their ability to bind the 5′-site DNA fragment, while the MBP-IntI1(R280K) could still bind the attI1 site (Fig. (Fig.4B).4B). However, the band pattern obtained with this mutant protein is weaker than that obtained with the wild-type integrase. For example, at a protein concentration of 250 nM MBP-IntI1(R280K) (lane 2), we observed the formation of complexes I, II, and III, with a stronger intensity for the fastest-migrating complexes, while the intensity of the fourth complex was very weak. At the same concentration of the wild-type protein, we observed the formation of all four complexes, with a stronger intensity for the slowest-migrating complexes and no unbound DNA. These results show that MBP-IntI1(R280K) binds the attI1 site with a lower affinity than the wild-type fusion protein. As shown in Fig. Fig.4C,4C, both MBP-IntI1(Y312F) and MBP-IntI1(Y312S) lead to the formation of four complexes that migrate similarity to those obtained with wild-type MBP-IntI1, as judged by the gel migration of these complexes. The band pattern observed shows that the binding affinity of these mutant proteins is the same as or even better than that of the wild-type protein.Relationships with other members of the family.
We found that MBP-IntI1 recombinase in which Arg-146 has been changed to lysine [MBP-IntI1(R146K)] by PCR mutagenesis cannot excise cassettes but can bind to the attI1 site with the same efficiency as the wild-type fusion protein. However, MBP-IntI1(R146I), MBP-IntI1(R146E), and MBP-IntI1(R146V) mutant proteins have completely lost both phenotypes. These findings are different from those for other members of the family. The only mutant protein of the lambda integrase at this residue [λ(R212Q)] binds the core site partially and is not able to catalyze in vivo or in vitro recombination (22). Mutants of the Cre recombinase with a change at this residue [Cre(R173K)] bind DNA as well as the wild-type protein but cannot catalyze in vivo or in vitro recombination (1). Mutants of Flp [Flp(R191K) and Flp(R191E)] bind FRT recombination sites as well as the wild-type protein but cannot carry out in vivo or in vitro recombination, except for the Flp(R191K) protein, which has shown slight activity in in vivo recombination (Table (Table2)2) (7, 14, 25). Therefore, the Cre(R173K) and Flp(R146K) mutants have the same phenotype as the MBP-IntI1(R146K) protein. However, the Flp(R191E) mutant protein shows efficient DNA binding while MBP-IntI1(R146E) does not bind to the attI1 site. We interpret these results according to the charge of the Arg-146 residue. The positively charged side chain of this residue makes contact with the DNA, which is negatively charged. This contact is probably important for the good conformation of the protein molecule in positioning the tyrosine residue to perform recombination. When this residue is exchanged for a lysine, DNA contacts are still able to take place because of the charge of the residue, but the side chain is smaller and the lysine is probably not able to position the tyrosine to catalyze recombination. We think that the charge of this residue is very important in the formation of DNA-protein complexes in the integron system, since all other MBP-IntI1 mutants tested are unable to bind DNA. This observation differs from those for Flp, because even when the wild-type residue was replaced by a negatively charged one, it could still bind DNA as well as the wild-type protein (Table (Table2).2).TABLE 2
Mutational analysis of IntI1 and corresponding residues of other recombinases from the Int familyRecombinase | Mutation | DNA binding | Recombination | Reference(s) |
---|---|---|---|---|
λInt | R212Q | Yesa | No | 22 |
λInt | Y342F | Yes | No | 22, 26 |
Flp | R191E | Yes | No | 7 |
Flp | R191K | Yes | Yes | 7, 14 |
Flp | R308G | Yes | No | 27 |
Flp | R308K | Yes | Yesa | 27 |
Flp | Y343F | Yes | No | 29 |
Flp | Y343S | Yes | No | 29 |
Cre | R173K | Yes | No | 1 |
P2 | R272K | NDb | No | 23 |
XerC | Y275F | Yes | No | 3 |
XerD | Y279F | Yes | No | 3 |
IntI1 | R146E | No | No | This study |
IntI1 | R146I | No | No | This study |
IntI1 | R146K | Yes | No | This study |
IntI1 | R146V | No | No | This study |
IntI1 | R280E | No | No | This study |
IntI1 | R280G | No | No | This study |
IntI1 | R280K | Yes | Yesa | This study |
IntI1 | Y312F | Yes | No | This study |
IntI1 | Y312S | Yes | No | This study |
20.
Biology Bulletin - In this study, physiological and molecular changes caused by different concentrations of tetraconazole were investigated in barley. The analyzes were carried out 7 days after the... 相似文献