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291.
H. Tsugawa Y. Otsuki M. Suzuki 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,97(7):1019-1026
A very simple and efficient transformation system for rice was established using a synthetic polycationic amino polymer (polycation).
Improvements in the culture conditions, especially filtration of the suspension cells before and after protoplast culture,
greatly contributed to a large yield of high-quality protoplasts and an increased ability of the cells to regenerate. Transformation
parameters, such as the ratio of DNA and polycation concentrations, preincubation of the DNA and polycation prior to DNA transfer,
and precentrifugation and resuspension of protoplasts before DNA transfer, were analyzed. Fertile transgenic plants containing
the bar gene were selected and shown to demonstrate resistance against high concentrations of bialaphos. Southern blot analysis showed
four to nine bands representing the bar gene in polycation-mediated transgenic rice plants compared with two to three bands in electroporation-mediated transgenic
rice plants. The regeneration efficiency of the polycation-mediated method was compared to that of the electroporation-mediated
method; while the polycation-mediated method tended to show a relatively lower regeneration rate, regenerants showed a normal
phenotype.
Received: 26 February 1998 / Accepted: 15 May 1998 相似文献
292.
Plastid transformation in Arabidopsis thaliana 总被引:33,自引:0,他引:33
Plastid transformation is reported in Arabidopsis thaliana following biolistic delivery of transforming DNA into leaf cells. Transforming plasmid pGS31A carries a spectinomycin resistance
(aadA) gene flanked by plastid DNA sequences to target its insertion between trnV and the rps12/7 operon. Integration of aadA by two homologous recombination events via the flanking ptDNA sequences and selective amplification of the transplastomes
on spectinomycin medium yielded resistant cell lines and regenerated plants in which the plastid genome copies have been uniformly
altered. The efficiency of plastid transformation was low: 2 in 201 bombarded leaf samples. None of the 98 plants regenerated
from the two lines were fertile.
Received: 13 February 1998 / Revision received: 24 April 1998 / Accepted: 5 June 1998 相似文献
293.
Transgenic Japanese lawngrass (Zoysia japonica Steud.) plants regenerated from protoplasts 总被引:14,自引:0,他引:14
Transgenic Japanese lawngrass (Zoysia japonica Steud.) plants were generated by means of polyethylene glycol (PEG)-mediated direct gene transfer into protoplasts. The plasmid
pBC1 was used to deliver the hygromycin phosphotransferase (hph) and β-glucuronidase (gus) genes into protoplasts. Selection with a high concentration (400 mg/l) of hygromycin yielded a number of resistant calli
and about 400 plants were generated. Polymerase chain reaction (PCR) and Southern hybridization analyses revealed that all
of then plants tested contained introduced genes. The gus gene regulated by the maize alcohol dehydrogenase-1 (Adh 1) promoter was expressed in the leaves and roots of transgenic Japanese lawngrass plants.
Received: 13 December 1996 / Revision received: 9 June 1997 / Accepted: 2 September 1997 相似文献
294.
Daniel K.Y. Solaiman 《Biotechnology Techniques》1998,12(11):829-832
An electroporation procedure for the transformation of Pseudomonas oleovorans was developed using a model plasmid, pCN51. The optimal electrotransformation was achieved with cells harvested at 45 to 60 min of growth and concentrated to a cell density of 5 OD600nm, plasmid concentration of 6 g per 100 l of cell suspension, and a 0.1-cm gap-width cuvette. Electroporation was performed at the settings of 250 , 25F and 2.5 kV. Transformation yields in the order of 103 colony-forming-unit per electroporation sample were obtained. This is a first report of the electroporation of the commercially valuable bacterium Ps. oleovorans. © Rapid Science Ltd. 1998 相似文献
295.
用BamH1和CIP处理pCB20质粒,Sau3Al部分降解枯草杆菌抗阿霉素突变株染色体DNA,T4连接酶连接载体和DNA片断,构成表达文库。用这个文库分别转化枯草杆菌BD224菌株感受态细胞和原生质体。 相似文献
296.
297.
E. M. Southgate M. R. Davey J. B. Power R. J. Westcott 《In vitro cellular & developmental biology. Plant》1998,34(3):218-224
Summary Techniques for transforming intact tissues of cereals were evaluated for their efficacy in transforming immature embryos and
Type II callus of maize (Zea mays L.). The techniques used were particle bombardment, tissue electroporation, tissue electrophoresis, and silicon carbide fibers.
Each method was assessed in terms of transient β-glucuronidase (GUS) expression. High levels of GUS expression were observed
in A188 Type II callus using both tissue electroporation and particle bombardment, with means of 417.8 and 954.5 blue expression
units (beu) per g fresh weight (FW) callus, respectively. Only particle bombardment resulted in high transient gene expression
in immature embryos, with a mean transformation frequency of 34.8 b.e.u. per embryo. Very low levels of GUS expression were
achieved with silicon carbide-mediated gene transfer, even when employing tissues used in the original publication (Black
Mexican Sweet suspension cells). GUS expression was not obtained following tissue electrophoretic gene delivery. 相似文献
298.
Analysis of mannose selection used for transformation of sugar beet 总被引:39,自引:0,他引:39
Joersbo Morten Donaldson Iain Kreiberg Jette Petersen Steen Guldager Brunstedt Janne Okkels Finn T. 《Molecular breeding : new strategies in plant improvement》1998,4(2):111-117
Various factors affecting mannose selection for the production of transgenic plants were studied using Agrobacterium tumefaciens-mediated transformation of sugar beet (Beta vulgaris L.) cotyledonary explants. The selection system is based on the Escherichia coli phosphomannose isomerase (PMI) gene as selectable gene and mannose as selective agent. Transformation frequencies were about 10-fold higher than for kanamycin selection but were only obtained at low selection pressures (1.0–1.5 g/l mannose) where 20–30% of the explants produced shoots. The non-transgenic shoots were eliminated during the selection procedure by a stepwise increase in the mannose concentration up to 10 g/l. Analysis of the transformed shoots showed that the PMI activity varied from 2.4 mU/mg to 350 mU/mg but the expression level was independent of the selection pressure. Complete resistance to mannose of transformed shoots was observed already at low PMI activities (7.5 mU/mg). Genomic DNA blot analysis confirmed the presence of the PMI gene in all transformants analysed. The possible mode of action of mannose selection compared to other selection methods is discussed. 相似文献
299.
Yokoi Shuji Higashi Sho-Ichi Kishitani Sachie Murata Norio Toriyama Kinya 《Molecular breeding : new strategies in plant improvement》1998,4(3):269-275
The chilling sensitivity of several plant species is closely correlated with the levels of unsaturation of fatty acids in the phosphatidylglycerol (PG) of chloroplast membranes. Plants with a high proportion of unsaturated fatty acids, such as Arabidopsis thaliana, are resistant to chilling, whereas species like squash with only a low proportion are rather sensitive to chilling. The glycerol-3-phosphate O-acyltransferase (GPAT) enzyme of chloroplasts plays an important role in determining the levels of PG fatty acid desaturation.A cDNA for oleate-selective GPAT of Arabidopsis under the control of a maize Ubiquitin promoter was introduced into rice (Oryza sativa L.) using the Agrobacterium-mediated gene transfer method. The levels of unsaturated fatty acids in the phosphatidylglycerol of transformed rice leaves were found to be 28% higher than that of untransformed controls. The net photosynthetic rate of leaves of transformed rice plants was 20% higher than that of the wild type at 17°C. Thus, introduction of cDNA for the Arabidopsis GPAT causes greater unsaturation of fatty acids and confers chilling tolerance of photosynthesis on rice. 相似文献
300.
Biolistic transfer of large DNA fragments to tobacco cells using YACs retrofitted for plant transformation 总被引:1,自引:0,他引:1
Mullen Jeffrey Adam Gerhard Blowers Alan Earle Elizabeth 《Molecular breeding : new strategies in plant improvement》1998,4(5):449-457
To determine whether large DNA molecules could be transferred and integrated intact into the genome of plant cells, we bombarded tobacco suspension cells with yeast DNA containing artificial chromosomes (YACs) having sizes of 80, 150, 210, or 550 kilobases (kb). Plant selectable markers were retrofitted on both YAC arms so that recovery of each arm in transgenic calli could be monitored. Stably transformed calli resistant to kanamycin (300 mg/L) were recovered for each size of YAC tested. Two of 12 kanamycin-resistant transformants for the 80 kb YAC and 8 of 29 kanamycin-resistant transformants for the 150 kb YAC also contained a functional hygromycin gene derived from the opposite YAC arm. Southern analyses using probes that spanned the entire 55 kb insert region of the 80 kb YAC confirmed that one of the two double-resistant lines had integrated a fully intact single copy of the YAC DNA while the other contained a major portion of the insert. Transgenic lines that contained only one selectable marker gene from the 80 kb YAC incorporated relatively small portions of the YAC insert DNA distal to the selectable marker. Our data suggest genomic DNA cloned in artificial chromosomes up to 150 kb in size have a reasonable likelihood of being transferred by biolistic methods and integrated intact into the genome of plant cells. Biolistic transfer of YAC DNA may accelerate the isolation of agronomically useful plant genes using map-based cloning strategies. 相似文献