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51.
53.
A low copy number cosmid 总被引:8,自引:1,他引:7
A low copy number cosmid was constructed by subcloning the pair of cos sites and the kanamycin resistance gene of pcos2EMBL into pGB2. The resulting cosmid, pPR691, has the pSC101 replicon and specifies resistance to kanamycin, spectinomycin, and streptomycin. pPR691 also carries restriction sites suitable for cloning partial Sau3A digests using the strategy of Bates and Swift (P. F. Bates and R. A. Swift, 1983, Gene 26, 137-146). A library of Salmonella typhimurium chromosomal DNA was made using this cosmid and the rfb gene cluster (map position 42) was isolated from this library. 相似文献
54.
盐胁迫下大豆根组织定量PCR分析中内参基因的选择 总被引:1,自引:0,他引:1
实时荧光定量PCR已广泛用于基因表达的分析, 适当的内参基因选择是获得准确分析结果的关键。在大豆(Glycine max)分子生物学研究中, 逆境响应基因和microRNA (miRNA)表达的内参辅助检测基因均有哪些目前尚不清楚。该研究选用不同盐梯度和时间点组合处理的大豆根组织为材料, 对已报道的其它条件下表达相对稳定的内参基因(ACT、ACT2/7、CYP2、ELF1A、ELF1B、F-Box、TUA和UBC2)以及miRNA内参基因(U6、miR1515a、miR1520c、miR1520d、miR171a和miR171b)的表达情况进行了全面检测; 并采用Δ-Ct、Bestkeeper、NormFinder和Genorm四种方法对检测结果进行了综合分析, 发现ELF1B和CYP2适合作为大豆根系盐胁迫响应基因研究的内参基因, miR1515a和U6适合作为盐胁迫下大豆根组织miRNA研究的内参。上述研究结果为大豆盐胁迫响应基因和miRNA表达及其进一步的功能研究奠定了基础。 相似文献
55.
Precise centromere mapping using a combination of repeat junction markers and chromatin immunoprecipitation-polymerase chain reaction 下载免费PDF全文
Luce AC Sharma A Mollere OS Wolfgruber TK Nagaki K Jiang J Presting GG Dawe RK 《Genetics》2006,174(2):1057-1061
Centromeres are difficult to map even in species where genetic resolution is excellent. Here we show that junctions between repeats provide reliable single-copy markers for recombinant inbred mapping within centromeres and pericentromeric heterochromatin. Repeat junction mapping was combined with anti-CENH3-mediated ChIP to provide a definitive map position for maize centromere 8. 相似文献
56.
Tsihlis ND Oustwani CS Vavra AK Jiang Q Keefer LK Kibbe MR 《Cell biochemistry and biophysics》2011,60(1-2):89-97
Nitric oxide (NO) limits formation of neointimal hyperplasia in animal models of arterial injury in large part by inhibiting vascular smooth muscle cell (VSMC) proliferation through cell cycle arrest. The ubiquitin-conjugating enzyme UbcH10 is responsible for ubiquitinating cell cycle proteins for proper exit from mitosis. We hypothesize that NO prevents VSMC proliferation, and hence neointimal hyperplasia, by decreasing levels of UbcH10. Western blotting and immunofluorescent staining showed that NO reduced UbcH10 levels in a concentration-dependent manner in VSMC harvested from the abdominal aortas of Sprague-Dawley rats. Treatment with NO or siRNA to UbcH10 decreased both UbcH10 levels and VSMC proliferation (P<0.001), while increasing UbcH10 levels by plasmid transfection or angiotensin II stimulation increased VSMC proliferation to 150% (P=0.008) and 212% (P=0.002) of control, respectively. Immunofluorescent staining of balloon-injured rat carotid arteries showed a ~4-fold increase in UbcH10 levels, which was profoundly decreased following treatment with NO. Western blotting of carotid artery lysates showed no UbcH10 in uninjured vessels, a substantial increase in the injury alone group, and a significant decrease in the injury+NO group (~3-fold reduction versus injury alone). Importantly, in vitro and in vivo, a marked increase in polyubiquitinated UbcH10 was observed in the NO-treated VSMC and carotid arteries, respectively, indicating that NO may be decreasing unmodified UbcH10 levels by increasing its ubiquitination. Central to our hypothesis, we report that NO decreases UbcH10 levels in VSMC in vitro and following arterial injury in vivo in association with increasing polyubiquitinated-UbcH10 levels. These changes in UbcH10 levels correlate with VSMC proliferation and neointimal hyperplasia, making UbcH10 a promising therapeutic target for inhibiting this proliferative disease. 相似文献
57.
Soomin Lee Zheng Li Dehua Meng Qinming Fei Libo Jiang Tengfei Fu Ze Wang Shuhao Liu Jian Zhang 《Acta biochimica et biophysica Sinica》2021,(11):1516-1526
Vascularization is an important early indicator of osteogenesis involving biomaterials.Bone repair and new bone formation are associated with extensive neovascu... 相似文献
58.
59.
转基因红花中角质细胞生长因子KGF-1的表达 总被引:3,自引:0,他引:3
通过构建重组表达质粒载体p139035S-KGF1和根癌农杆菌介导在红花(Carthamus tinctorius)中表达角质细胞生长因子(KGF-1)。从侵染到诱导生根共需要14周, 转化率达0.1%。红花子叶在潮霉素筛选培养基上培养4–5周后便可获得丛生芽, 再生芽移入含潮霉素的伸长生根培养基, 培养4–8周可诱导生根。通过PCR、Southern blot、RT-PCR及Western blot检测证明目的基因KGF-1已经整合到红花细胞的染色体中, 实现了KGF-1外源蛋白在红花中的成功表达, 为开发KGF-1蛋白新的生产途径奠定了基础。 相似文献
60.
Mitochondrial impairment is hypothesized to contribute to the pathogenesis of chronic cholestatic liver diseases. Mitofusin 2 (Mfn2) regulates mitochondrial morphology and signaling and is involved in the development of numerous mitochondrial-related diseases; however, a functional role for Mfn2 in chronic liver cholestasis which is characterized by increased levels of toxic bile acids remain unknown. Therefore, the aims of this study were to evaluate the expression levels of Mfn2 in liver samples from patients with extrahepatic cholestasis and to investigate the role Mfn2 during bile acid induced injury in vitro. Endogenous Mfn2 expression decreased in patients with extrahepatic cholestasis. Glycochenodeoxycholic acid (GCDCA) is the main toxic component of bile acid in patients with extrahepatic cholestasis. In human normal hepatocyte cells (L02), Mfn2 plays an important role in GCDCA-induced mitochondrial damage and changes in mitochondrial morphology. In line with the mitochondrial dysfunction, the expression of Mfn2 decreased significantly under GCDCA treatment conditions. Moreover, the overexpression of Mfn2 effectively attenuated mitochondrial fragmentation and reversed the mitochondrial damage observed in GCDCA-treated L02 cells. Notably, a truncated Mfn2 mutant that lacked the normal C-terminal domain lost the capacity to induce mitochondrial fusion. Increasing the expression of truncated Mfn2 also had a protective effect against the hepatotoxicity of GCDCA. Taken together, these findings indicate that the loss of Mfn2 may play a crucial role the pathogenesis of the liver damage that is observed in patients with extrahepatic cholestasis. The findings also indicate that Mfn2 may directly regulate mitochondrial metabolism independently of its primary fusion function. Therapeutic approaches that target Mfn2 may have protective effects against hepatotoxic of bile acids during cholestasis. 相似文献