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121.
Molecular cloning of cDNA for human prostatic acid phosphatase 总被引:1,自引:0,他引:1
A human liver cDNA library in λgt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, λ Hap21 and λ Hap22 were further characterized: clone λHap21 contained a 0.8-kb cDNA insert and clone λHap22 a 1.8–2.0-kb insert. XbaI digestion of λHap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone 1 Hap22 contained all the genes carried by λ gt11(lac 5cI857nin 5Sam 100) and the 2-kb insert. An Escherichia coli(λHap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(λHap22) lysate revealed that the non-induced λHap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-β-galactosidase and was produced only upon induction with IPTG. These results indicated that AHap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity. 相似文献
122.
Ahmed Landoulsi Patrick Hughes Renee Kern Masamichi Kohiyama 《Molecular & general genetics : MGG》1989,216(2-3):217-223
Summary Plasmid DNA containing the replication origin of the Escherichia coli chromosome (oriC) has been shown to be inefficient as a template for DNA synthesis in vitro when isolated from dam mutants. here, we extend this study to hemimethylated oriC plasmids and to replication in dam-3 mutant enzyme extracts. The results show that: (1) hemimethylated oriC plasmids replicate with the same low efficiency as nonmethylated DNA; (2) DNA synthesis starts at oriC regardless of the methylated state of the template; (3) replication in dam-3 enzyme extracts is inefficient because this strain is deficient in DnaA protein; and (4) consistent with this observation, the copy number of the oriC plasmid pFH271 is reduced in the dam-3 mutant. However, we have found that low DnaA protein levels in dam-3 mutants are not sufficient to explain the reduced transformation efficiency of oriC plasmids. We suggest that there must exist in vivo inhibitory factors not present or present in low quantities in vitro which specifically recognize the hemimethylated or nonmethylated forms of the oric region. 相似文献
123.
Kazuyuki Tao Kozu Makino Shuji Yonei Atsuo Nakata Hideo Shinagawa 《Molecular & general genetics : MGG》1989,218(3):371-376
Summary Treatment of Escherichia coli and Salmonella typhimurium cells with a low dose of hydrogen peroxide induces expression of a large number of genes, and confers resistance to oxidative stresses. The oxyR gene encodes a positive regulatory protein for a subset of these genes involved in the defense against oxidative damage. We cloned a DNA fragment that contains the E. coli oxyR region on a plasmid vector, and analyzed the nucleotide sequence of the gene. The amino acid sequence of OxyR protein, deduced from the nucleotide sequence, shows a high degree of homology to the sequences of a number of bacterial activator proteins including LysR, cysB, IlvY, MetR and NodD. The product of the oxyR gene identified by the maxicell procedure was a 34 kDa protein, which agrees with the size predicted from the nucleotide sequence of the gene. 相似文献
124.
Hiroshi Taniguchi Takuya Tokida Hiroshi Fujita Hiraku Itikawa 《Molecular & general genetics : MGG》1989,217(2-3):317-323
Summary IndnaK7(Ts) mutant cells, scission of DNA strands occurred after temperature shift up. When cells at 30°C were labeled with [3H]-thymidine and then shifted to 46° or 49°C for 20 min, the profiles of sedimentation of thier cellular DNA in an alkaline
sucrose gradient revealed a decrease in the size of DNA to a quarter of that at 30°C in the mutant, but not in wild-type cells.
The level of manganese-containing superoxide dismutase (MnSOD) in the mutant was about twice that in wild-type cells, even
at the permissive temperature, implying increased production of superoxide radical anion, which may cleave DNA strands directly
or indirectly in the mutant. Moderate increase in the MnSOD level on temperature shift up was observed in both strains. These
results indicated that some components of the DnaK protein participate in protection of cellular membrane functions from thermal
damage resulting from elevated production of the superoxide anion radical. 相似文献
125.
A series of eight experiments was conducted using large pots to (1) find the most effective date, site, concentration of K-solution
and K-salt for foliar K-fertilization of maize plants (Zea mays, L.) grown with sufficient K-supply in soil, (2) explain why maize responded to the K-treatment, and (3) examine the influence
of various levels of N and P supplies on the effectiveness of K-fertilizer via the leaves.
A single spraying on sweet maize and field maize on any day between 50% tasselling date to 10 days after tasselling shortened
maturity date, increased grain yield, stover yield, grain-stover ratio, absorption of N, P, K, Ca and Mg, sweetness of young
grain (of sweet maize), and crude protein content of grain. However spraying on the third day after 50% tasselling was most
effective. The second application later than 7 days after the 50% tasselling date suppressed the effects of spraying on the
most effective date. In application of many repetitive sprayings covering the most effective date, a spraying program with
late spraying could reduce grain yield. KNO3, 2.5% KNO3-solution, and applications on all aerial parts were found to be the most effective. Increases in grain yield for spraying
on all aerial parts, spraying on ear leaf only, spraying on all leaves above ear leaf and applying K to soil were 74%, 51%,
41% and 23%, respectively. The foliar K-fertilization affected maize by stimulating chlorophyll synthesis and not by increasing
leaf area. A balance in N and K supplies was determined to be effective for the K-fertilization. 相似文献
126.
Developing cotyledons of the common bean, Phaseolus vulgaris L., transport within their secretory system (endoplasmic reticulum and Golgi apparatus) the abundant vacuolar proteins, phaseolin and phytohemagglutinin. To identify proteins that may play a role in vacuolar targeting, we treated cotyledon microsomal fractions with a bifunctional crosslinking reagent, dithiobis(succinimidyl propionate), isolated protein complexes with antibodies to phaseolin and phytohemagglutinin, and analysed the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis. This allowed us to identify a protein of Mr=9000 (P-9000) that was crosslinked to both phaseolin and phytohemagglutinin. P-900 is abundantly present in the endoplasmic reticulum. The aminoterminus of P-9000 shows extensive sequence identity with the amino-terminus of PA1 (Mr=11 000), a cysteine-rich albumin whose processing products accumulate in the vacuoles of pea (Pisum sativum L.) cotyledons. Like PA1, P-9000 is synthesized as a pre-proprotein that is posttranslationally processed into smaller polypeptides. The possible functions of P-9000 are discussed.Abbreviations DSP
dithiobis(succinimidyl propionate)
- EDTA
ethylenediaminetetraacetic acid
- ER
endoplasmic reticulum
- kDa
kilodalton
- Mr
relative molecular mass
- PHA
phytohemagglutinin
- SDS
sodium dodecylsulfate
- PAGE
polyacrylamide gel electrophoresis 相似文献
127.
128.
Total polyadenylated RNA from ripening or germinating Ricinus communis L. endosperm was translated in rabbit reticulocyte lysate in the absence or presence of canine pancreatic microsomes. The products were immunoprecipitated using antibodies raised againts Triton X-114-extracted integral membrane proteins of protein bodies or glyoxysomes. While the proteins of proteinbody membranes were found to insert co-translationally into added microsomes, this was not observed in the case of glyoxysomal proteins. This observation was confirmed using antibodies raised against a purified glyoxysome membrane protein, alkaline lipase. These results indicate that different routes exist for the insertion of membrane proteins into the two organelles. In both cases membrane-protein insertion does not appear to be accompanied by proteolytic processing.Abbreviations anti-PB
antiserum to integral protein-body membrane proteins
- anti-G
antiserum to integral glyoxysomal membrane proteins
- anti-L
antiserum to alkaline lipase
- ER
endoplasmic reticulum
- Mr
relative molecular mass
- mRNA
poly(A)-rich messenger RNA
- PAGE
polyacrylamide gel electrophoresis
- poly(A)
polyadenylic acid
- SDS
sodium dodecyl sulphate 相似文献
129.
When pea (Pisum sativum L.) embryos were cultured on low osmotica, with or without added abscisic acid (ABA), there was very little change in the
total mRNA translation products resolved by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).
The only marked alteration was an increase in production of two low-molecular-weight proteins. The purification and partial
characterisation of these two ABA-responsive seed proteins (ABR17 and ABR18) is described. Both proteins were purified to
homoeneity, as judged by SDS-PAGE, from embryos cultured in the presence of ABA. Antisera were raised against both proteins.
Each serum cross-reacted with the other protein, indicating that the proteins are closely related. Their apparent molecular
masses (Mrs) were estimated to be 17200 (ABR17) and 18100 (ABR18) by SDS-PAGE, and 26000 by gel filtration. Both proteins were heterogeneous
on isoelectric focusing. Neither protein was detected (by immunoblotting or immunoprecipitation of cell-free translation products)
in embryos grown in vivo at early to mid-development stages but both were present in embryos late in development. These proteins
appear to be produced late in seed development but are capable of being induced early in development by culturing embryos
in vitro and are markedly enhanced by ABA. 相似文献
130.
Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin,fusicoccin 总被引:1,自引:0,他引:1
The high-affinity fusicoccin-binding protein (FCBP) was solubilized from plasma-membrane vesicles prepared from leaves of Vicia faba L. by aqueous two-phase partitioning. Conditions for the solubilization of intact FCBP-radioligand complexes were worked out. About 60–70% of the complexes can be solubilized with 50–60 mM nonanoyl-N-methylglucamide in the presence of 1 mg· ml-1 soybean phosphatidylcholine, type IV S, and 20% (v/v) glycerol at pH 5.5. The slow dissociation of the radioligand, 9-nor-fusicoccin-8-alcohol-[3H] from the FCBP at low temperatures permits the purification of FCBP-radioligand complexes at 4–10° C by fast protein liquid chromatography on anion-exchange and gel permeation columns. The FCBP, extracted from plasma membranes with cholate and chromatographed in the presence of this detergent, gave an apparent molecular mass (Mr) of 80±20 kDa on gel permeation columns under the conditions used. By comparison of the elution profiles of the fraction most enriched in FCBP-radioligand complexes with polypeptide patterns obtained on sodium dodecyl sulfate-polyacrylamide gels, a polypeptide with an Mr of approx. 34kDa co-separated with the radioactivity profile. A second, faint band of approx. 31 kDa was sometimes also observed co-electrophoresing. Photoaffinity labeling of plasma-membrane vesicles with the new compound 9-nor-8[(3,5-[3H]-4-azidobenzoy)ethylenediamine]-fusicoccin ([3H]ABE-FC) and subsequent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis labeled a single band with an Mr of 35±1 kDa. Labeling in this band was strongly reduced when the membranes were incubated with [3H]ABE-FC in the presence of 0.1–1 M fusicoccin. From our data, we conclude (i) that the 34-35-kDa polypeptide represents the FCBP and (ii) that in detergent extracts of plasma membranes this polypeptide is probably present as a di- or trimeric structure.Abbreviations ABE-FC
[(4-azidobenzoyl)-ethylenediamine]-fusicoccin
- ABE-NHS
(4-azidobenzoyl)-N-hydroxysuccinimide ester
- FC
fusicoccin
- FCBP
fusicoccin-binding protein
- FCol
9-norfusicoccin-8-alcohol
- MAB
monoclonal antibody
- Mega-9(10)
nonanoyl(decanoyl)-N-methylglucamide
- Mr
apparent molecular mass
- PMSF
phenylmethyl-sulfonyl fluoride
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- TCA
trichloroacetic acid
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol 相似文献