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991.
Rapid and reliable detection of 11 food-borne pathogens using thin-film biosensor chips 总被引:1,自引:0,他引:1
Sulan Bai Jinyi Zhao Yaochuan Zhang Wensheng Huang Shi Xu Haodong Chen Liu-Min Fan Ying Chen Xing Wang Deng 《Applied microbiology and biotechnology》2010,86(3):983-990
Traditional methods for identifying food-borne pathogens are time-consuming and laborious, so it is necessary to develop innovative
methods for the rapid identification of food-borne pathogens. Here, we report the development of silicon-based optical thin-film
biosensor chips for sensitive detection of 11 food-borne pathogens. Briefly, aldehyde-labeled probes were arrayed and covalently
attached to a hydrazine-derivatized chip surface, and then, biotinylated polymerase chain reaction (PCR) amplicons were hybridized
with the probes. After washing and brief incubation with an antibiotin immunoglobulin G–horseradish peroxidase conjugate and
a precipitable horseradish peroxidase substrate, biotinylated chains bound to the probes were visualized as a color change
on the chip surface (gold to blue/purple). Highly sensitive and accurate examination of PCR fragment targets can be completed
within 30 min. This assay is extremely robust, sensitive, specific, and economical and can be adapted to different throughputs.
Thus, a rapid, sensitive, and reliable technique for detecting 11 food-borne pathogens was successfully developed. 相似文献
992.
Molecular biologic techniques applied to the microbial prospecting of oil and gas in the Ban 876 gas and oil field in China 总被引:3,自引:0,他引:3
Fan Zhang Yuehui She Yong Zheng Zhifeng Zhou Shuqiong Kong Dujie Hou 《Applied microbiology and biotechnology》2010,86(4):1183-1194
Currently, molecular biologic techniques achieve a great development in studies of soil samples. The objective of this research
is to improve methods for microbial prospecting of oil and gas by applying culture-independent techniques to soil sampled
from above a known oil and gas field. Firstly, the community structure of soil bacteria above the Ban 876 Gas and Oil Field
was analyzed based on 16S rRNA gene clone libraries. The soil bacteria communities were consistently different along the depth;
however, Chloroflexi and Gemmatimonadetes were predominant and methanotrophs were minor in both bacteria libraries (DGS1 and DGS2). Secondly, the numbers of methane-oxidizing
bacteria, quantified using a culture-dependent procedure and culture-independent group-specific real-time PCR (RT-PCR), respectively,
were inconsistent with a quantify variance of one or two orders of magnitude. Special emphasis was given to the counting advantages
of RT-PCR based on the methanotrophic pmoA gene. Finally, the diversity and distribution of methanotrophic communities in the soil samples were analyzed by constructing
clone libraries of functional gene. All 508-bp inserts in clones phylogenetically belonged to the methanotrophic pmoA gene with similarities from 83% to 100%. However, most of the similarities were below 96%. Five clone libraries of methanotrophs
clearly showed that the anomalous methanotrophs (Methylosinus and Methylocystis) occupy the studied area. 相似文献
993.
J.‐M. Fan J. Luo G.‐P. Zhang L. Chen M. Teng M.‐F. Yang L. Wang C.‐Q. Wang 《Letters in applied microbiology》2010,51(1):11-17
Aims: Identification and characterization of Japanese encephalitis virus (JEV) envelope protein gene from swine. Methods and Results: Genomic RNA was separated from JEV isolated strain Henan‐09‐03, and used as templates for cDNA synthesis of E gene. The cDNA of E gene was amplified by RT‐PCR and cloned into the pMD19‐T‐Vector and confirmed by sequencing. The cloned gene was then subcloned into the pET‐32a and was introduced into Escherichia coli BL21 (DE3) for expression. The E protein was purified by Ni chelating column‐based affinity chromatography. The molecular weight of expressed protein was about 50 kDa. Compared with the published sequence of SA14 ( AF495589 ), the homology of the nucleotide sequence was 98% and the seven mutations resulting in amino acid substitutions at Leu 36 Ser, Leu107 Val, Ala167 Thr, Asn 230 Ser, Leu 340 Pro, Asn 430 Ile, Phe 448 Leu. Phylogenetic analysis of the E sequence of isolated strain classified it within genotype III of the JEV. The result of Western blotting indicated that the antigenicity of the protein was specific. Conclusions: The stable expression of the protein and the analysis of its antigenic specificity provide the foundation for developing the ELISA early stage diagnosis kit. Significance and Impact of the Study: As coating antigen, the recombinant E protein served a good source in the indirect ELISA method for the detection of JEV antibody. 相似文献
994.
IscA is a key member of the iron-sulfur cluster assembly machinery in prokaryotic and eukaryotic organisms; however, the physiological function of IscA still remains elusive. In the present paper we report the in vivo evidence demonstrating the iron-binding activity of IscA in Escherichia coli cells. Supplement of exogenous iron (1 μM) in M9 minimal medium is sufficient to maximize the iron binding in IscA expressed in E. coli cells under aerobic growth conditions. In contrast, IscU, an iron-sulfur cluster assembly scaffold protein, or CyaY, a bacterial frataxin homologue, fails to bind any iron in E. coli cells under the same experimental conditions. Interestingly, the strong iron-binding activity of IscA is greatly diminished in E. coli cells under anaerobic growth conditions. Additional studies reveal that oxygen in medium promotes the iron binding in IscA, and that the iron binding in IscA in turn prevents formation of biologically inaccessible ferric hydroxide under aerobic conditions. Consistent with the differential iron-binding activity of IscA under aerobic and anaerobic conditions, we find that IscA and its paralogue SufA are essential for the iron-sulfur cluster assembly in E. coli cells under aerobic growth conditions, but not under anaerobic growth conditions. The results provide in vivo evidence that IscA may act as an iron chaperone for the biogenesis of iron-sulfur clusters in E. coli cells under aerobic conditions. 相似文献
995.
Liyue Wang Fan Gong Xiaoyan Dong Wei Zhou Qiutang Zeng 《Molecular and cellular biochemistry》2010,337(1-2):159-166
Endothelium-derived nitric oxide (NO) is a cytoprotective molecule to prevent endothelial cells (ECs) from apoptosis. CREB-binding protein (CBP) is involved in the apoptotic pathway in several tumor cells, however, little is known whether CBP is associated with apoptosis in ECs and the apoptotic effect of CBP on ECs is regulated by NO. Therefore, the purpose of the present study was to investigate whether silencing CBP expression could affect the sensitivity of ECs toward apoptotic stimuli and determined the role of NO. In this study, we found that when CBP expression was silenced by RNA interference, ECs were more prone to apoptosis under serum deprivation, whereas the apoptosis was not significantly induced in the serum-containing condition. The increased apoptosis is paralleled by a reduction of NO, and the apoptosis was reversed by NO donors, suggesting an important role of NO. Furthermore, CBP silencing decreased NO production by downregulating the endothelial NO synthase (eNOS) expression in a dose-dependent manner. These results indicated that CBP silencing is associated with decreased eNOS expression and NO production, and therefore concomitantly increased the sensitivity of ECs toward apoptosis. 相似文献
996.
Z. W. Wang X. Y. Li Z. L. Tang S. L. Yang Z. Z. Ying T. Fu B. Fan Y. L. Mu H. Ao K. Li 《Molecular biology reports》2010,37(7):3393-3400
F-box proteins are quite significant ubiquitin-proteasome pathway regulators in eukaryotic cells. FBXO40, a member of this large family, alters its expression pattern in muscle atrophy. Here we isolated most of the verified porcine
FBXO40 coding sequence (CDS) (2258 bp) and assigned it to the porcine chromosome 13q4.1-4.6 by using the INRA-Minnesota porcine
radiation hybrid panel, and we also explored the tissue expression distributions, which is relatively high in longissimus
dorsi muscle, heart, low in kidney, small intestine, brain, hypophysis, lymphonode, thymus, spleen, large intestine, ovary,
stomach, and undetectable in testis, liver, uterus and thyroid gland. Inferring phylogenetic tree was constructed to study
the evolutionary implications. Moreover, a HindII (HincII)-RFLP (A/C) polymorphism in 3′-untranslated region (3′-UTR) of porcine FBXO40 gene was demonstrated by sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. Statistical analysis
result of this polymorphism showed that the allele A was predominant in all detected indigenous breeds, but C in western introduced
commercial breeds. The SNP was further analyzed in our experimental pig population including Tongcheng, Landrace, Large White,
and crossbreds of Large White × (Landrace × Tongcheng) and Landrace × (Large White × Tongcheng). The association analysis
results indicated that the A/C base substitution was associate with some hematological indexes, the hemoglobin concentration
(P < 0.0001), mean corpuscular volume hemoglobin concentration (P = 0.0002) and mean corpuscular volume (P = 0.0138). 相似文献
997.
Yaping Xin Linsen Zan Yongfeng Liu Hongyu Liu Wanqiang Tian Yueyuan Fan Lei Huang 《Molecular biology reports》2010,37(6):3043-3049
Six Y-STR loci (UMN0929, UMN0108, UMN0920, INRA124, UMN2404 and UMN0103) were analyzed using 576 healthy and unrelated males
and 10 females of the Qinchuan cattle population in Chinese Shaanxi Province. Allele frequency, gene diversity, the polymorphic
information content, and the number of effective gene were calculated. All loci were in accordance with the Hardy–Weinberg
equilibrium (P > 0.05). The population data were compared with published data of other cattle breeds, suggesting that Qinchuan cattle were
originated primarily from Bos Taurus. Results are valuable for individual identification, paternity testing, and origin analysis of Qinchuan cattle breed. 相似文献
998.
Vallisneria natans and Vallisneria spinulosa are two morphologically very similar and sympatrically dominant submerged macrophytes in lakes of the middle-lower reaches of the Yangtze River. Genetic variation was compared based on a total of 196 individuals from six V. natans populations and 201 individuals from seven V. spinulosa populations. Using eight ISSR primers, a total of 139 and 129 DNA fragments were generated with 121 being polymorphic in V. natans and 99 in V. spinulosa. The two species maintained higher genetic variation both at the species and population levels in comparison with other aquatic macrophytes. A higher level of genetic diversity among populations was found in V. natans than in V. spinulosa: the percentage of polymorphic loci (PPL) in V. natans was 52-62% vs. 38-47% in V. spinulosa; gene diversity (H) was 0.21 in V. natans vs. 0.17 in V. spinulosa.Both an analysis of molecular variance (AMOVA) and F-estimation (FST) showed that most of the total genetic variation resided within populations of both species (AMOVA: 85% and 80%; FST: 0.132 and 0.202), indicating low genetic differentiation between populations. Principal coordinates analysis (PCA) indicated evident gene flow between populations of both species. The outcrossing reproductive mode and pervasive gene flow might have played important roles in maintaining high genetic diversity and in shaping low population differentiation of the two Vallisneria species, while the extent of clonal growth might account for the different levels of population divergence between them. 相似文献
999.
Jiayi Wang Xiangfan Liu Huacheng Wu Peihua Ni Zhidong Gu Yongxia Qiao Ning Chen Fenyong Sun Qishi Fan 《Nucleic acids research》2010,38(16):5366-5383
Long non-coding RNA (lncRNA), highly up-regulated in liver cancer (HULC) plays an important role in tumorigenesis. Depletion of HULC resulted in a significant deregulation of several genes involved in liver cancer. Although up-regulation of HULC expression in hepatocellular carcinoma has been reported, the molecular mechanisms remain unknown. In this study, we used in vivo and in vitro approaches to characterize cancer-dependent alterations in the chromatin organization and find a CREB binding site (encompassing from −67 to −53 nt) in the core promoter. Besides, we also provided evidence that PKA pathway may involved in up-regulation of HULC. Furthermore, we demonstrated HULC may act as an endogenous ‘sponge’, which down-regulates a series of microRNAs (miRNAs) activities, including miR-372. Inhibition of miR-372 leads to reducing translational repression of its target gene, PRKACB, which in turn induces phosphorylation of CREB. Over-expression of miR-372 decreases the association of CREB with the proximal promoter, followed by the dissociation of P300, resulting in a change of the histone ‘code’, such as in deacetylation and methylation. The study elucidates that fine tuning of HULC expression is part of an auto-regulatory loop in which it’s inhibitory to expression and activity of miR-372 allows lncRNA up-regulated expression in liver cancer. 相似文献
1000.
Guo-Liang Qian Jia-Qin Fan De-Feng Chen Yue-Jing Kang Bing Han Bai-Shi Hu Feng-Quan Liu 《Biological Control》2010,52(1):17-23
An N-acyl homoserine lactonase gene aiiA, transcribed by a strong and constitutive Escherichia coli promoter Plpp (Accession No. EU723847), was transformed into Lysobacter enzymogenes strain OH11, creating strain OH11A. The N-acyl-homoserine lactone (AHL)-degradation assay showed that transformant OH11A acquired the ability to degrade AHL molecules produced by Agrobacterium tumefaciens, Pectobacterium carotovorum, Pseudomonas syringae pv. tomato strain DC3000 and Acidovorax avenae subsp. citrulli. Pathogenicity tests showed that while the parental strain OH11 did not reduce P. carotovorum infection, the transformant OH11A caused a strong reduction of Pectobacterium virulence on Chinese cabbage and cactus, whereas strain OH11A did not seem to interfere with the normal growth of this pathogen in cabbages. In antimicrobial activity assays, strain OH11A and OH11 showed similar antimicrobial activity against Phytophthora capsici and Sclerotinia sclerotiorum. This work provided a new strategy for developing genetically engineered multi-functional L. enzymogenes strains that possessed the ability to biologically control fungal pathogens and reduce bacterial pathogenicity. 相似文献