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81.
Morphologically normal and fertile transgenic plants of mungbean with two transgenes, bar and α-amylase inhibitor, have been developed for the first time. Cotyledonary node explants were transformed by cocultivation
with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pKSB that carried bialaphos resistance (bar) gene and Phaseolus vulgaris α-amylase inhibitor-1 (αAI-1) gene. Green transformed shoots were regenerated and rooted on medium containing phosphinothricin (PPT). Preculture and
wounding of the explants, presence of acetosyringone and PPT-based selection of transformants played significant role in enhancing
transformation frequency. Presence and expression of the bar gene in primary transformants was evidenced by PCR-Southern analysis and PPT leaf paint assay, respectively. Integration
of the Phaseolus vulgaris α-amylase inhibitor gene was confirmed by Southern blot analysis. PCR analysis revealed inheritance of both the transgenes
in most of the T1 lines. Tolerance to herbicide was evidenced from seed germination test and chlorophenol red assay in T1 plants. Transgenic plants could be recovered after 8–10 weeks of cocultivation with Agrobacterium. An overall transformation frequency of 1.51% was achieved. 相似文献
82.
83.
CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity 总被引:1,自引:0,他引:1
Blake Carrington Gaurav K. Varshney Shawn M. Burgess Raman Sood 《Nucleic acids research》2015,43(22):e157
CRISPR/Cas9 has emerged as a versatile genome-engineering tool that relies on a single guide RNA (sgRNA) and the Cas9 enzyme for genome editing. Simple, fast and economical methods to generate sgRNAs have made targeted mutagenesis routine in cultured cells, mice, zebrafish and other model systems. Pre-screening of sgRNAs for target efficacy is desirable both for successful mutagenesis and minimizing wasted animal husbandry on targets with poor activity. Here, we describe an easy, quick and cost-effective fluorescent polymerase chain reaction (PCR)-based method, CRISPR Somatic Tissue Activity Test (CRISPR-STAT), to determine target-specific efficiency of sgRNA. As a proof of principle, we validated our method using 28 sgRNAs with known and varied levels of germline transmission efficiency in zebrafish by analysis of their somatic activity in injected embryos. Our data revealed a strong positive correlation between the fluorescent PCR profiles of the injected embryos and the germline transmission efficiency. Furthermore, the assay was sensitive enough to evaluate multiplex gene targeting. This method is easy to implement by laboratories with access to a capillary sequencer. Although we validated the method using CRISPR/Cas9 and zebrafish, it can be applied to other model systems and other genome targeting nucleases. 相似文献
84.
Raman Dhariwal Vijay Gahlaut Bhaganagare R. Govindraj Dharmendra Singh Saloni Mathur Shailendra Vyas Rajib Bandopadhyay Jitendra Paul Khurana Akhilesh Kumar Tyagi Kumble Vinod Prabhu Kunal Mukhopadhyay Harindra Singh Balyan Pushpendra Kumar Gupta 《Functional & integrative genomics》2015,15(2):233-245
85.
Gopinath KP Kathiravan MN Srinivasan R Sankaranarayanan S 《Bioresource technology》2011,102(4):3687-3693
In this study, it was attempted to evaluate the influences and also recommended some elimination methods for inhibitory effects offered by salts and heavy metal ions. Congo red dye solution treated with mutant Pseudomonas sp. was taken as a model system for study. The salts used in this study are NaCl, CaCl2 and MgSO4·7H2O. Though the growth was inhibited at concentrations above 4 g/l, toleration was achieved by acclimatization process. In case of heavy metal ions, Cr (VI) showed low inhibition up to 500 mg/l of concentration, compared to Zn (II) and Cu (II). It was due to the presence of chromium reductase enzyme which was confirmed by SDS-PAGE. Zn (II) and Cu (II) ion inhibitions were eliminated by chelation with EDTA. The critical ion concentrations obtained as per Han-Levenspiel model for Cr (VI), Zn (II) and Cu (II) were 0.8958, 0.3028 and 0.204 g/l respectively. 相似文献
86.
Hong Q Bakshi RK Palucki BL Park MK Ye Z He S Pollard PG Sebhat IK Liu J Guo L Cashen DE Martin WJ Weinberg DH MacNeil T Tang R Tamvakopoulos C Peng Q Miller RR Stearns RA Chen HY Chen AS Strack AM Fong TM MacIntyre DE Wyvratt MJ Nargund RP 《Bioorganic & medicinal chemistry letters》2011,21(8):2330-2334
We report the discovery of piperazine urea based compound 1, a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist. Compound 1 shows anti-obesity efficacy without potentiating erectile activity in the rodent models. 相似文献
87.
Wang S Singh RD Godin L Pagano RE Hubmayr RD 《American journal of physiology. Lung cellular and molecular physiology》2011,300(4):L560-L568
We present plasma membrane (PM) internalization responses of type I alveolar epithelial cells to a 50 mosmol/l increase in tonicity. Our research is motivated by interest in ATI repair, for which endocytic retrieval of PM appears to be critical. We validated pharmacological and molecular tools to dissect the endocytic machinery of these cells and used these tools to test the hypothesis that osmotic stress triggers a pathway-specific internalization of PM domains. Validation experiments confirmed the fluorescent analogs of lactosyl-ceramide, transferrin, and dextran as pathway-specific cargo of caveolar, clathrin, and fluid-phase uptake, respectively. Pulse-chase experiments indicate that hypertonic exposure causes a downregulation of clathrin and fluid-phase endocytosis while stimulating caveolar endocytosis. The tonicity-mediated increase in caveolar endocytosis was associated with the translocation of caveolin-1 from the PM and was absent in cells that had been transfected with dominant-negative dynamin constructs. In separate experiments we show that hypertonic exposure increases the probability of PM wound repair following micropuncture from 82 ± 4 to 94 ± 2% (P < 0.01) and that this effect depends on Src pathway activation-mediated caveolar endocytosis. The therapeutic and biological implications of our findings are discussed. 相似文献
88.
Barnacles are some of the major inhabitants of intertidal zones and have calcite-based exoskeleton to anchor and armor their tissues. Structural characterization studies of the specie Ambhibalanus reticulatus were performed to understand the construction of the exoskeleton which forms a light-weight yet stiff structure. The parietal shell is constructed of six compartments to yield a truncated cone geometry, which is neatly fixed onto the basal shell that attaches the organism to the substrate surface. The connections among the different compartments happen through sutured edges and also have chemical interlocking to make the junctions impermeable. Also, the shell parts are furnished with hollow channels reducing the overall mass of the construction. The structure and functions of different parts of the exoskeleton are identified and outlined. Finally, the mechanical properties such as modulus, hardness and fracture toughness of the exoskeleton obtained by indentation techniques are discussed. 相似文献
89.
90.
Gao S Babu R Lu Y Martinez C Hao Z Krivanek AF Wang J Rong T Crouch J Xu Y 《PloS one》2011,6(1):e16101
Development of a seed DNA-based genotyping system for marker-assisted selection (MAS) has provided a novel opportunity for understanding aberrant reproductive phenomena such as hetero-fertilization (HF) by observing the mismatch of endosperm and leaf genotypes in monocot species. In contrast to conventional approaches using specific morphological markers, this approach can be used for any population derived from diverse parental genotypes. A large-scale experiment was implemented using seven F(2) populations and four three-way cross populations, each with 534 to 1024 individuals. The frequency of HF within these populations ranged from 0.14% to 3.12%, with an average of 1.46%. The highest frequency of HF in both types of population was contributed by the pollen gametes. Using three-way crosses allowed, for the first time, detection of the HF contributed by maternal gametes, albeit at very low frequency (0.14%-0.65%). Four HF events identified from each of two F(2) populations were tested and confirmed using 1032 single nucleotide polymorphic markers. This analysis indicated that only 50% of polymorphic markers can detect a known HF event, and thus the real HF frequency can be inferred by doubling the estimate obtained from using only one polymorphic marker. As expected, 99% of the HF events can be detected by using seven independent markers in combination. Although seed DNA-based analysis may wrongly predict plant genotypes due to the mismatch of endosperm and leaf DNA caused by HF, the relatively low HF frequencies revealed with diverse germplasm in this study indicates that the effect on the accuracy of MAS is limited. In addition, comparative endosperm and leaf DNA analysis of specific genetic stocks could be useful for revealing the relationships among various aberrant fertilization phenomena including haploidy and apomixis. 相似文献