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Nagy ZB Gárdonyi M Mészáros A Varga-Orvos Z Solomon RG Tamás L 《Molecular biotechnology》2007,37(3):206-211
Site-directed PCR-based mutagenesis methods are widely used to generate mutations. All published methods work on DNA clones
carrying the target sequence. However, DNA clones are not always available. We have previously published a RT-PCR-based site-directed
mutagenesis method starting from total RNA to overcome this problem. In this article, we report an improvement of our previous
method to facilitate introduction of multiple mutations into a target sequence. We demonstrate the efficacy and feasibility
of this strategy by mutation of the human β-actin gene. BamHI restriction endonuclease cleavage sites were generated within the gene to assist screening. Using three mutagenic primers
in a single RT-PCR reaction, seven different clones were produced carrying three single and four multiple mutations. An investigation
of the effect of the cycle number and elongation time of the PCR reactions revealed that both have an influence on the ratio
of clones carrying single and multiple mutations. An optimized protocol was established for efficient multiple site-directed
mutagenesis. 相似文献
155.
Ekman J Kosonen M Jokela S Kolari M Korhonen P Salkinoja-Salonen M 《Journal of industrial microbiology & biotechnology》2007,34(3):203-211
Colored biofilms cause problems in paper industry. In this work we used real-time PCR to detect and to quantitate members
of the genus Meiothermus from the process samples and end products from 24 machines manufacturing pulp, paper and board in four countries. The results
obtained from 200 samples showed the importance of members of the genus Meiothermus as ubiquitous biofoulers in paper machines. This genus was the dominant biofouler in some mills. From ≤104 to 1011 copies of Meiothermus 16S rRNA genes were found per gram of process deposit (wet weight). Meiothermus spp. were found in paper and board products with colored defects and connection between deposit-forming microbes and end-product
spots was shown. 16S rRNA gene sequences of 29 biofilm producing bacterial isolates from different mills were determined.
Based on sequence data, 25 of the isolates were assigned to the genus Meiothermus, with Meiothermus silvanus and M. ruber as the most frequent species. 相似文献
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157.
Rad51, a eukaryotic homolog of RecA, is an important protein involved in DNA recombination and repair. We have characterized rad51 of Pneumocystis carinii and Pneumocystis murina. rad51 is a single copy gene that encodes a 1.2 kb mRNA, which contains an open reading frame encoding 343 amino acids. Rad51 from Pneumocystis showed high homology to those from yeast. ATP binding motifs GEFRTGKS and LLIVD, similar to those of Saccharomyces cerevisiae and Schizosaccharomyces pombe, are conserved in Pneumocystis Rad51. The recombinant protein when expressed in E. coli showed DNA-dependent ATPase activity. Since Rad51 is a key enzyme in DNA repair and recombination, it potentially plays an important role in the recombination process leading to antigenic variation and thereby resistance to host immune responses in Pneumocystis. 相似文献
158.
Several types of disintegrins have been isolated from Crotalus spp rattlesnakes, including RGD disintegrins, and PIII-SVMPs. We isolated six cDNAs from snake venom glands using RT-PCR. Three RGD disintegrins (atroxatin, mojastin, and viridistatin) and three PIII-SVMPs (catroriarin, scutiarin, and viristiarin) cDNAs were isolated from the rattlesnakes Crotalus atrox, Crotalus scutulatus scutulatus, and Crotalus viridis viridis, respectively. Atroxatin and Viridistatin shared 90% amino acid identity to each other, and 87% identity to Mojastin. Scutiarin and Viristiarin were identical. All PIII-SVMPs isolated in this study shared the highest amino acid identity with Catrocollastatin. cDNA and protein sequences for RGD disintegrins, one MVD disintegrin, and PIII-SVMPs of the genus Crotalus (present in the NCBI database), were used in phylogenetic analysis. Neighbor-joining analysis of PIII-SVMP and RGD/MVD disintegrin-coding DNA sequences showed that these groups of genes separate into separate clades. A Phi(ST) pairwise comparison and Analysis of Molecular Variance (AMOVA) between PIII-SVMPs and RGD/MVD disintegrins showed significant genetic differences. Mutations observed in ten of the cDNAs analyzed did not affect Cys-coding sequences. Our K(A)/K(S) data suggest that rapid evolution occurred between the genes coding for PIII-SVMPs resulting, in the production of RGD disintegrin-coding genes. However, once these genes diverged, mutations in the PIII-SVMP-coding genes were accumulated less frequently. 相似文献
159.
Mariele Porto Carneiro Le?o Patricia Vieira Tiago Fernando Dini Andreote Welington Luiz de Araújo Neiva Tinti de Oliveira 《Genetics and molecular biology》2015,38(1):86-92
The entomopathogenic fungi of the genus Metarhizium have several
subtilisin-like proteases that are involved in pathogenesis and these have been used
to investigate genes that are differentially expressed in response to different
growth conditions. The identification and characterization of these proteases can
provide insight into how the fungus is capable of infecting a wide variety of insects
and adapt to different substrates. In addition, the pr1A gene has
been used for the genetic improvement of strains used in pest control. In this study
we used quantitative RT-PCR to assess the relative expression levels of the
pr1A gene in M. anisopliae and M.
acridum during growth in different culture conditions and during
infection of the sugar cane borer, Diatraea saccharalis Fabricius.
We also carried out a pathogenicity test to assess the virulence of both species
against D. saccharalis and correlated the results with the pattern
of pr1A gene expression. This analysis revealed that, in both
species, the pr1A gene was differentially expressed under the growth
conditions studied and during the pathogenic process. M. anisopliae
showed higher expression of pr1A in all conditions examined, when
compared to M. acridum. Furthermore, M. anisopliae
showed a greater potential to control D. saccharalis. Taken
together, our results suggest that these species have developed different strategies
to adapt to different growing conditions. 相似文献
160.