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1.
绿色荧光蛋白及其在分子生物学上的应用 总被引：3，自引：0，他引：3
许多海洋无脊椎动物体内都含有绿色荧光蛋白,这种蛋白质结构很特殊,在受到蓝光或紫外线刺激时可以发射绿色荧光,并不需要任何协同因子、底物,适合用作普遍的报告标记,尤其适合于活体细胞或组织。而且它发出的荧光稳定,检测简单,结果真实可靠。并且GFP对光漂白、氧化剂、还原荆以及其他许多化学试剂具有极强的稳定性．易于构建载体。它独特的性质引起了生物学界的广泛关注。本文简要地概述了绿色荧光蛋白及其在分子生物学上的应用,包括蛋白融合,细胞筛选,定位标记,基因表达调控,计算细胞生长速度等。 相似文献
2.
Embryogenic calli were induced from the seeds of creeping bentgrass (Agrostis palustris Huds.) cv. Regent and colonial bentgrass (Agrostis Tenuis Sibth. Fl. Oxen.) cv. Tiger. The embryogenic calli were precultured on fresh medium for 4-7 days and then co-cultivated with Agrobacterium tumefaciens, LBA4404, which contains plasmid vector-pSBGM harboring bar coding region, synthetic green fluorescent protein (sGFP) coding region and matrix attachment region (MAR). After 3 days of co-cultivation, the calli were washed thoroughly and transferred to MS medium containing 2 mg/L of 2, 4-D, 12-15 mg/L phosphinothricin (PPT) and 250 mg/L of cefotaxime. After 2-3 months of selection, the actively growing calli of 'Regent' and 'Tiger' were transferred to MS medium with 12-15 mg/L PPT and 250 mg/L cefotaxime for regeneration. The putative transformants were maintained on MS medium with 3 mg/L PPT for long period but control died within 1 month. After establishing in greenhouse, the transformants also showed strong resistance to 0.4% of herbicide Basta but control plants died within 2 weeks. Under confocal microscope, both young leaves and roots showed significant GFP expression. PCR analysis revealed the presence of a DNA fragment of GFP gene at the expected size (380 bp) in the transformants and its absence in a randomly selected control plant. 相似文献
3.
William S. Maki 《Learning & behavior》1975,3(4):312-316
In a study of sustained attention (“vigilance”), pigeons performed a conditional discrimination in a 3-key operant chamber. Pecking a white center key initiated a 0.2- or 2.0-sec cue (a red or green disk). The side keys then displayed white disks, and a peck on the right or left key was reinforced depending on whether the preceding cue was red or green. Pecks on the white center key initiated the cue according to one of two schedules of cue production (FR 1 or VI 7.5 sec). Control of side key choices by 0.2-sec cues was disturbed by transition from FR 1 to VI 7.5, and recovered after the schedule of cue production changed from VI 7.5 back to FR 1. Control of choices by 2.0-sec cues was not affected by changing schedules of cue production. Rates of pecking the cue were higher than rates of cue-producing responses. 相似文献
4.
Wang JY Wu XY Zhang Z Du XF Chai RY Liu XH Mao XQ Qiu HP Wang YL Lin FC Sun GC 《Journal of Zhejiang University. Science. B》2008,9(10):802-810
The peroxisomal matrix proteins involved in many important biological metabolism pathways in eukaryotic cells are encoded by nucleal genes, synthesized in the cytoplasm and then transported into the organelles. Targeting and import of these proteins depend on their two peroxisomal targeting signals (PTS1 and PTS2) in sequence as we have known so far. The vectors of the fluorescent fusions with PTS, i.e., green fluorescence protein (GFP)-PTSI, GFP-PTS2 and red fluorescence protein (RFP)-PTS1, were constructed and introduced into Magnaporthe oryzae Guy11 cells. Transformants containing these fusions emitted fluorescence in a punctate pattern, and the locations of the red and green fluorescence overlapped exactly in RFP-PTS1 and GFP-PTS2 co-transformed strains. These data indicated that both PTS 1 and PTS2 fusions were imported into peroxisomes. A probable higher efficiency of PTSI machinery was revealed by comparing the fluorescence backgrounds in GFP-PTS1 and GFP-PTS2 transformants. By introducing both RFP-PTS1 and GFP-PTS2 into △mgpex6 mutants, the involvement of MGPEX6 gene in both PTS1 and PTS2 pathways was proved. In addition, using these transformants, the inducement of peroxisomes and the dynamic of peroxisomal number during the pre-penetration processes were investigated as well. In summary, by the localization and co-localization of PTSI and PTS2, we provided a useful tool to evaluate the biological roles of the peroxisomes and the related genes. 相似文献
5.
柴明良 汪炳良 KIM Jae-yeoul LEE Jong-min KIM Doo-hwan 《Journal of Zhejiang University. Science. B》2003,(3)
INTRODUCTIONCreepingbentgrass (AgrostispalustrisHuds.)isanoutstandingcool seasonspeciesavailableforuseongolfcourseputtinggreens,tees,andcloselymowedfairways.Althoughco lonialbentgrass (AgrostistenuisSibth .Fl.Ox en .)isnotwelladaptedtotheverylowmowingheig… 相似文献
6.
A 21-day laboratory incubation experiment was conducted to investigate the impact of pesticides (insecticide, herbicide, fungicide)
on paddy field soil health under controlled moisture (flooded soil) and temperature (25°C) environment. The electron transport
system (ETS)/Dehydrogenase activity showed negative correlation with pesticides concentrations, decreased with increase of
pesticide concentration. The higher doses (5 to 10 times field rates) of pesticides significantly inhibited ETS activity,
while lower rates failed to produce any significant reducing effect on the control. The toxicity of pesticides in decreasing
the ETS activity was in the order: insecticide > fungicide > herbicide, irrespective of their rates of application. The pesticides
increased the soil phenol content, which increased with increasing concentration of agrochemicals. The pesticide application
did not produce any significant change in soil protein content. The response of biomass phospholipid content was nearly similar
to that of ETS activity. The phospholipid content decreased with the addition of pesticides in the order insecticide > fungicide
> herbicide and the toxicity was in the order: 10 FR (field rate) > 5 FR > 1.0 FR > 0.5 FR > control.
Project (No. G1999011809) supported by the National Key Basic Research Foundation (973) of China
Ph. D. student at Zhejiang University, from Ministry of Agriculture (Punjab), Government of Pakistan 相似文献
7.
Schedule-induced polydipsia was studied using a behavioral contrast paradigm. Food pellets were delivered to food-deprived rats on a response-independent FT 1-min schedule. Licking on a tube produced water on a MULT FR 10 FR 10, MULT FR 10 EXT, or MIXED FR 10 EXT for three rats (Experiment 1) and on a MULT VI VI, MULT VI EXT, or MIXED VI EXT schedule for three other rats (Experiment 2). On the FR schedules, rats could drink more water by increasing lick rates, but on the VI schedules the amount of drinking was fixed by the schedule parameters and was relatively unaffected by lick rates. Relative to MULT FR FR, positive polydipsia contrast was clearly demonstrated on MULT and MIXED FR EXT; but relative to MULT VI VI, contrast was not demonstrated on MULT and MIXED VI EXT. These data suggest that polydipsia contrast occurs only if increased licking permits increased drinking. 相似文献
8.
Fu Y Wang SQ Liu YP Wang GP Wang JT Gong SS 《Journal of Zhejiang University. Science. B》2008,9(4):299-305
Objective: To evaluate the transduction efficiency of a recombinant adenovirus carrying the gene for green fluorescent protein (Ad-GFP) into the primary cultures of fetal neural stem cells (NSCs) by the expression of GFP. Methods: The Ad-GFP was constructed by homologous recombination in bacteria with the AdEasy system; NSCs were isolated from rat fetal hippocampus and cultured as neurosphere suspensions. After infection with the recombinant Ad-GFP, NSCs were examined with a fluorescent microscopy and a flow cytometry for their expression of GFP. Results: After the viral infection, flow cytometry analysis revealed that the percentage of GFP-positive cells was as high as 97.05%. The infected NSCs sustained the GFP expression for above 4 weeks. After differentiated into astrocytes or neurons, they continued to express GFP efficiently. Conclusion: We have successfully constructed a viral vector Ad-GFP that can efficiently infect the primary NSCs. The reporter gene was showed fully and sustained expression in the infected cells as well as their differentiated progenies. 相似文献
9.
INTRODUCTION Soil-borne pathogens, including Pythium spp. and Fusarium spp., cause significant yield losses in horticulture and agriculture crops (Mao et al., 1997). Current practices for controlling plant diseases are based largely on disease resistant crops, cultivation management in fields and application of synthetic pesticides (Elizabeth and Emmert, 1999). Biological control using antagonistic microbes to reduce the use of chemical pesticides in a system of integrated plantdisease … 相似文献
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11.
Embryogenic calli were induced from the seeds of creeping bentgrass (Agrostis palustris Huds.) cv. Regent and colonial bentgrass (Agrostis Tenuis Sibth. Fl. Oxen.) cv. Tiger. The embryogenic calli were precultured on fresh medium for 4–7 days and then co-cultivated withAgrobacterium tumefaciens, LBA4404. which contains plasmid vector-pSBGM harboring bar coding region, synthetic green fluorescent protein (sGFP) coding
region and matrix attachment region (MAR). After 3 days of co-cultivation, the calli were washed thoroughly and transferred
to MS medium containing 2 mg/L of 2, 4-D, 12–15 mg/L phosphinothricin (PPT) and 250 mg/L of cefotaxime. After 2–3 months of
selection, the actively growing calli of ‘Regent’ and ‘Tiger’ were transferred to MS medium with 12–15 mg/L PPT and 250 mg/L
cefotazime for regeneration. The putative transformants were maintained on MS medium with 3 mg/L PPT for long period but control
died within 1 month. After establishing in greenhouse, the transformants also showed strong resistance to 0.4% of herbicide
Basta but control plants died within 2 weeks. Under confocal microscope, both young leaves and roots showed significant GFP
expression. PCR analysis revealed the presence of a DNA fragment of GFP gene at the expected size (380 bp) in the transformants
and its absence in a randomly selected control plant. 相似文献
12.
Xingchi KAN Yingsheng CHEN Bingxu HUANG Shoupeng FU Wenjin GUO Xin RAN Yu CAO Dianwen XU Ji CHENG Zhanqing YANG Yanling XU 《Journal of Zhejiang University. Science. B》2021,22(11):929
Inflammation plays an important role in the development of acute lung injury (ALI). Severe pulmonary inflammation can cause acute respiratory distress syndrome (ARDS) or even death. Expression of proinflammatory interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS) in the process of pulmonary inflammation will further exacerbate the severity of ALI. The purpose of this study was to explore the effect of Palrnatine (Pa) on lipopolysaccharide (LPS)-induced mouse ALI and its underlying mechanism. Pa, a natural product, has a wide range of pharmacological activities with the potential to protect against lung injury. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed to detect the expression and translation of inflammatory genes and proteins in vitro and in vivo. Immunoprecipitation was used to detect the degree of P65 translocation into the nucleus. We also used molecular modeling to further clarify the mechanism of action. The results showed that Pa pretreatment could significantly inhibit the expression and secretion of the inflammatory cytokine IL-1β, and significantly reduce the protein level of the proinflammatory protease iNOS, in both in vivo and in vitro models induced by LPS. Further mechanism studies showed that Pa could significantly inhibit the activation of the protein kinase B (Akt)/nuclear factor-κB (NF-κB) signaling pathway in the LPS-induced ALI mode and in LPS-induced RAW264.7 cells. Through molecular dynamics simulation, we observed that Pa was bound to the catalytic pocket of Akt and effectively inhibited the biological activity of Akt. These results indicated that Pa significantly relieves LPS-induced ALI by activating the Akt/NF-κB signaling pathway. 相似文献
13.
目的:我们的目标是从源于胚胎体(EBs)的小鼠胚胎干细胞(mESCs)中分离运动神经元样细胞前体(MNLCPs),以用于发展针对运动神经元疾病的药物筛选试验和移植疗法.天然Shh蛋白(或Shh通路激动剂)和维甲酸诱导的胚胎体中,MNLCPs和未分化细胞的含量是不确定的.如果不把未分化的细胞从细胞培养中充分去除,其可能会干涉药物筛选试验或在移植后增生.我们开发了一种以密度梯度离心法为基础的富集MNLCPs的方法.方法:我们用Wichterle等人2008年改进的方法,将mESCs( HBG3:eGFP:HB9)扩大和分化.通过化学和酶学的无研磨处理,含有绿色荧光蛋白的MNLCPs和未分化细胞的胚胎体被小心轻轻地离解成单细胞.利用OptiprepTM8％～20％逐步梯度离心技术将MNLCPs回收.拥有绿色荧光蛋白的MNLCPs的含量由流式细胞仪检测.结果:我们的结果表明,在胚胎体形成前,mESCs在明胶包被的培养板上生长,其分化为MNLCPs的能力减少.比较mESCs在明胶,明胶与PMEFs,及PMEFs包被的培养板上的生长发现,mESCs在PMEFs包被的培养板上产生含绿色荧光蛋白的MNLCPs的得率为(54.1±11.0)％(x-±s;n=12),在明胶包被的培养板上的得率为(2.8±1.1)％(x-±s;n=9).用密度梯度离心法获得的含绿色荧光蛋白的MNLCPs的平均含量为(87.7±5.5)％(x-±s;n=3).结论:我们的数据表明,不使用细胞分选器,无研磨解离和密度梯度离心法也能用于富集具有高存活率的MNLCPs.有必要对MNLCPs在体外、体内和表型上进行进一步的生理学意义(如神经轴突的生长及形成神经肌肉接头的能力)上的鉴定. 相似文献
14.
报导了大孔型交联的聚乙烯苄四乙烯五胺树脂对Cr(V1)的吸附、洗脱条件,吸附速率常数及活化能、反应热力学函数、Freundlich常数等,并讨论了吸附机理.该树脂对微量(Cr(V1)的富集、回收或除去均有较好的效果. 相似文献
15.
Rats were trained on fixed ratio (FR) schedules requiring either 5 or 10 leverpresses to produce reinforcement and an intertrial interval (ITI). Half of the Ss at each ratio requirement were extinguished on an FR 5 and half on an FR 10 schedule of ITI presentation. Fewer foodcup approaches were made on the FR 10 than on the FR 5 extinction schedule, regardless of acquisition FR. Leverpresses per approach were fewer on the FR 5 than on the FR 10 extinction schedule and were fewer following FR 5 than following FR 10 acquisition. Data suggested the existence of interoceptive as well as exteroceptive stimulus control of foodcup approach and were discussed in terms of their implications for a response-unit account of extinction. 相似文献
16.
Zhang Xin Zhang Bing-xin Zhang Zhen Shen Wei-feng Yang Ching-hong Yu Jing-quan Zhao Yu-hua 《Journal of Zhejiang University. Science. B》2005,6(8):770-777
Fusarium head blight (FHB) caused byFusarium graminearum is a devastating disease that results in extensive yield losses to wheat and barley. A green fluorescent protein (GFP) expressing
plasmid pRP22-GFP was constructed for monitoring the colonization of two biocontrol agents,Brevibacillus brevis ZJY-116, on the spikes of barley and their effect on suppression of FHB. Survival and colonization of theBrevibacillus brevis ZJY-1 andBacillus subtilis ZJY-116 strains on spikes of barley were observed by tracking the bacterial transformants with GFP expression. Our field
study revealed that plasmid pRP22-GFP was stably maintained in the bacterial strains without selective pressure. The retrieved
GFP-tagged strains showed that the bacterial population fluctuation accorded with that of the rain events. Furthermore, both
biocontrol strains gave significant protection against FHB on spikes of barley in fields. The greater suppression of barley
FHB disease was resulted from the treatment of barley spikes with biocontrol agents before inoculation withF. graminearum.
Project supported by the National Natural Science Foundation of China (No. 30230250) and Science and Technology Committee
of Zhejiang Province (No. 2003C22029), China 相似文献
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18.
Proapoptotic and pronecrosis effect of different truncated hepatitis C virus core proteins 总被引：2，自引：0，他引：2
Yan XB Chen Z Luo DH Xu XY Wu W Zhou LF 《Journal of Zhejiang University. Science. B》2005,6(4):295-300
Objective: To study the roles of different truncated hepatitis C virus (HCV) core proteins (CORE) in the pathogenesis of HCV persistent infection and hepatocellular carcinoma (HCC) and to assess intracellular localization in transiently transfected cells. Methods: Seven truncated CORE-GFP (green fluorescent protein) fusion protein expression plasmids were constructed,which contained HCV CORE sequences derived from tumor tissues (BT) and non-tumor tissues (BNT) from one patient infected with HCV. Amino acid (aa) lengths were BT: 1-172 aa, 1-126 aa, 1-58 aa, 59-126 aa, 127-172 aa; BNT: 1-172 aa and C191:1-172 aa respectively. Subcellular localization of CORE-GFP was analyzed by con-focal laser scanning microscope. Apoptosis and necrosis were quantified by flow cytometry. Results: Different truncated CORE-GFP localized mainly in the cytoplasm, but nuclear staining was also observed. HCV CORE could induce apoptosis and necrosis, and different truncated COREs could induce cell apoptosis and necrosis at different levels. Among the same length 1-172 aa of BT, BNT and C191, the cell apoptosis and necrosis percentage of BT is highest, and C191 is the lowest (BT＞BNT＞C191). To the different fragment COREs of BT,N-terminal of CORE induced apoptosis and necrosis higher, compared with that of C-terminal (1-172 aa＞1-126 aa＞1-58aa＞ 127-172 aa＞59-126 aa). Conclusion: These results suggest HCV CORE could induce apoptosis and necrosis of cells, which might play an important role in the pathogenesis of HCV persistent infection and HCC and the different CORE domains of different HCV quasi-species might have some difference in their pathogenesis. 相似文献
19.
陈家越 《宁德师专学报(自然科学版)》2012,24(1):43-46
考察了自制的印刷碳电极活化后对去甲肾上腺素的电化学催化氧化作用研究,结果表明,在3×10-7～1.0×10-4mol/L的浓度范围内,响应电流与去甲肾上腺素的浓度呈线性关系,检出限为1×10-7mol/L.该修饰电极可直接应用于重酒石酸去甲肾上腺素的样品分析检测,结果令人满意. 相似文献
20.
Following barpress training with different terminal fixed ratios (FR), rats were given the interpolated experiences of runway acquisition and extinction (as part of another experiment) followed by 2 months of vacation. Then they were tested in FR 10 barpress reacquisition, FR 10 barpress extinction, consistently reinforced runway reacquisition, and a second runway extinction. In a start (response initiation) measure, resistance to extinction during the FR 10 extinction and in the second runway extinction was positively related to the terminal FR values of the initial barpress training, an indication of highly durable differential persistence effects attributable to the initial training to different terminal fixed ratios of barpress responding. 相似文献