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The delivery of proteins instead of DNA into plant cells allows for a transient presence of the protein or enzyme that can be useful for biochemical analysis or genome modifications. This may be of particular interest for genome editing, because it can avoid DNA (transgene) integration into the genome and generate precisely modified “nontransgenic” plants. In this work, we explore direct protein delivery to plant cells using mesoporous silica nanoparticles (MSNs) as carriers to deliver Cre recombinase protein into maize (Zea mays) cells. Cre protein was loaded inside the pores of gold-plated MSNs, and these particles were delivered by the biolistic method to plant cells harboring loxP sites flanking a selection gene and a reporter gene. Cre protein was released inside the cell, leading to recombination of the loxP sites and elimination of both genes. Visual selection was used to select recombination events from which fertile plants were regenerated. Up to 20% of bombarded embryos produced calli with the recombined loxP sites under our experimental conditions. This direct and reproducible technology offers an alternative for DNA-free genome-editing technologies in which MSNs can be tailored to accommodate the desired enzyme and to reach the desired tissue through the biolistic method.Introducing DNA-modifying enzymes rather than DNA-based expression cassettes is an attractive alternative for genetic engineering and genome-editing applications such as gene targeting or site-specific recombination. It offers a transient presence of the enzymes, and the process can be coordinated with high levels of enzymatic activity at the time and sites of the desired DNA recombination events. Many DNA-metabolizing enzymes (endonucleases, transposases, and topoisomerases), when delivered in an unrestrained manner, show adverse effects on cell viability. Delivery in the form of protein or RNA may help to mitigate these effects (Cui et al., 2011; Sander et al., 2011; Watanabe et al., 2012). In addition, by introducing proteins, one can avoid the need to remove the protein-encoding DNA fragments from the engineered plant genome. This may help shorten the time from laboratory to field for future improved germplasms.Site-specific recombinases such as Cre or FLP have been widely used in genetic engineering applications (Sorrell and Kolb, 2005). The 38-kD Cre enzyme specifically binds to and recombines the 34-bp loxP sequences, allowing the removal, integration, or inversion of the DNA fragment flanked by these sequences (for review, see Wang et al., 2011). There are a number of established methodologies designed to provide the Cre recombinase activity for site-specific recombination in eukaryotic cells that do not involve the delivery of DNA. These methods include lipofection (Baubonis and Sauer, 1993), microinjection of protein or mRNA (de Wit et al., 1998; Luckow et al., 2009), electroporation of protein or mRNA (Kolb and Siddell, 1996; Ponsaerts et al., 2004), or using modified microorganisms for Cre delivery to their host cells (Vergunst et al., 2000; Koshy et al., 2010). Another strategy that has been used is the incubation or injection of tissues/cell cultures with cell-permeant Cre, a modified Cre protein fused to protein transduction domains or cell-penetrating peptides (Jo et al., 2001; Will et al., 2002; Lin et al., 2004; Nolden et al., 2006).For biotechnological applications in plant sciences, protein delivery systems have been developed, including microinjection (Wymer et al., 2001), protein immobilization to gold particles (Wu et al., 2011), and protein transduction through cell-penetrating peptides (for review, see Chugh et al., 2010). The cell-penetrating peptides were shown to enable intracellular delivery of the Cre recombinase protein to rice (Oryza sativa) callus tissues (Cao et al., 2006). Nanobiotechnology is offering an attractive alternative, since nanoparticles can be precisely tailored to deliver a particular biomolecule to the cell, tissue, or organism of interest when needed (for review, see Du et al., 2012). Mesoporous silica nanoparticles (MSNs) are particularly suited for this purpose. These porous nanoparticles are formed by a matrix of well-ordered pores that confers high loading capacity of molecules like proteins (for review, see Popat et al., 2011). Additionally, surfaces of MSNs can be readily modified, permitting the customization of nanoparticles to particular experimental needs (for review, see Trewyn et al., 2007). In our previous studies, it was shown that MSNs can be used for the codelivery of DNA and chemicals (Torney et al., 2007) as well as DNA and proteins (Martin-Ortigosa et al., 2012a) to plant cells via biolistics. To improve MSN performance as a projectile, gold plating of MSN surfaces was performed, increasing nanoparticle density and, subsequently, the ability to pass through the plant cell wall upon bombardment (Martin-Ortigosa et al., 2012b).In this work, the Cre recombinase enzyme was loaded into the pores of gold-plated MSNs and delivered through the biolistic method to maize (Zea mays) cells containing loxP sites integrated into chromosomal DNA (Lox-corn; Fig. 1A). Lox-corn expressed the glyphosate acetyltransferase gene (gat) and the Anemonia majano cyan fluorescent protein gene (AmCyan1) flanked by loxP sites. The MSN-released Cre enzyme recombined the loxP sites, thus removing the DNA fragment flanked by these sequences. Such excisions led to the expression of a variant of Discosoma sp. red fluorescent protein gene (DsRed2) and the loss of the selectable marker gene (Fig. 1A). Visual selection was used to recover the recombination events. Subsequently, fertile maize plants were regenerated from the recombined events and DNA analyses confirmed the recombination events. To our knowledge, this is the first time that MSNs have been used for the delivery of a functional recombinase into plant tissues, leading to successful genome editing.Open in a separate windowFigure 1.A, Schematic representation of the MSN-based bombardment technology. Cre protein is loaded into the pores of gold-plated MSN (Cre-6x-MSN) and subsequently bombarded onto immature embryos of a transgenic maize line carrying a loxP construct (Lox-corn). The parental transgenic Lox-corn tissues are blue fluorescence and herbicide resistant because they harbor a cassette with the glyphosate acetyltransferase (gat) selection gene and the AmCyan1 (cyan) marker gene flanked by the loxP sites. The DsRed2 (dsred) gene for the expression of a red fluorescent protein is placed downstream of the cassette. Once Cre recombinase is released inside the cell, it performs the recombination, excising gat-AmCyan1 genes and leading to the expression of the DsRed2 gene, switching the cell fluorescence pattern from blue to red. P, Promoter; T, terminator. UBINTRF, CYANF, and DSRED2R are primers for DNA analysis. B, Transmission electron microscope image showing the typical hexagonal shape and the well-ordered pore structure of a 6x-MSN. C, Scanning electron microscope image showing gold nanoparticle deposition (white dots) in all surfaces of 6x-MSN. D, Western blot showing Cre protein loading and release dynamics from 6x-MSN. The protein loading is almost immediate, even though some protein can be detected in the buffer even after 1 h of loading. For the release, some Cre protein can be observed after 24 h of incubation. Most of the protein remains in the 6x-MSN pellet. C+, 400 ng of Cre protein; Empty, a lane with no protein loading. The bands observed in the Empty lane were the spillover from the neighboring Pellet lane, which represents Cre-loaded 6x-MSN after the release experiment resuspended in Laemmli loading buffer (see “Materials and Methods”).  相似文献   
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Key message

Twenty-seven QTLs were identified for rice seed vigor, in which 16 were novel QTLs. Fifteen elite parental combinations were designed for improving seed vigor in rice.

Abstract

Seed vigor is closely related to direct seeding in rice (Oryza sativa L.). Previous quantitative trait locus (QTL) studies for seed vigor were mainly derived from bi-parental segregating populations and no report from natural populations. In this study, association mapping for seed vigor was performed on a selected sample of 540 rice cultivars (419 from China and 121 from Vietnam). Population structure was estimated on the basis of 262 simple sequence repeat (SSR) markers. Seed vigor was evaluated by root length (RL), shoot length (SL) and shoot dry weight in 2011 and 2012. Abundant phenotypic and genetic diversities were found in the studied population. The population was divided into seven subpopulations, and the levels of linkage disequilibrium (LD) ranged from 10 to 80 cM. We identified 27 marker–trait associations involving 18 SSR markers for three traits. According to phenotypic effects for alleles of the detected QTLs, elite alleles were mined. These elite alleles could be used to design parental combinations and the expected results would be obtained by pyramiding or substituting the elite alleles per QTL (apart from possible epistatic effects). Our results demonstrate that association mapping can complement and enhance previous QTL information for marker-assisted selection and breeding by design.  相似文献   
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Vascular smooth muscle cells (VSMCs) are exposed to mechanical cyclic stretch in vivo, which play important roles in maintenance of vascular homeostasis and regulation of pathological vascular remodeling. Reversible protein phosphorylation is crucial for intracellular signaling transduction. However, the dynamic phosphorylated profile induced by cyclic stretch in VSMCs is still unclear. Using the stable isotope labeling by amino acid in cell culture, VSMCs were labeled and exposed to 10% physiological cyclic stretch in vitro at 1.25 Hz for 0 min, 15 min, 30 min, 1 h and 6 h, respectively. Using TiO2 beads and liquid chromatography tandem mass spectrometry, the temporal phosphoproteomic profiles in response to cyclic stretch were then detected. Bioinformatics analysis including fuzzy c-means clustering, functional classifications, and Ingenuity Pathway Analysis were applied to further reveal the potential mechanotranduction networks. The results indicated that protein kinase C (PKCs) family, Rho-associated coiled-coil containing protein kinase 1 (ROCK1) and Akt may participate in cyclic-stretch induced VSMC functions. Cyclic stretch repressed the expression of ROCK1, while it had no significant effect on the phosphorylation of PKCα/βII, PKCζ/λ and PKCδ/θ. PKCθ was activated first at short time-phase (15 min and 30 min), and again at long time-phase (6 h, 12 h and 24 h). The activation of p-PKCμ was immediate and short-term, similar to p-Akt. Our present in vitro work hence revealed that cyclic stretch activates complex mechanotransduction networks, suggesting that novel mechanoresponsive molecules, i.e., PKCθ, PKCμ, and ROCK1, may participate in the mechanotransduction and modulation VSMC functions.  相似文献   
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The absorption of phospholipid may improve the fluidity of membrane and enzyme activities. Phospholipids also play a role in promoting Caveolae formation and membrane synthesis. Caveolin-1 has a significant effect on signaling pathways involved in regulating cell proliferation and stress responsiveness. Thus, we can speculate that Caveolin-1 could affect the sense of environmental stress. We use Chang liver cell line to investigate the ability of Caveolin-1 to modulate the cellular response to ethanol injury. Caveolin-1 downregulate cells (Cav-1?/?) were established by stable transfecting with psiRNA-CAV1 plasmids, which were more sensitive to toxic effects of ethanol than the untransfected parental cells (WT). Releasing of ALT and electric conductivity were changed significantly in Cav-1?/? cells compared with WT. Caveolin-1 gene silencing could obviously down-regulate the activities of protein kinase C-α (PKC-α) and phospho-p42/44 MAP kinase, indicating cell proliferation and self-repairing abilities were inhibited. However, the levels of Caveolin-1 and PKC-α were increased by phosphatidylcholine administration. The results indicated that the inhibition of lipid peroxidation by phosphatidylcholine could lead to the prevention of membrane disruption, which closely correlated with the level of Caveolin-1. Since the protective effects of phosphatidylcholine against ethanol-induced lipid peroxidation might be regulated by phospholipid-PKC-α signaling pathway, related with Caveolin-1, the potential effects of phosphatidylcholine on membranes need to be verified.  相似文献   
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Designed retroaldolases have utilized a nucleophilic lysine to promote carbon–carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (> 10− 3 s− 1) and kcat/KM (11–25 M− 1 s− 1) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the > 105-fold rate accelerations that were achieved are within 1–3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat = 106 to 108) and an extensively evolved computational design (kcat/kuncat > 107). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches.  相似文献   
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