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11.
Efforts to control viral diseases of grapevine include the production of certified material and development of virus-resistant transgenic grapevines. However, effective antiviral agents, once the viruses have infected the plants, are still lacking. This study shows that a crude garlic extract has significant antiviral activity against grapevine viruses. Replication of grapevine leafroll-associated virus 2 (GLRaV-2) was obviously inhibited in grapevine cv. Cabernet Sauvignon calli treated with diluted (1:100) garlic extract. The relative RNA levels of GLRaV-2 and grapevine fleck virus (GFkV) in cv. Summer Black grapevine in in vitro-grown plantlets 10 days after treatment with diluted (1:100) garlic extract were about 22% and 20%, respectively, of that in controls. The viral RNA accumulation of GLRaV-2, GFkV, grapevine virus A (GVA), grapevine fanleaf virus (GFLV) and grapevine rupestris stem pitting-associated virus (GRSPaV) in field-grown grapevine cv. Centennial Seedless plants sprayed with diluted (1:100) garlic extract were about 31–40%, 26–38%, 18–31%, 17–42% and 15–18%, respectively, of that in controls. Moreover, the garlic extract treatment led to a significant decrease in viral RNA accumulation of GLRaV-3, GLRaV-2, GVA, GFkV, GFLV, GRSPaV and grapevine Pinot Gris virus in pot-grown grapevine cv. Shine Muscat plants, and viral disease symptoms in these plants were obviously attenuated. In addition, this extract significantly induced expression of pathogenesis-related protein genes and stimulated activity of antioxidant enzymes in grapevines. Taken together, these results indicate that the crude garlic extract acts as a significant inhibitor against a broad range of grapevine viruses.  相似文献   
12.
甜樱桃花芽不同发育时期内参基因的筛选与验证   总被引:1,自引:0,他引:1  
为了筛选甜樱桃花芽不同发育时期均稳定表达的内参基因,以甜樱桃桑提娜和黔樱一号不同发育时期花芽为材料,通过qRT-PCR技术检测28S rRNA、EF1-a1、EF1-a2、UBC、RPL13、18S rRNA、RSP3、CYP40、ACT2和α-TUB3等10个常用看家基因的表达水平,并利用GeNorm、NormFinder和BestKeeper综合评价其表达稳定性。结果表明,EF-1a2和RSP3在所有样品中稳定性最好。分别以EF-1a2、RSP3及EF-1a2+RSP3作为内参基因检测不同发育时期花芽生长素运输载体AUX1基因及生长素响应因子ARF基因的表达模式,该2个基因在不同内参基因标定下表达模式相同。表明EF-1a2、RSP3及EF-1a2+RSP3可作为甜樱桃花芽不同发育时期的内参基因。  相似文献   
13.
【目的】构建柑橘脉突病毒(citrus vein enation virus,CVEV)侵染性克隆,为从分子水平解析其致病机理打下基础。【方法】利用SMARTer? RACE(rapid amplification of cDNA ends)试剂盒对CVEV的5′序列进行RACE,并依据序列分析结果及CVEV分离株VE-1保守序列,设计CVEV基因组全长cDNA扩增引物。以CVEV毒源植株的总RNA为模板,通过EV25-F/EV5983-R引物扩增CVEV基因组全长cDNA。利用In-Fusion重组连接线性化pXT1和CVEV全长cDNA。通过菌液PCR及测序分析鉴定CVEV基因组全长cDNA克隆。通过农杆菌介导的真空浸润接种摩洛哥酸橙(Citrus aurantium)、邓肯葡萄柚(C. paradisi)、尤力克柠檬(C.limon)、枳柚(C. paradisi×Poncirus trifoliata)、Rusk枳橙(P. trifoliata×C. sinensis)、枣阳小叶枳(P. trifoliata),进一步通过RT-PCR检测、症状观察鉴定所构建CVEV全长cDNA克隆的侵染性。【结果】建立了CVEV的基因组全长RT-PCR扩增体系,获得基于双元载体pXT1的CVEV基因组全长cDNA克隆10个。随机选取的6个全长cDNA克隆CVEV1901—CVEV1906的序列一致性为99.35%。其中,CVEV1901基因组全长5 983 nt,由5个开放阅读框、5′端207 nt和3′端198 nt的两个非翻译区、以及ORF2和ORF3之间122 nt的基因间隔区组成。序列分析结果显示,CVEV1901与浙江分离株XZG及四川SM分离株的序列一致性分别为99.98%和99.11%;与西班牙VE-1分离株、美国加州VE701分离株和日本IBK分离株基因组序列一致性在96.89%—98.61%;与同属中豌豆耳突花叶病毒(pea enation mosaic virus)和紫花苜蓿耳突病毒(alfalfa enamovirus)的序列一致性约90%。通过农杆菌介导的真空浸润将CVEV1901接种至6个不同的柑橘品种,接种后120 d的RT-PCR检测结果表明摩洛哥酸橙、邓肯葡萄柚、尤力克柠檬、枳柚、Rusk枳橙和枣阳小叶枳阳性植株/接种植株(阳性率)分别为16/17(94.12%)、12/14(85.71%)、16/21(76.19%)、15/19(78.95%)、13/14(92.86%)和0/18(0)。其中,部分摩洛哥酸橙出现典型CVEV侵染症状,叶片侧脉和支脉产生耳状小突起,叶背有相应的凹陷;部分邓肯葡萄柚和尤力克柠檬出现叶片皱缩现象。【结论】建立了CVEV的基因组全长RT-PCR扩增体系,获得了CVEV基因组全长cDNA侵染性克隆,通过农杆菌介导的真空浸润接种可引起摩洛哥酸橙、邓肯葡萄柚和尤力克柠檬的CVEV侵染症状。  相似文献   
14.
辣(甜)椒是我国栽培面积最大的蔬菜作物,年播种面积约2.2×106 hm2,其中甜椒约5×105hm2。生产上传统病害如疫病、病毒病等依然严峻,近年来辣椒轻斑驳病毒(PMMoV,烟草花叶病毒属)等新型流行病害爆发,严重制约甜椒生产;同时,消费者对品种的品质、多样性提出更高要求。笔者课题组先后从国内外引进甜(辣)椒种质资源1 400余份,通过鉴定、评价,筛选出具有抗病毒病、白粉病和果大、皮薄、光泽度好、品质优等优良性状的种质资源120余份。构建了与抗TMV(L3L4)、抗番茄斑点萎蔫病毒TSWV(Tsw)等重要性状紧密连锁的分子标记,辅助育种准确率达90%以上。通过常规育种技术和分子标记相结合,创制出含有抗PMMoV(P0,1,2L3,且兼具抗TMV、ToMV、PMMoV(P0,1,2)、CMV和疫病,果大、果实均一度高、光泽度好等综合性状优良、配合力高的甜椒骨干亲本‘0516’,以其为骨干亲本,培育出4个新一代优质、多抗、适应不同生态区的新品种‘中椒105号’‘中椒106号’‘中椒107号’‘中椒108号’。上述系列新品种含有L 3,抗TMV、PMMoV(P0,1,2),兼抗CMV和疫病;果实形状大小、色泽、整齐度等商品品质,Vc等营养品质显著提高。  相似文献   
15.
正已知有3种病毒可在自然条件下侵染竹类植物,即竹花叶病毒(bamboo mosaic virus,BaMV)~[1]、樱桃坏死锈斑驳病毒(cherry necrotic rusty mottle virus,CNRMV)和苹果茎沟病毒(apple stem grooving virus,ASGV)~[2,3]。其中,BaMV是最早在巴西的金竹(Bambusa vulgaris Cv.)和孝顺竹  相似文献   
16.
Cf-2/Rcr3~(pim)基因型番茄不仅能够抵御番茄叶霉菌的侵染,而且对马铃薯金线虫的寄生也有一定的抑制效果。为挖掘根结线虫的新抗性资源,本研究采用室内人工接种法测定了Cf-0/Rcr3~(pim)、Cf-2/Rcr3-3和Cf-2/Rcr3~(pim)基因型番茄品系对南方根结线虫的抗感性。抗性评价结果显示,Cf-0/Rcr3~(pim)品系对南方根结线虫表现高感,Cf-2/Rcr3-3品系为中感,而Cf-2/Rcr3~(pim)品系则为感病。与Cf-0/Rcr3~(pim)和Cf-2/Rcr3-3基因型相比,Cf-2/Rcr3~(pim)基因型番茄品系虽然对南方根结线虫侵染的敏感性略低,但是不能阻止线虫在根系上的大量繁殖,不适于根结线虫的防控应用。  相似文献   
17.
AIM: To investigate the effect of Linc00152 on the viability, apoptosis and radiosensitivity of cervical cancer cells. METHODS: RT-qPCR was used to detect the expression levels of Linc00152 and microRNA-376c-3p(miR-376c-3p) in human cervical cancer HeLa cells and SiHa cells, and normal cervical Ect1/E6E7 cells. The cervical cancer HeLa cells with low Linc00152 expression or miR-376c-3p over-expression were established. MTT assay, flow cytometry, colony formation assay and Western blot were used to determine the cell viability, apoptosis, radiosensitivity and related protein expression. The dual-luciferase reporter assay was used to verify the regulatory relationship between Linc00152 and miR-376c-3p in the HeLa cells. RESULTS: Compared with the Ect1/E6E7 cells, Linc00152 was up-regulated in the HeLa cells and SiHa cells, and miR-376c-3p was down-regulated (P < 0.05). Low expression of Linc00152 or over-expression of miR-376c-3p inhibited the viability of HeLa cells, induced apoptosis, enhanced the radiosensitivity, inhibited the protein expression of cyclin D and Bcl-2, and promoted the protein expression of P21 and Bax (P < 0.05). Linc00152 negatively regulated miR-376c-3p expression in the HeLa cells, and inhibition of miR-376c-3p expression reversed the effect of low expression of Linc00152 on HeLa cell viability, apoptosis and radiosensitivity. CONCLUSION: Linc00152 is highly expressed in the cervical cancer cells. Linc00152 affects the viability, apoptosis and radiosensitivity of HeLa cells by targeting miR-376c-3p, which is a potential diagnosis and treatment target for cervical cancer.  相似文献   
18.
AIM To investigate the effect of sulodexide (SDX) on high glucose-induced damage in retinal microvascular endothelial cells. METHODS (1) High-fat diet combined with intraperitoneal injection of streptozocin were used to induce type 2 diabetes mellitus (DM) followed by injection of saline or SDX in C57BL/6J male mice. Retinal microvascular leakage and density, and the protein levels of NLRP3 inflammasome-related proteins, zonula occludens-1 (ZO-1) and NADPH oxidase 4 (NOX4) were measured. (2) Human retinal microvascular endothelial cells (HRMECs) were treated with normal glucose or high glucose with or without SDX, and were further transfected with siRNA to knock down NOX4, or infected by adenovirus to over-express NOX4. The protein levels of ZO-1, VE-cadherin (VE-Cad), NOX4 and NLRP3 inflammasome-related proteins as well as the level of reactive oxygen species (ROS) were detected. RESULTS Treatment with SDX increased the protein level of ZO-1, attenuated retinal leakage and NLRP3 inflammasome activation, and enhanced the density of microvasculature and the number of ganglion cells in diabetic retinas. The protein levels of ZO-1 and VE-Cad were decreased, while the levels of NOX4, NLRP3 inflammasome-related proteins and ROS generation were increased in high glucose-treated HRMECs. Silencing of NOX4 inhibited high glucose-induced increases in NLRP3 inflammasome and ROS generation, and decreases in the protein levels of ZO-1 and VE-Cad. Over-expression of NOX4 significantly increased the levels of NLRP3 inflammasome-related proteins and ROS generation in HRMECs, and reduced the protein levels of ZO-1 and VE-Cad. Treatment with SDX partly reversed NOX4 over-expression-induced changes. CONCLUSION SDX alleviates hyperglycemia-induced retinal microvascular endothelial injury via inhibiting NOX4/ROS/NLRP3 pathways.  相似文献   
19.
AIM To investigate the effect of exosomes secreted by mouse melanoma cells on the expression of Ras-related C3 botulinum toxin substrate 1 (Rac1) protein in fibroblasts. METHODS Ultracentrifugation was adopted to separete exosomes secreted by mouse melanoma B16-F10 cells. The morphological structure of exosomes was observed by negative-staining electron microscopy. The size distribution of exosomes was determined by nanoparticle tracking analysis (NTA). The exosomal markers, tumor susceptibility gene 101 (Tsg101) and tyrosinase-related protein 2 (Tyrp2), were identified by Western blot. Laser confocal microscopy was used to observe the process that mouse embryonic fibroblasts (MEF) took in exosomes during co-culture. Immunocytochemical staining and Western blot were used to detect the expression of Rac1 protein in MEF. RESULTS B16-F10 cell exosomes showed a typical tea tray-like structure, with a size range of 141~255 nm, and expressed protein markers Tsg101 and Tyrp2. The results of laser confocal microscopy showed that compared with co-culture at 0 h, a small number of exosomes appeared in the MEF at 12 h, and a large number of exosomes accumulated in the MEF after co-cultured for 24 and 36 h. Western blot analysis showed that compared with co-culture at 0 h, the expression of Rac1 protein in the MEF was significantly increased at 24 h and 36 h of co-culture (P<0.01). The results of immunocytochemical staining showed that compared with co-culture at 0 h, the positive expression level of Rac1 in the MEF cells was significantly increased at 12 h, 24 h and 36 h of co-culture (P<0.05 or P<0.01). CONCLUSION Intake of exosomes secreted by mouse melanoma cells promotes the expression of Rac1 protein in fibroblasts.  相似文献   
20.
AIMTo investigate whether Rho-associated coiled-coil kinase (ROCK) is involved in high glucose-induced apoptosis of primary cardiomyocytes by regulating PI3K/Akt signaling pathway. METHODSPrimary Wistar rat cardiomyocytes were cultured and identified by α-sarcomeric actin (α-SCA) immunohistochemistry. Cardiomyocytes were treated with 5.5, 33 and 40 mmol/L glucose for 48 h. The cell viability was measured by MTT assay, and the mRNA expression of ROCK1 and ROCK2 in the cardiomyocytes was detected by RT-qPCR. Flow cytometry was used to analyze the apoptosis of the cardiomyocytes. The protein levels of ROCK1, ROCK2, cleaved caspase-3, Bcl-2, PI3K, Akt and p-Akt were determined by Western blot. In order to confirm the regulatory effect of ROCKs on PI3K/Akt signaling pathway, the cells were divided into control group (5.5 mmol/L glucose), high glucose group (33 mmol/L glucose) and high glucose+Y27632 (ROCK inhibitor) group. Western blot was used to detect the protein levels of ROCK1, ROCK2, PI3K, Akt and p-Akt. RESULTSAfter 48 h of high glucose exposure, the values of relative cell viability in 33 and 40 mmol/L glucose groups were (79.71±2.43)% and (68.41±7.49)%, respectively, both of which were significantly decreased compared with normal control group (P<0.05). After 48 h of high glucose exposure, the relative mRNA levels of ROCK1 and ROCK2 in 33 and 40 mmol/L glucose groups were significantly increased compared with normal control group (P<0.05). Compared with normal control group, the apoptotic rate in 33 and 40 mmol/L glucose groups was increased significantly (P<0.05). Compared with normal control group, the protein expression of ROCK1, ROCK2 and cleaved caspase-3 in 33 and 40 mmol/L glucose groups was increased (P<0.05), while the protein expression of Bcl-2 was decreased (P<0.05). No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed, while the protein level of p-Akt in 33 and 40 mmol/L glucose groups was decreased compared with normal control group (P<0.05). Compared with high glucose group, the expression of ROCK1 and ROCK2 was decreased in high glucose+Y27632 group. No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed. Compared with normal control group, the protein level of p-Akt in high glucose group was decreased, and the protein level of p-Akt in high glucose+Y27632 group was increased significantly compared with high glucose group. CONCLUSION Under high glucose environment, ROCK may reduce the level of p-Akt by inhibiting the PI3K/Akt signaling pathway, thus promoting the apoptosis of cardiomyocytes.  相似文献   
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