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黄国存 张寒霜 高鹏 李俊兰 朱生伟 孙敬三HUANG Guo-cun ZHANG Han-shuang GAO Peng LI Jun-lan ZHU Sheng-wei SUN Jing-san 《遗传》2001,23(2):131-200
以绿色荧光蛋白GFP基因为报道基因,用花粉管通道和农杆菌介导的转化方法将外源基因导入棉花(Gossypium hirsutumL.),分别获得转化幼胚、幼苗和转化愈伤组织。用手持紫外灯结合显微镜检术能够快速地对转化子进行活体筛选鉴定,比用GUS检测方法有明显的优越性。本研究不但为花粉管通道转化法的可行性提供了新的证据,同时也建立了GFP用于棉花基因工程研究的检测技术体系。
Abstract:With the Green Fluorescent Protein gene (GFP) as a reporter gene, the transgenic embryos, seedlings and calli of cotton(Gossypium hirsutum L.) were obtained by the method of pollen tube pathway and Agrobacterium-mediated techniques separately. The GFP gene under the control of the 35s Cauliflower Mosaic Virus promoter produced bright?green fluorescence easily detectable and screenable in cotton tissue by fluorescence microscopy and a hand-held ultraviolet lamp. The screenable marker aided and facilated the rapid segregation of individual transformation events, drastically reduced the quantity of tissue to be handled. The GFP can be screened in vivo without destroying the materials, so it is more practical and useful than GUS. The use of GFP could advance the development of cotton gene engineering. 相似文献
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绿色荧光蛋白 总被引:26,自引:0,他引:26
来源于水母Aequorea victoria的绿色荧光蛋白(green fluorescent protein, GFP)现已成为在生物化学和细胞生物学中研究和开发应用得最广泛的蛋白质之一. 其内源荧光基团在受到紫外光或蓝光激发时(λmax=395 nm, 小峰在479 nm)可高效发射清晰可见的绿光. GFP的高分辨率晶体结构为了解和研究蛋白质结构和光谱学功能关系提供了一个极好的机会. GFP已成为一个监测在完整细胞和组织内基因表达和蛋白质定位的理想标记. 通过突变和蛋白质工程构建的GFP嵌合蛋白在生理指示剂、生物传感器、光化学领域以及生产发光纤维等方面展示了广阔前景. 相似文献
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绿色荧光蛋白在生命科学研究中的应用 总被引:7,自引:0,他引:7
贺竹梅 李华平 李宝健 李志芳 黄定华HE Zhu-mei LI Hua-ping Li Bao-jian LI Zhi-fang Huang Ding-hua 《遗传》1998,20(5):43-46
近年来,随着水母Aequoreavictoria来源的绿色荧光蛋白(Greenfluorescentprotein,GFP)在各种异源细胞,如细菌、霉菌、线虫、酵母、果蝇、昆虫细胞、哺乳动物细胞及植物细胞中的表达,GFP作为一种新型的报告物在生物学界... 相似文献
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Variants of Green Fluorescent Protein GFPxm 总被引:1,自引:0,他引:1
Luo WX Cheng T Guan BQ Li SW Miao J Zhang J Xia NS 《Marine biotechnology (New York, N.Y.)》2006,8(5):560-566
As research progresses, fluorescent proteins useful for optical marking will evolve toward brighter, monomeric forms that
are more diverse in color. We previously reported a new fluorescent protein from Aequorea macrodactyla, GFPxm, that exhibited many characteristics similar to wild-type green fluorescent protein (GFP). However, the application
of GFPxm was limited because GFPxm expressed and produced fluorescence only at low temperatures. To improve the fluorescent
properties of GFPxm, 12 variants were produced by site-directed mutagenesis and DNA shuffling. Seven of these mutants could
produce strong fluorescence when expressed at 37°C. The relative fluorescence intensities of mutants GFPxm16, GFPxm18, and
GFPxm19 were higher than that of EGFP (enhanced GFP) when the expression temperature was between 25 and 37°C, and mutants
GFPxm16 and GFPxm163 could maintain a high fluorescence intensity even when expressed at 42°C. Meanwhile, at least 4 mutants
could be successfully expressed in mammalian cell lines. The fluorescence spectra of 6 of the 12 mutants had a progressive
red shift. The longest excitation-emission maximum was at 514/525 nm. In addition, 3 of the 12 mutants had two excitation
peaks including an UV-excitation peak, while another mutant had only one UV-excitation peak. 相似文献
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Use of Green Fluorescent Protein To Tag and Investigate Gene Expression in Marine Bacteria 总被引:5,自引:3,他引:5 下载免费PDF全文
Serina Stretton Somkiet Techkarnjanaruk Alan M. McLennan Amanda E. Goodman 《Applied microbiology》1998,64(7):2554-2559
Two broad-host-range vectors previously constructed for use in soil bacteria (A. G. Matthysse, S. Stretton, C. Dandie, N. C. McClure, and A. E. Goodman, FEMS Microbiol. Lett. 145:87–94, 1996) were assessed by epifluorescence microscopy for use in tagging three marine bacterial species. Expression of gfp could be visualized in Vibrio sp. strain S141 cells at uniform levels of intensity from either the lac or the npt-2 promoter, whereas expression of gfp could be visualized in Psychrobacter sp. strain SW5H cells at various levels of intensity only from the npt-2 promoter. Green fluorescent protein (GFP) fluorescence was not detected in the third species, Pseudoalteromonas sp. strain S91, when the gfp gene was expressed from either promoter. A new mini-Tn10-kan-gfp transposon was constructed to investigate further the possibilities of fluorescence tagging of marine bacteria. Insertion of mini-Tn10-kan-gfp generated random stable mutants at high frequencies with all three marine species. With this transposon, strongly and weakly expressed S91 promoters were isolated. Visualization of GFP by epifluorescence microscopy was markedly reduced when S91 (mini-Tn10-kan-gfp) cells were grown in rich medium compared to that when cells were grown in minimal medium. Mini-Tn10-kan-gfp was used to create an S91 chitinase-negative, GFP-positive mutant. Expression of the chi-gfp fusion was induced in cells exposed to N′-acetylglucosamine or attached to chitin particles. By laser scanning confocal microscopy, biofilms consisting of microcolonies of chi-negative, GFP+ S91 cells were found to be localized several microns from a natural chitin substratum. Tagging bacterial strains with GFP enables visualization of, as well as monitoring of gene expression in, living single cells in situ and in real time. 相似文献
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绿色荧光蛋白及其应用 总被引:1,自引:0,他引:1
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,在受到激发时可以发射绿色或蓝色荧光。虽然对它的研究从本世纪六十年代才开始,但是它独特的性质逐渐引起了生物学界的广泛关注。本文将就绿色荧光蛋白的结构、性质及其应用前景作一综述。 相似文献
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Green fluorescent protein (GFP) and its homologs are widely used as fluorescent markers of gene expression and for determination of protein localization and motility in living cells. In particular, based on GFP and GFP-like proteins a number of techniques have been developed that can be used either to estimate protein mobility in living cells, or to introduce a distinctive fluorescent signal in order to track the movement of labeled molecules directly. Considerable progress in the development of such technologies in the last two or three years motivates us to reevaluate the present scope of biotechnological instruments in studies of protein movement in cells. 相似文献
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绿荧光蛋白(GFP)研究进展 总被引:6,自引:1,他引:5
GFP作为一种全新的标记基因,在生物学的各研究领域得到了广泛的应用,本文概述了的近年来相关方面的研究进展和重要应用,以及尚存在的不足。 相似文献
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绿色荧光蛋白的发光机制 总被引:1,自引:0,他引:1
从多管水母(Aequoreavictoria)中分离纯化的绿色荧光蛋白(GFP)是由238个氨基酸残基组成的单链多肽,分子量约27kD,1992年其cDNA被克隆[1]。1994年重组野生型GFP(WtGFP)在异源细胞中表达[2]。野生型GFP被紫外光和蓝光激发后能发出绿色荧光,最大荧光吸收/激发峰在395nm,在475nm有一个肩峰,荧光发射峰为508nm。GFP的结构和光致荧光非常稳定,而且因GFP生色团的形成是自催化的,检测GFP的光致荧光不需要外加底物和辅因子,便于活体观察[2]。如今… 相似文献
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Green Fluorescent Protein Purification by Organic Extraction 总被引:1,自引:0,他引:1
Alexander V. Yakhnin Leonid M. Vinokurov Alexey K. Surin Yuli B. Alakhov 《Protein expression and purification》1998,14(3):382-386
Green fluorescent protein (GFP) is widely used as an excellent reporter molecule in biochemistry and cell biology. Some biochemical and immunological assays require high-purity GFP. However, the majority of current procedures for GFP purification include multiple time-consuming chromatography steps with a low yield of the desired product or require tag-containing proteins. An alternative method is described for the GFP purification without affinity extensions using organic extraction yielding a highly homogeneous protein indistinguishable in spectroscopic properties from that purified by previous methods. 相似文献
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Prachayasittikul V Isarankura Na Ayudhya C Boonpangrak S Galla HJ 《The Journal of membrane biology》2004,200(1):47-56
The Green Fluorescent Protein (GFP) is a useful marker to trace the expression of cellular proteins. However, little is known about changes in protein interaction properties after fusion to GFP. In this study, we present evidence for a binding affinity of chimeric cadmium-binding green fluorescent proteins to lipid membrane. This affinity has been observed in both cellular membranes and artificial lipid monolayers and bilayers. At the cellular level, the presence of Cd-binding peptide promoted the association of the chimeric GFP onto the lipid membrane, which declined the fluorescence emission of the engineered cells. Binding affinity to lipid membranes was further investigated using artificial lipid bilayers and monolayers. Small amounts of the chimeric GFP were found to incorporate into the lipid vesicles due to the high surface pressure of bilayer lipids. At low interfacial pressure of the lipid monolayer, incorporation of the chimeric Cd-binding GFP onto the lipid monolayer was revealed. From the measured lipid isotherms, we conclude that Cd-binding GFP mediates an increase in membrane fluidity and an expansion of the surface area of the lipid film. This evidence was strongly supported by epifluorescence microscopy, showing that the chimeric Cd-binding GFP preferentially binds to fluid-phase areas and defect parts of the lipid monolayer. All these findings demonstrate the hydrophobicity of the GFP constructs is mainly influenced by the fusion partner. Thus, the example of a metal-binding unit used here shines new light on the biophysical properties of GFP constructs.This revised version was published online in June 2005 with a corrected cover date. 相似文献
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绿色荧光蛋白(GFP)是海洋生物水母(Aequorea victoria)体内的一种发光蛋白,分子量27kD,由238个氨基酸组成。该蛋白65~67位Ser-Tyr-Gly三种氨基酸环化加氧形成特殊的生色团结构。野生型GFP发光较弱,而且gfp-cDNA含有隐蔽型剪切位点,而加工改造的GFP在植物中能够正常表达并且加强了荧光信号。GFP作为新的报告基因和遗传标记被广泛应用于植物研究之中。 相似文献
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《Bioscience, biotechnology, and biochemistry》2013,77(4):646-649
In this study, we confirmed the ability of the 2-kb promoter fragment of the chicken ovalbumin gene to drive tissue-specific expression of a foreign EGFP gene in chickens. Recombinant lentiviruses containing the EGFP gene were injected into the subgerminal cavity of 539 freshly laid embryos (stage X). Subsequently the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Twenty-four chicks (G0) were hatched and screened for EGFP with PCR. Two chicks were identified as transgenic birds (G1), and these founders were mated with wild-type chickens to generate transgenic progeny. In the generated transgenic hens (G2), EGFP was expressed specifically in the tubular gland of the oviduct. These results show the potential of the chicken ovalbumin promoter for the production of biologically active proteins in egg white. 相似文献
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应用阳离子脂质体介导法,将含绿色荧光蛋白(GFP)基因的质粒pEGFP-N1转染到培养成单层的草鱼肾细胞(CIK)中,通过荧光倒置显微镜和特异性RT-PCR方法检测GFP的表达.在荧光倒置显微镜下可见CIK细胞的胞质和胞核均呈现绿色荧光,且细胞核的绿色荧光强度强于细胞质.转染细胞中的转录产物经RT-PCR扩增后,凝胶电泳鉴定出与GFP基因片段分子量大小一致的条带,经测序证明其为GFP基因序列.结果表明,GFP基因可以在草鱼CIK细胞内高效率成功表达,为构建以GFP为报告基因的真核重组质粒及研究草鱼出血病DNA疫苗奠定了重要的基础. 相似文献