一种道路照明光源的光视光效模型研究

文章依据视觉功效法建立基于中间视觉道路照明的行车视觉行为反应时间测试系统,获取受测试数据集;根据人眼对光谱敏感度适应效应理论,对实验测试数据进行分析处理,构建了基于行车视觉行为反应时间的光视光效模型,获得了中间视觉下光视光效率曲线;采用建立的模型给出了基于反应时间的光源节能性评价计算分析方法,并对道路照明传统白色和黄白色光源与设计的白绿色光源进行了光视光效性能分析与节能评价。结果表明,在中间视觉下设计的白绿色光源光视性能更好,且光视效能更高。

基于道路照明标准的LED光源配光图像清晰度研究

通过分析人眼的视觉结构特点和道路照明亮度标准,提出一种基于三基色的配光模型。对模型的综合验证表明,在相同亮度情况下,R、G、B的三刺激值是呈非线性增大的,并且绿色刺激值最大。通过计算图像清晰度和SP的值,说明LED白绿光用作道路照明效果更佳。

Human Pro-insulin Transgenic Calf Derived from Somatic Cell Nuclear Transfer

The current study was undertaken to evaluate the possibility of producing a human pro-insulin transgenic cow by means of somatic cell nuclear transfer （SCNT）. A double selection system, Neomycin resistance （Neo^r） gene and enhanced green fluorescent protein （EGFP） gene linked through an inner ribosomal entry site （IRES） sequence directed by a Cytomegalovirus （CMV） promoter, was used for enrichment and selection of the transgenic cells and preimplantation embryos. Transgenes were introduced into bovine fetal fibroblast cells （BFF） cultured in vitro through electroporation （900 V/cm, 5 ms）. Transgenic bovine fibroblast cells （TBF） were enriched through addition of G418 in culture medium （800 μg/mL）. Before being used as a nuclear donor, the TBF cells were either cultured in normal conditions （10% FBS） or treated with serum starvation （0.5% FBS for 2-4 days） followed by 10 hours recovery for G1 phase synchronization. Transgenic cloned embryos were produced through GFP-expressing cell selection and SCNT. The results were the percentage of blastocyst development following SCNT was lower using TBF than BFF cells （23.2% VS 35.2%, P 〈 0.05）. No difference in the percentage of cloned blastocysts between the two groups of transgenic nuclear donor of normal and starvation cultures were observed （23.2% VS 18.9%, P 〉 0.05）. Two to four GFP-expressing blastocysts were transferred into the uterus of each synchronised recipient. One pregnancy from of seven recipients （21 embryos） was confirmed by rectum palpation 60 days after embryo transfer and one recipient has given birth to a calf at term. PCR and DNA sequencing analysis confirmed that the calf was produced using human proinsulin transgenic animal.

Construction of Fusion Expression Vector of α-galactosidase-EGFP in Cucumber

[Objective] The research aimed to construct the fusion protein expression vector of α-galactosidase-EGFP （enhanced green fluorescent protein） in cucumber controlled by CaMV35S promoter.[Method] CaMV35S promoter sequence and the coding region of EGFP were amplified by polymerase chain reactions （PCR） with vector pCambia 1303 as the template.Using reverse transcript PCR technology,with total RNAs of cucumber as template,the coding region of acid α-galactosidase Ⅰ in cucumber was amplified.The above three fragments were inserted into the multiple cloning sites of expression vector pCambia 1381c.The fusion expression vector of α-galactosidase-EGFP located at the C-terminal of the target genes was constructed.[Result] After enzyme digestion and sequencing,the fusion expression of α-galactosidase-EGFP in cucumber was constructed successfully.[Conclusion] The research laid the experimental basis for further study on the subcellular localization of α-galactosidase in cucumber.

Construction and expression of GFP conjugated MIM-I-BAR

To achieve a visible inverse Bin-amphiphysin-Rvs （I-BAR）domain recombinant of missing in metastasis （MIM） protein,the green fluorescent protein （GFP）encoding gene was cloned at the terminal of MIM-I-BAR as a probe.The DNA was successfully constructed on a 6xHis-tagged prokaryotic expression plasmid.The non-GFP labeled MIM-I-BAR encoding plasmid was also constructed as a control. Being successfully transformed into BL21 （DE3 ）cells,the GFP-conjugated MIM-I-BAR （MIM-I-BAR-GFP ） exhibits strong visible fluorescence,and the expression product can be easily detected by visual inspection, a fluorescence microscope, Western blot or ultraviolet and visible spectrophotometer. Moreover, examination of expression efficiency under various culture conditions revealed that the MIM-I-BAR-GFP gene has a high protein yield at 10 ℃,but not at the culture temperature of 37 ℃.This property is much different from that of the non-fluorescent MIM-I-BAR gene. This optimal expression condition is also proved to be feasible for protein production in midi-scale. The fluorescent recombinant MIM-I-BAR-GFP protein can serve as a useful tool in scientific research, biomedical application and pharmaceutical development.

Colonization Pattern of Azospirillum brasilense Yu62 on Maize Roots

Plasmid pVK1001 which carried the gfp gene of GFPmut2, a mutant of GFP, was introduced into Azospirillum brasilense Yu62 by electroporation. Maize seedlings were inoculated with the GFP-labelled baeteria and grown gnotobiotically in flask with semi-solid agar medium. Observations were performed with confocal laser scanning microscopy (CLSM) and electron microscopy, respectively, at 8 d and 12 d after inoculation. Confocal laser scanning microscopy showed that A. brasilense Yu62 could penetrate into the cortex tissue, colonizing in the intercellular spaces of the parenchyma cells of the cortex tissue. Transmission and scanning electron microscopy (TEM) showed that the majority of the bacteria colonized on the root surface and only a minority of them resided in the root interior.

Application of GFP Gene in the Study of Insect-Resistant Transgenic Plants

用合成的cry1Ac基因与绿色荧光蛋白基因 (GFP)构成融合蛋白基因 ,然后和改造的GNA基因构建双价抗虫基因植物表达载体pBGbfg ,经根癌农杆菌介导转化了烟草。在紫外灯照射下 ,观察到转基因植株叶片中有较强的绿色荧光 ;经抗虫试验、PCR、Southernblot和Westernblot等检测 ,表明该重组植物表达载体能够在转基因植物中有效表达外源基因 ,转基因植株绿色荧光的表型与其抗虫性密切相关。从而成功地建立了以绿色荧光蛋白基因与抗虫基因组成的融合基因转化系统 ,简化了抗虫转基因植物筛选程序 ,有助于快速获得双价抗虫转基因植株。

Actin Visualization in Living Immature Pollen of Rice Using a GFP-Mouse Talin Fusion Protein

Green fluorescent protein (GFP) fused to the F_actin binding domain of mouse talin labels the actin cytoskeleton in the immature pollen of stable transformed rice (Oryza sativa L.) plants. Actin microfilaments could be visualized only in the late_developmental stage of the immature pollen. During this developmental stage, microfilaments, initially composed of very short fibrils, develop into a very complex and novel network that sometimes totally and sometimes partially encloses the vegetative nucleus and the spherical shaped generative cell in the central cytoplasm of the immature pollen. The behavior of the actin microfilamentous structure throughout the late_developmental stage of the immature pollen is extremely dynamic, and the likelihood of this structure in generating forces for vegetative nucleus and generative cell movement in the immature pollen has been discussed. No actin filaments were visualized in the spherical generative cells.

F-Actin Visualization in Generative and Sperm Cells of Living Pollen of Rice Using a GFP-Mouse Talin Fusion Protein

Green fluorescent protein (GFP) fused to the F-actin binding domain of mouse talin labels the actin cytoskeleton in the living generative and sperm cells of a third generation transgenic rice (Oryza sativa L.) plant, A005-G-T-1-2. Observations were made on pollen at four major developmental stages, viz. I. uni-nucleate microspore stage; II. early bi-cellular pollen stage; III. late bi-cellular pollen stage; and IV. tri-cellular pollen stage. At each of these developmental stages vegetative nucleus, generative nucleus/ cell, and sperm cells were seen undergoing continuous and coordinated motion and migration. These movements seemed to be influenced by associated microfilament networks existing in the pollen. Based on these observations we propose that it is the interaction between the microfilament networks (usually one existing in the central cytoplasm and another in the cortex) that controls the dynamic movement of the vegetative nucleus, generative nucleus/cell and sperm cells. Furthermore, we have also observed that there is an array of microfilaments (oriented mostly parallel to the long axis of the cell) existing in the generative and sperm cells. As far as we are aware, this is the first report showing the existence of microfilaments in living generative and sperm cells of rice pollen. The implication and significance of the existence of microfilaments in generative and sperm cells in rendering self-propelled motion of these cells in relation to their passage and movement in the pollen tube and embryo sac for fertilization were discussed.

Localization of Two GFP_tagged Tobacco Plastid Division Protein NtFtsZs in Escherichia coli

Two plastid division genes, NtFtsZ1 and NtFtsZ2 isolated from Nicotiana tabacum L. were fused with gfp and expressed in Escherichia coli . The regular localizations of full length NtFtsZs∶GFP along the filamentous bacteria indicated that the NtFtsZs could recognize the potential division sites in E. coli and be polymerized with heterogeneous FtsZ from bacteria. The overexpression of NtFtsZs ∶ gfp inhibited the division of host strain cells and resulted in the long filamentous bacterial morphology. These results suggested that eukaryotic ftsZs have similar function to their prokaryotic homologs. Meanwhile, the different deletions of motifs of NtFtsZs are also employed to investigate the functions of these proteins in E. coli . The results showed that the C_terminal domains of NtFtsZs were related to the correct localization of NtFtsZs in E. coli and the N_terminal domains of NtFtsZs were responsible for the polymerization of homogeneous and heterogeneous FtsZ proteins. The significance of these results in understanding the functions of NtFtsZs in plastid division were discussed.

Response of Chlorophyll Fluorescence Characteristic of Trifolium repens L. to Water Stress

[Objective] The aim was to study response of chlorophyll fluorescence parameters in Trifolium repens L.leaves to water stress from perspective of physiology.[Method] With T.repens cultivar ＂Haifa＂ as tested material,three soil water content levels were set to culture plants,including 75% （no stress,CK）,50% （moderate stress,LD） and 25% （severe stress,HD）,the effects of water stress on chlorophyll fluorescence parameters were determined.[Result] The results showed that the chlorophyll fluorescence parameters changed a little when the relative soil moisture content was 75% and 50%,while the chlorophyll fluorescence parameters such as conversion efficiency of primary light energy （Fv/Fm） of PS Ⅱ,the photochemical quenching coefficient （qP） and the quantum yield of PS Ⅱ electron transport （φPS Ⅱ） decreased respectively when the relative soil moisture content was 25%.[Conclusion] When T.repens in severe drought conditions,the physiological functions were damaged,showing symptoms of drought injury.

溶杆菌SNNU513基因gfp标记及在玉米根部定殖

溶杆菌属细菌在植物病害生物防治中有着广阔的应用潜力。本文以本实验室从中药材远志分离筛选的溶杆菌属新菌株Lysobacter sp.SNNU513为材料,筛选出高效制备该菌株感受态细胞Inoue法,电击转化条件为场强20 kV/cm、电脉冲时间5 ms时可将含绿色荧光蛋白基因gfp的质粒pGLO导入该菌株感受态细胞中,重组菌SNNU513-pGLO能高效、稳定表达绿色荧光蛋白基因,与出发菌株的生长特性、抑菌活性等生物学特性差异不显著。将成功构建的重组菌SNNU513-pGLO用于检测该菌株在玉米根部的定殖规律,结果表明,定殖量从表皮到韧皮部有明显减少趋势。

Gene transfer into primary cultures of fetal neural stem cells by a recombinant adenovirus carrying the gene for green fluorescent protein

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 success- fully 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.

Ameliorated stress related proteins are associated with improved cardiac function by sarcoplasmic reticulum calcium ATPase gene transfer in heart failure

Background Previous studies showed that overexpression of sarco-endoplasmic retieulum calcium ATPase （SERCA2a） in a variety of heart failure （HF） models was associated with greatly enhanced cardiac performance. However, it still undefined the effect of SERCA2a overexpression on the systemic inflammatory response and neuro-hormonal factors. Methods A rapid right ventricular pacing model of experimental HF was used in beagles. Then the animals underwent recombinant adeno-associated vires 1 （rAAV1） mediated gene trans- fection by direct intra-myocardium injection. HF animals were randomized to receive the SERCA2a gene, enhanced green fluorescent protein （control） gene, or equivalent phosphate buffered saline. Thirty days after gene delivery, the cardiac function was evaluated by echocardiographic testing. The protein level of SERCA2a was measured by western blotting. The proteomic analysis of left ventricular （LV） sample was determined using two-dimensional （2-D） gel el^ctrophoresis and MALDI-TOF-MS. The serum levels of the systemic inflammatory and neuro-hormonal factors were assayed using radioimmunoassay kits. Results The cardiac function improved after SERCA- 2a gene transfer due to the significantly increased SERCA2a protein level. Beagles treated with SERCA2a had significantly decreased serum levels of the inflammatory markers （interleukin-6 and tumor necrosis factor-a） and neuro-hormonal factors （brain natriuretic peptide, endothelin-1 and angiotensin II） compared with HF animals. The myocardial proteomic analysis showed that haptoglobin heavy chain, heat shock protein （alpha-crystallin-related, B6） were down-regulated, and galectin-1 was up-regulated in SERCA2a group compared with HF group, companied by up-regulated contractile proteins and NADH dehydrogenase. Conclusions These findings demonstrate that regional intramyocardial injections of rAAV 1-SERCA2a vectors may improve global LV function, correlating with reverse activation of the systemic inflammatory, excessive neuroendocrine factors and the stress-associated myocardial proteins, suggesting that the beneficial effects of SERCA2a gene transfer may involve the attenuation of stress-associated reaction.

Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein （GFP） was expressed in C. reinhardtii under the control of promoters： RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae： RBCS2 mediated strain Tran-Ⅰ and HSP70A-RBCS2 mediated strain Tran-Ⅱ. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-Ⅱ was at least double of that in Tran-Ⅰ. In addition, a threefold increase of GFP in Tran-Ⅱ was induced by heat shock at 40℃. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.

Surface Display of Domain Ⅲ of Japanese Encephalitis Virus E Protein on Salmonella Typhimurium by Using an Ice Nucleation Protein

A bacterial cell surface display technique based on an ice nucleation protein has been employed for the development of live vaccine against viral infection. Due to its ubiquitous ability to invade host cells, Salmonella typhimurium might be a good candidate for displaying viral antigens. We demonstrated the surface display of domain III of Japanese encephalitis virus E protein and the enhanced green fluorescent protein on S. typhimurium BRD509 using the ice nucleation protein. The effects of the motif in the ice nucleation protein on the effective display of integral protein were also investigated. The results showed that display motifs in the protein can target integral foreign protein on the surface of S. typhimurium BRD509. Moreover, recombinant strains with surface displayed viral proteins retained their invasiveness, suggesting that the recombinant S. typhimurium can be used as live vaccine vector for eliciting complete immunogenicity. The data may yield better understanding of the mechanism by which ice nucleation protein displays foreign proteins in the Salmonella strain.

Microbubble-enhanced ultrasound exposure improves gene transfer in vascular endothelial cells

AIM： To explore the effects of ultrasound exposure combined with microbubble contrast agent （SonoVue） on the permeability of the cellular membrane and on the expression of plasrnid DNA encoding enhanced green fluorescent protein （pEGFP） transfer into human umbilical vein endothelial cells （HUVECs）. METHODS： HUVECs with fluorescein isothiocyanatedextran （FD500） and HUVECs with pEGFP were exposed to continuous wave （1.9 MHz, 80.0 mW/cm^2） for 5 min, with or without a SonoVue. The percentage of FD500 taken by the HUVECs and the transient expression rate of pEGFP in the HUVECs were examined by fluorescence microscopy and flow cytornetry, respectively. RESULTS： The percentage of FDS00-positive HUVECs in the group of ultrasound exposure combined with SonoVue was significantly higher than that of the group of ultrasound exposure alone （24.0%± 5.5% vs 66.6% ± 4.1%, P 〈 0.001）. Compared with the group of ultrasound exposure alone, the transfection expression rate of pEGFP in HUVECs was markedly increased with the addition of SonoVue （16.1% ± 1.9% vs 1.5% ± 0.2%, P 〈 0.001）. No statistical significant difference was observed in the HUVECs survival rates between the ultrasound group with and without the addition of SonoVue （94.1% ± 2.3% vs 91.1% ± 4.1% ）. CONCLUSION： The cell membrane permeability of HUVECs and the transfection efficiency of pEGFP into HUVECs exposed to ultrasound are significantly increased after addition of an ultrasound contrast agent without obvious damage to the survival of HUVECs. This non- invasive gene transfer method may be a useful tool for clinical gene therapy of hepatic tumors.

GFP tracking transcriptional activity of endogenous p53: vector construction and application in genotoxicity detection

Objective: To establish a sensitive and specific system for genotoxicity detection in vivo. Methods: Endogenous p53 tracer vector p53RE was constructed by using green fluorescent protein (GFP) as a reporter to trace p53 transcriptional activity under the control of base SV40 early promoter. The tracer cells 3T3-REG were established by transfecting NIH3T3 cells with tracer vector and treated with ultraviolet, H2O2 and adriamycin to induce DNA damage and the subsequent endogenous p53 expression. The GFP expression and its green fluorescence in the tracer cells were observed and measured with fluorescent microscope and FACS. Results: The GFP expression increased rapidly after various treatment and reached the maximum 1 h later, and decreased gradually afterwards. FACS analysis showed that GFP expression in 3T3-REG tracer cells was consistent with the concentration of H2O2, while that in 3T3-SVG cells, which were transfected with control vector pSV-GFP. hardly increased in response to the treatment. Conclusion: GFP tracing p53 transcriptional activity is a sensitive and specific method for genotoxicity detection.

Cloning, expression, purification, and characterization of LC-1 ScFv with GFP tag

Total RNA was isolated from the hybridoma cell line （LC- 1 ）, which secretes anti-lung adenocarcinoma monoclonal antibody, and was transferred into cDNA. Based on the FRl （framework region l） and FR4 conserved regions of LC-1 gene, the variable regions of heavy chain （Vh） and light chain （Vl） were amplified, and the Vh and modified Vl were connected to single chain Fv （ScFv） by SOE-PCR （splice overlap extension PCR）. The modified ScFv was fused with green fluorescent protein （GFP） and introduced into E. coli JM109. The fusion protein induced by lPTG （Isopropylthiogalactoside） was about 57000 on a 10% SDS-PAGE gel （10% Sds Polyacrylamide Gel Electrophoresis）, and primarily manifested as inclusion bodies. The renatured protein purified by Ni-NTA Superflow resins showed ability to bind to antigen on SPC-A-l lung adenocarcinoma. In addition, the induced host cells fluoresced bright green under 395 nm wavelength, which indicated that the expected protein with dual activity was expressed in the prokaryotic system. The ScFv with GFP tag used in this research can be applied as a new reagent to detect immunological dye, and provide a feasible way to detect adenocarcinoma in a clinical setting.

Construction of a chloroplast protein interaction network and functional mining of photosynthetic proteins in Arabidopsis thaliana

Chloroplast is a typical plant cell organelle where photosynthesis takes place. In this study, a total of 1 808 chloroplast core proteins in Arabidopsis thaliana were reliably identified by combining the results of previously published studies and our own predictions. We then constructed a chloroplast protein interaction network primarily based on these core protein interactions. The network had 22 925 protein interaction pairs which involved 2 214 proteins. A total of 160 previously uncharacterized proteins were annotated in this network. The subunits of the photosynthetic complexes were modularized, and the functional relationships among photosystem Ⅰ （PSI）, photosystem Ⅱ （PSII）, light harvesting complex of photosystem Ⅰ （LHC Ⅰ） and light harvesting complex of photosystem Ⅰ （LHC Ⅱ） could be deduced from the predicted protein interactions in this network. We further confirmed an interaction between an unknown protein AT1G52220 and a photosynthetic subunit PSI-D2 by yeast two-hybrid analysis. Our chloroplast protein interaction network should be useful for functional mining of photosynthetic proteins and investigation of chloroplast-related functions at the systems biology level in Arabidopsis.