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91.
Cytoplasmic determinants that specify the fate of endoderm, muscle and epidermis cells are known to be localized in specific
areas of fertilized eggs of ascidians. The presence of such cytoplasmic determinants in unfertilized eggs was demonstrated
in previous studies, but no information has yet been proved about their distribution. To investigate the distribution of cytoplasmic
determinants in unfertilized eggs, we devised a method for distinguishing the polarity of unfertilized eggs using vital staining
and we performed cytoplasmic-transfer experiments by fusing blastomeres and cytoplasmic fragments from various identified
regions of unfertilized eggs. Cytoplasmic fragments, that contained cortical and subcortical material, from five different
positions along the animal-vegetal axis were prepared, and they were fused with a4.2 (presumptive-epidermis) or A4.1 (non-epidermis)
blastomeres. The ectopic development of endoderm, muscle and epidermis cells that was promoted by the transplanted cytoplasm
was assessed by examining the expression of alkaline phosphatase (ALP), myosin and epidermis-specific antigen, respectively.
Differentiation of endoderm and muscle was observed at higher frequencies as cytoplasmic fragments closer to the vegetal pole
were transplanted. Conversely, formation of epidermis was observed at higher frequencies as cytoplasmic fragments closer to
the animal pole were transplanted. The results suggest that, in cortical and subcortical regions of unfertilized ascidian
eggs, endoderm and muscle determinants are widely distributed along a gradient, with maximum activity at the vegetal pole,
whilst epidermis determinants are also distributed along a gradient but with maximum activity at the animal pole.
Recieved: 10 June 1996 / Accepted: 12 September 1996 相似文献
92.
Larkin PJ Gibson JM Mathesius U Weinman JJ Gartner E Hall E Tanner GJ Rolfe BG Djordjevic MA 《Transgenic research》1996,5(5):325-335
We report an improved method for white clover (Trifolium repens) transformation usingAgrobacterium tumefaciens. High efficiencies of transgenic plant production were achieved using cotyledons of imbibed mature seed. Transgenic plants were recovered routinely from over 50% of treated cotyledons. Thebar gene and phosphinothricin selection was shown to be a more effective selection system thannptII (kanamycin selection) oraadA (spectinomycin selection). White clover was transformed with the soybean auxin responsive promoter, GH3, fused to the GUS gene (-glucuronidase) to study the involvement of auxin in root development. Analysis of 12 independent transgenic plants showed that the location and pattern of GUS expression was consistent but the levels of expression varied. The level of GH3:GUS expression in untreated plants was enhanced specifically by auxin-treatment but the pattern of expression was not altered. Expression of the GH3:GUS fusion was not enhanced by other phytohormones. A consistent GUS expression pattern was evident in untreated plants presumably in response to endogenous auxin or to differences in auxin sensitivity in various clover tissues. In untreated plants, the pattern of GH3:GUS expression was consistent with physiological responses which are regarded as being auxin-mediated. For the first time it is shown that localised spots of GH3:GUS activity occurred in root cortical tissue opposite the sites where lateral roots subsequently were initiated. Newly formed lateral roots grew towards and through these islands of GH3:GUS expression, implying the importance of auxin in controlling lateral root development. Similarly, it is demonstrated for the first time that gravistimulated roots developed a rapid (within 1 h) induction of GH3:GUS activity in tissues on the non-elongating side of the responding root and this induction occurred concurrently with root curvature. These transgenic plants could be useful tools in determining the physiological and biochemical changes that occur during auxin-mediated responses. 相似文献
93.
检测香蕉花叶心腐病病原CMV的PAS—ELISA方法的改进 总被引:1,自引:0,他引:1
作为一种快速、敏感的免疫学方法,ELISA已被广泛地用于植物病毒的研究与应用性检测方面。在实验室中用PAS-ELISA检测香蕉花叶心腐病病原CMV已被证明是一种获得无CMV组培苗的有效手段。在香蕉无病毒组培苗工厂化大批量生产中,我们应用此法作为质量控制的手段之一,剔除病苗,保证产品不带CMV,获得一定成效。但同时我们也发现,香蕉组织中含有引起非特异性吸附反应的物 相似文献
94.
利用单克隆抗体免疫磁珠吸附方法分离脐血CD34+细胞,并观察了IL3/GMCSF融合蛋白(PIXY321)对脐血CD34+细胞的刺激作用。PIXY321对脐血CD34+细胞扩增作用大于IL3和GMCSF单独及联合应用。在液体培养条件下,每毫升20ngPIXY321可有效地扩增脐血造血祖细胞,适宜扩增时间为5-8天,扩增后造血祖细胞的数量可达扩增前的8-10倍,从而初步建立了一种简单可行的脐血造血细胞扩增方法。 相似文献
95.
96.
吖啶橙失活处理应用于不对称融合初步探讨 总被引:1,自引:0,他引:1
马铃薯栽培种和野生种叶肉原生质体经过吖啶橙(AO)处理后,暗培养两天,再光照4h而失活.失活程度取决于AO处理时间、AO浓度、光照时机等.四倍体失活浓度高于二倍体,处理后马上光照失活效果最强烈,8d后光照其小细胞团同未光照处理相差不大。AO失活的栽培种NO7同罗丹明6G(R6G)失活的Solanumbulbocastanum融合后可以发生代谢互补恢复分裂,已得到假定的杂种小愈伤组织. 相似文献
97.
Marc Pauly Isabelle Kayser Martine Schmitz Fernand Ries François Hentges Mario Dicato 《Journal of molecular evolution》1995,41(6):974-978
The mdr1 gene, first member of the human multidrug-resistance gene family, is a major gene involved in cellular resistance to several drugs used in anticancer chemotherapy. Its product, the drug-excreting P-glycoprotein, shows a bipartite structure formed by two similar adjacent halves. According to one hypothesis, the fusion of two related ancestral genes during evolution could have resulted in this structure. The DNA sequence analysis of the introns located in the region connecting the two halves of the human mdr1 gene revealed a highly conserved poly(CA) · poly (TG) sequence in intron 15 and repeated sequences of the Alu family in introns 14 and 17. These repeated sequences most likely represent molecular fossils of ancient DNA elements which were involved in such a recombination event.
Correspondence to: M. Pauly 相似文献
98.
Affinity tag AG consisting of immunoglobulin G (lgG)-binding domains of protein A from Staphylococcus aureus (EDABC) and those of protein G from Streptococcus strain G148 (C2C3) were used to facilitate immobilization of beta-galactosidase (betagal) from Escherichia coli. Poly(methylmethacrylate/N-isopropylacrylamide/methacrylic acid) [P(MMA/NIPAM/MAA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] latex particles, which show thermosensitivity, were used as support materals to prepare affinity adsorbents. Human gamma-globulin (HgammaGb), whose major fraction is lgG, was used as an affinity ligand and was covalently immobilized onto the both latex particles by the carbodiimide method under various conditions. A fusion protein, AGbetagal, was immobilized at pH 7.3 by the specific binding of affinity tag to these affinity adsorbents. The amount of adsorbed AGbetagal per unit amount of immobilized HgammaGb, namely, efficiency of ligand utilization, was strongly affected by the type of latex particles and pH value for HgammaGb immobilization. The efficiency of ligand utilization was maximum in the affinity adsorbents prepared at pH 6.0 to 7.0, and that in the HgammaGb-P(MMA/NIPAM/MAA) latex particles was high. This result could be explained by the conformation and orientation of immobilized HgammaGb molecules. Immobilized AGbetagal retained approximately 75% of its activity in solution and the binding is stable enough to allow repeated use. These results clearly demonstrate that combination of the affinity tag AG and the affinity adsorbents, based on the thermosensitive latex particles, offers a simple and widely applicable method for preparation of immobilized enzyme with high activity. (c) 1995 John Wiley & Sons, Inc. 相似文献
99.
Design, synthesis, expression, and characterization of the genes for mouse Fc gamma RIIb1 and Fc gamma RIIb2 cytoplasmic regions. 下载免费PDF全文
L. Chen N. L. Thompson G. J. Pielak 《Protein science : a publication of the Protein Society》1997,6(5):1038-1046
The cytoplasmic regions of the mouse low-affinity Fc gamma RII isoforms, mFc gamma RIIb1, and mFc gamma RIIb2, play a key role in signal transduction by mediating different cellular functions. mFc gamma RIIb1 has a 94-residue cytoplasmic region, whereas mFc gamma RIIb2 has a 47-residue cytoplasmic region. Genes encoding the cytoplasmic regions of mFc gamma RIIb1 (b1-94) and mFc gamma RIIb2 (b2-47) were designed, synthesized, and expressed as fusion proteins in Escherichia coli. A sequence-specific protease, thrombin, was used to release the b1-94 peptide, which was purified by using HPLC. The b2-47 peptide was synthesized chemically. CD spectropolarimetry was employed to examine the secondary structures of b1-94 and b2-47. These studies were conducted in aqueous solution, in mixtures of water and trifluoroethanol or methanol, and as a function of temperature. The results indicate that the b1-94 and b2-47 structures are sensitive functions of the solvent environment, and that nonaqueous solvents induce significant alpha-helical structure. 相似文献
100.
The maltose binding protein (MBP or MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides. It is used widely as a carrier protein for the production of recombinant fusion proteins. The melting of recombinant MBP was studied by differential scanning and titration calorimetry and fluorescence spectroscopy under different solvent conditions. MBP exhibits a single peak of heat absorption with a delta(Hcal)/delta(HvH) ratio in the range of 1.3-1.5, suggesting that the protein comprises two strongly interacting thermodynamic domains. Binding of maltose resulted in elevation of the Tm by 8-15 degrees C, depending of pH. The presence of ligand at neutral pH, in addition to shifting the melting process to higher temperature, caused it to become more cooperative. The delta(Hcal)/delta(HvH) ratio decreased to unity, indicating that the two domains melt together in a single two-state transition. This ligand-induced merging of the two domains appears to occur only at neutral pH, because at low pH maltose simply stabilized MBP and did not cause a decrease of the delta(Hcal)/delta(HvH) ratio. Binding of maltose to MBP is characterized by very low enthalpy changes, approximately -1 kcal/mol. The melting of MBP is accompanied by an exceptionally large change in heat capacity. 0.16 cal/K-g, which is consistent with the high amount of nonpolar surface--0.72 A2/g--that becomes accessible to solvent in the unfolded state. The high value of delta Cp determines a very steep delta G versus T profile for this protein and predicts that cold denaturation should occur above freezing temperatures. Evidence for this was provided by changes in fluorescence intensity upon cooling the protein. A sigmoidal cooperative transition with a midpoint near 5 degrees C was observed when MBP was cooled at low pH. Analysis of the melting of several fusion proteins containing MBP illustrated the feasibility of assessing the folding integrity of recombinant products prior to separating them from the MBP carrier protein. 相似文献