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Pseudomonas sp. strains, isolated from soil, utilized toluene as their sole carbon source through ameta cleavage pathway. Strains metabolizing toluene through anortho cleavage pathway were selected from the wild typemeta strain. Theortho pathway strains were subjected to chemostat selection to obtain a fast-growing strain with doubling time reduced from 14 to 1.2 h. Benzoale and antibiotics enrichment selection procedures were utilized to select a blocked mutant. The blocked mutant grew on acetate as its sole carbon source and oxidatively converted toluene tocis, cis-muconic acid. Double-blocked and muconate-permeable mutants were also selected to reduce reversion frequency and to enhance muconic acid production. In shake-flask experiments, muconic acid at 3.5 g/l was obtained after 2 days of fermentation. In a 14 l fermenter, muconic acid was produced at 45 g/l in 4 days of controlled fed-batch fermentation. The oxidative bioconversion process was also demonstrated in a 1500 l fermenter.
Résumé Des souches dePseudomonas sp., isolées du sol, ont utilisé le toluène comme seule source de carbone par la vole de la rupture de cycle enmeta. On a sélectionné des souches métabolisant le toluène par la voie de la rupture de cycle enortho, à partir de la souche sauvage de typemeta. Les souches de la voieortho ont été soumises à la sélection en chémostat pour obtenir des souches à croissance rapide dont le temps de doublement est rédult de 14 à 1.2 h. Les procédures de sélection par enrichissement sur benzoale et antibiotiques ont été utilisées pour sélectionner un mutant bloqué. Le mutant bloqué croît sur acétate comme seule source de carbone et convertit le toluène par voie oxydative en acidecis,cis-muconique. On a également sélectionné des mutants doublement bloqués et perméables au muconate pour réduire la lréquence de réversion et pour augmenter la production d'acide muconique. En expérimentation en flacons agités, on a obtenu 3.5 g/l d'acide muconique après 2 jours de fermentation. En fermentuer de 14 l, on a produit 45 g/l d'acide muconique en 4 jours de fermentation contrôlée en milieu non renouvelé à allmentation étagée. Le processus de bioconversion oxydative a également été démontré en fermenteur de 1500 l.相似文献
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Protein Tyrosine Kinase Activity and Its Endogenous Substrates in Rat Brain: A Subcellular and Regional Survey 总被引:21,自引:11,他引:10
The rat CNS contains high levels of tyrosine-specific protein kinases that specifically phosphorylate the tyrosine-containing synthetic peptide poly(Glu80,Tyr20). The phosphorylation of this peptide is rapid and occurs with normal Michaelis-Menten kinetics. Using this peptide to assay for enzyme activity, we have measured the protein tyrosine kinase activity in homogenates from various regions of rat CNS. A marked regional distribution pattern was observed, with high activity present in cerebellum, hippocampus, olfactory bulb, and pyriform cortex, and low activity in the pons/medulla and spinal cord. The distribution of protein tyrosine kinase activity was examined in various subcellular fractions of rat forebrain. The majority of the activity was associated with the particulate fractions, with enrichment in the crude microsomal (P3) and crude synaptic vesicle (LP2) fractions. Moreover, the subcellular distribution of pp60csrc, a well-characterized protein tyrosine kinase, was examined by immunoblot analysis using an affinity-purified antibody specific for pp60csrc. The subcellular distribution of pp60csrc paralleled the overall protein tyrosine kinase activity. In addition, using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were demonstrated on immunoblots of homogenates from the various regions and the subcellular fractions. The immunoblots revealed numerous phosphotyrosine-containing proteins that were present in many of the CNS regions examined and were associated with specific subcellular fractions. The differences in tyrosine-specific protein kinase activity, and in phosphotyrosine-containing proteins, observed in various regional areas and subcellular fractions may reflect specific functional roles for protein tyrosine kinase activity in mammalian brain. 相似文献
7.
Cell-specific expression of pyruvate, pi dikinase : in situ mRNA hybridization and immunolocalization labeling of protein in wheat seed 总被引:2,自引:0,他引:2
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Pyruvate, Pi dikinase (PPDK) is a key enzyme in the C4 photosynthetic pathway. However, its metabolic role in C3 plants remains uncertain. Northern blot analyses of PPDK mRNAs from wheat leaves and seeds probed with maize PPDK cDNA indicates the presence of organ-specific mRNAs. Immunofluorescent labeling of protein in wheat seed demonstrate that the PPDK polypeptide and the ribulose-1, 5-bisphosphate carboxylase small subunit polypeptide are localized predominantly in the aleurone layer and the chlorophyllous pericarp tissue, respectively. This differential distribution of the two polypeptides in wheat seed is paralleled by the differential localization of the their mRNAs as revealed by in situ hybridization. These results suggest a distinct role of cytoplasmic PPDK in seeds, which is different from the well established role in C4 photosynthesis. 相似文献
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Y. Shaaltiel N. -H. Chua S. Gepstein J. Gressel 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1988,75(6):850-856
Summary The genetics of paraquat-resistance in Conyza bonariensis was studied. Reciprocal crosses were prepared between resistant and sensitive individuals. The enzymes of the pathway that detoxifies superoxide to innocuous oxygen species involved in resistance were evaluated in the F1 and F2 generations. All F1 plants were as resistant as the resistant parent, irrespective of parental sex, demonstrating dominance and excluding maternal inheritance. The activities of superoxide-dismutase, ascorbate-peroxidase and glutathione-reductase in the F1 were constitutively as high as in the resistant parent. Resistance in the F2 generation was distributed in a 31 ratio (resistant to sensitive). Leaves from F2 plants were removed for a resistance assay and enzyme immuno-assays of single plants were performed. The high levels of superoxide-dismutase and glutathione-reductase, the two enzymes for which antibodies were available, were similar in resistant individuals to the levels in the resistant parent; the levels were low in the susceptible individuals. These results indicate either a very tight linkage, or more probably, that one dominant nuclear gene controls resistance by pleiotropically controlling the levels of enzymes of the activeoxygen detoxification pathway. 相似文献
9.
Radiolabelled calmodulin has previously been used to screen cDNA expression libraries to isolate calmodulin-binding proteins.
We have modified this technique for the isolation of plant calmodulin-binding proteins. [35S]-methionine was used instead of the inorganic [35S]-sulfate, or125I used in previous methods. In addition, theE. coli pET expression system was chosen to obtain high levels of recombinant calmodulin at the time of labelling. The procedure
thus takes into account both the specific activity of the probe and the amount of protein necessary for screening a large
number of filters. Here we describe in detail a procedure for the production and purification of [35S]-recombinant calmodulin and the use of the radiolabelled protein as a probe to screen plant cDNA expression libraries. The
[35S]-labeled calmodulin probe easily detects the λICM-1 phage encoding a partial mouse calmodulin-dependent protein kinase II
that was previously isolated using a [125I]-calmodulin probe (Sikela and Hahn, 1987). Subsequently, a tobacco root cDNA expression library was screened and a positive
clone encoding a calcium-dependent calmodulin-binding protein was isolated. 相似文献
10.
Rong-Xiang Fang Zhen Pang Dong-Ming Gao Ke-Qiang Mang Nam-Hai Chua 《Plant molecular biology》1991,17(6):1255-1257