基于CRISPR/Cas9系统对干酪乳杆菌进行红色荧光蛋白标记

本实验利用CRISPR/Cas9系统对干酪乳杆菌(Lactobacillus casei) LC2W进行红色荧光蛋白(red fluorescent protein,RFP)标记,用于研究干酪乳杆菌在肠道内的分布和定植状况,评价其作为益生菌的功能。首先,基于本实验室已有的干酪乳杆菌CRISPR/Cas9编辑质粒pLCNICK-1628构建重组质粒pLCNICK-1628-RFP,电转入干酪乳杆菌LC2W感受态细胞中,使干酪乳杆菌基因组中的LC2W-1628基因被红色荧光蛋白基因替换,从而使干酪乳杆菌LC2W能表达出红色荧光蛋白。得到红色荧光标记的干酪乳杆菌LC2W突变株后,测定了其荧光强度-OD600标准曲线,发现RFP在干酪乳杆菌LC2W中能稳定表达。     

siRNA对多头绒泡菌丝氨酸/精氨酸蛋白激酶表达的沉默

SR蛋白激酶（serine/arginine protein-specific kinase,SRPK）是一类特异磷酸化剪接因子SR蛋白的激酶家族,可调节SR蛋白的选择型剪接,影响SR蛋白在核内的分布与定位,在pre-mRNA剪接调控上具有重要的作用。目前对于多头绒泡菌SRPK的功能仍不清楚。为了进一步研究PSRPK的功能,设计可转录小干扰RNA（siRNA）的寡核苷酸序列含U6启动子的载体pSIREN-RetroQ定向连接,构建了真核表达质粒pSIREN-PSRPK-1,pSIREN-PSRPK-2,pSIREN-PSRPK-3,pSIREN-PSRPK-4,pSIREN-PSRPK-5及对照质粒pSIREN-PSRPK-Neg。把编码PSRPK基因的cDNA与红色荧光蛋白表达载体pDsRed-N1重组,构建了表达PSRPK-红色荧光融合蛋白的表达质粒pP-SRPK-DsRed。将真核表达质粒和质粒pPSRPK-DsRed共转染HEK293细胞。转染后72h通过倒置荧光显微镜观察,结果表明,pSIREN-PSRPK-2和pSIREN-PSRPK-5可以有效沉默红色荧光融合蛋白的表达;进一步采用RT-PCR和Northern点杂交分析表明,pSIREN-PSRPK-2和pSIREN-PSRPK-5对红色荧光融合蛋白mRNA表达具有明显的抑制作用与倒置荧光显微镜下观察到的结果一致。这为进一步研究多头绒泡菌SRPK的功能奠定了实验基础。     

兔Oct4启动子驱动RFP基因表达载体的构建及验证

转录因子OCT4在维持和调控胚胎干细胞的多能性中发挥着重要的作用。Oct4基因启动子驱动标志蛋白的表达对研究胚胎干细胞多能性和建立iPs细胞有重要意义。由于GFP在慢病毒转染过程中常用作转染标记,计划构建兔Oct4基因启动子（rOct4）驱动红色荧光蛋白表达的载体,这将有利于兔ES细胞和iPS细胞制备的研究。通过PCR方法扩增rOct4,构建了rOct4驱动RFP基因的表达载体rOct4-RFP。经转染小鼠ES细胞验证正确后,将rOct4-RFP质粒转染兔成纤维细胞系获得rOct4-RFP成纤维细胞系。经过酶切和测序验证,证明rOct4-RFP构建成功,而且能够在小鼠Es细胞系E14中表达细胞红色荧光蛋白,并受细胞分化状态的调控。通过脂质体介导的基因转移、抗性筛选和PCR鉴定建立了rOct4-RFP转基因成纤维细胞系。     

板栗疫病菌绿色荧光与红色荧光蛋白共定位载体的构建

低毒病毒-板栗疫病菌组合是研究病毒与宿主相互作用的一个优秀的模式系统.我们构建了含绿色荧光蛋白基因gfp的载体pCPXHY2GFP与含红色荧光蛋白基因rfp的载体pCPXG418RFP,并用于转化野生型菌株EP155,获得了以潮霉素为筛选标记、表达绿色荧光蛋白的转化株pCPXHY2GFP/EP155和以G418为筛选标记、表达红色荧光蛋白的转化株pCPXG418RFP/EP155.将载体pCPXG418RFP转化pCPXHY2GFP/EP155,获得的转化株能观察到绿色荧光蛋白与红色荧光蛋白共定位的现象.板栗疫病菌绿色荧光与红色荧光共定位载体pCPXHY2GFP与pCPXG418RFP的构建,为深入研究病毒与宿主相互作用的分子机制提供了强有力的研究材料.     

利用斑马鱼胚胎快速鉴定真核质粒中目的基因表达的研究

目的建立利用斑马鱼胚胎快速鉴定真核质粒中目的基因表达的实验体系。方法选20枚斑马鱼受精卵,在显微镜下每隔1h记录胚胎的发育情况。另选250枚单细胞期斑马鱼胚胎,平均分成5组,一组胚胎作为对照,剩余4组分别向胚胎的单细胞内注射pEGFP-N1(真核表达质粒)、pCMV-DsRed-Express2(真核表达质粒)、pET28-GFP(原核表达质粒)、pET28-RFP(原核表达质粒)质粒,在不同时间点连续观察绿色荧光及红色荧光的表达情况。另选600枚单细胞期斑马鱼胚胎,平均分成3组,一组胚胎作为对照,一组向胚胎单细胞内注射pEGFP-N1质粒,另外一组向胚胎单细胞内注射pEGFP-N1-MUC1外源基因融合重组质粒,注射4h后在荧光显微镜下观察绿色荧光的表达情况,并用RT-PCR的方法检测目的基因MUC1mRNA的转录情况。结果注射pEGFP-N1、pCMV-DsRed-Express2真核表达质粒的胚胎,注射4h后分别观察到很强的绿色荧光及红色荧光;注射pET28-GFP、pET28-RFP原核表达质粒的胚胎,10h内都未观察到绿色荧光及红色荧光;注射pEGFP-N1-MUC1外源基因融合质粒,注射4h后同样...     

多头绒泡菌类p34cdc2蛋白的定位研究

目前关于动物和酵母细胞中p34cdc2的定位研究结果尚存在分歧,而关于该蛋白在植物细胞中的定位尚不清楚.以多头绒泡菌( Physarum polycephalum )S期、G2早期、G2中期、G2晚期、前期、中期和后末期的原质团和细胞核为材料进行免疫印迹,发现原质团和细胞核都含有一种分子量约34 kD的类p34cdc2蛋白,该蛋白在原质团和细胞核中的含量在整个细胞周期进程中基本保持稳定.以抗p34cdc2单克隆抗体为探针的免疫电镜结果显示,类p34cdc2蛋白既分布于细胞核也分布于细胞质中,在细胞核中主要与染色体和核仁结合.经抗p34cdc2单克隆抗体处理后,多头绒泡菌的有丝分裂启始迟滞约2 h.结果表明,多头绒泡菌类p34cdc2蛋白存在于细胞核和细胞质中,与细胞有丝分裂密切相关,其含量在细胞周期进程中基本保持稳定.     

温度对大肠埃希菌Mn-SOD基因启动子调控条件的优化

目的 构建大肠埃希菌BL21 Mn-SOD-RFP报告基因载体,并探讨温度对大肠埃希菌Mn-SOD基因启动子的调控.方法 利用重组PCR技术,构建以Mn-SOD启动子调控的红色荧光蛋白(RFP)报告基因载体,将融合基因与T载体连接导入大肠埃希菌中,在不同温度(20、37、40和45℃)培养不同时间(13、20、30、37、44和54 h)后,利用荧光显微镜和荧光光度计观察大肠埃希菌表达RFP的情况.结果 正确构建Mn-SOD-RFP融合基因,重组PCR结果与测序结果完全一致;不同温度不同时间诱导后,Mn-SOD启动子在37℃,培养30～ 37 h表达的红色荧光蛋白最多.结论 成功构建该报告基因载体,并完成温度、时间对其调控的优化,为更进一步研究其他因素对SOD基因启动子的调控机制奠定基础.     

多头绒泡菌类p34^cdc2蛋白的定位研究

目前关于动物和酵母细胞中p34cdc2 的定位研究结果尚存在分歧 ,而关于该蛋白在植物细胞中的定位尚不清楚。以多头绒泡菌 (Physarumpolycephalum)S期、G2早期、G2中期、G2晚期、前期、中期和后末期的原质团和细胞核为材料进行免疫印迹 ,发现原质团和细胞核都含有一种分子量约 34kD的类p34cdc2 蛋白 ,该蛋白在原质团和细胞核中的含量在整个细胞周期进程中基本保持稳定。以抗p34cdc2 单克隆抗体为探针的免疫电镜结果显示 ,类p34cdc2 蛋白既分布于细胞核也分布于细胞质中 ,在细胞核中主要与染色体和核仁结合。经抗p34cdc2 单克隆抗体处理后 ,多头绒泡菌的有丝分裂启始迟滞约 2h。结果表明 ,多头绒泡菌类p34cdc2 蛋白存在于细胞核和细胞质中 ,与细胞有丝分裂密切相关 ,其含量在细胞周期进程中基本保持稳定。     

利用GFP/RFP双荧光指示载体鉴定特异性启动子功能

在基因表达定位或启动子调控模式的研究中, 多以gusA作为报告基因。但由于部分组织中高内源GUS背景活性或转化手段的限制, 使判断基因表达定位或调控时存在很大误差。为了解决上述问题, 本实验将报道基因绿色荧光蛋白(GFP)和红色荧光蛋白(RFP)融合构建双荧光标记瞬时表达载体pBI221-RFP/GFP。该载体以CaMV35S启动子驱动GFP确定转化效率, 通过鉴定阳性个体的红色荧光活性分析目的基因或启动子的表达模式。并通过番茄E8和西瓜AGPL1果实特异启动子验证了该载体在启动子调控模式研究中的应用可行性。结果表明pBI221-RFP/GFP是一个可以在基因和启动子功能验证中应用的高效瞬时表达载体。     

hTEM8-N-RFP融合蛋白真核表达载体的构建及在HEK293F细胞中表达

目的:构建人肿瘤内皮标志物8(hTEM8)胞外区(N端)与红色荧光蛋白(RFP)融合表达载体并在HEK293F细胞中表达,为进一步研究hTEM8的相互作用蛋白及其在肿瘤血管形成过程中的机制奠定实验基础。方法:以质粒pDsRed-Express-C1和重组质粒pcDNA3.1(+)-hTEM8/Fc为模板,PCR扩增RFP和hTEM8-N基因片段,先后插入真核表达载体pcDNA3.1(+)中,构建重组表达载体pcDNA3.1(+)-hTEM8-N-RFP,转染HEK293F细胞,通过荧光显微镜观察融合蛋白在转染细胞中的的表达,并用G418对转染的细胞进行加压筛选,Western blot检测hTEM8-N-RFP融合蛋白在转染细胞中的表达。结果:DNA测序、酶切鉴定的结果显示,表达载体pcDNA3.1(+)-hTEM8-N-RFP构建成功,且序列正确。转染后经荧光显微镜观察到HEK293F细胞中有红色荧光,经加压筛选单克隆后,在荧光显微镜下观察到稳定表达红色荧光的细胞株,Western blot检测到融合蛋白hTEM8-N-RFP在真核细胞HEK293F中获得表达。结论:成功构建了pcDNA3.1(+)-hTEM8-N-RFP真核表达载体,并在HEK293F细胞中表达,为后期研究hTEM8的相互作用蛋白和其生理功能奠定了良好的基础。     

Involvement of extracellular cAMP-specific phosphodiesterase in control of motile activity of Physarum polycephalum plasmodium

Possible involvement of extracellular cAMP-specific phosphodiesterase in the control of cell motile behavior has been investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the autooscillatory mode of motility. It was found that the rate of the hydrolysis of 10 mM cAMP by a partially purified preparation of cAMP-specific phosphodiesterase secreted by the plasmodium in the course of migration decreases 20-30 times under the action of 1 mM dithiothreitol. In the presence of 1-5 mM of this strong reducing agent, the onset of the plasmodium spreading and the transition to the stage of migration were delayed in a concentration-dependent manner. In accordance with the morphological pattern of motile behavior, the duration of the maintenance of high frequency autooscillations, which normally precede the increase in the rate of the spreading and appear also in response to the application of attractants at spatially uniform concentrations, strongly increased by the action of dithiothreitol. The results obtained suggest that the autocrine production of cAMP and extracellular cAMP-specific phosphodiesterase is an important constituent of the mechanism controlling the motile behavior of the Physarum polycephalum plasmodium.     

多头绒泡菌中动蛋白的免疫化学鉴定

动蛋白(kinesin)是一种微管系统的运动蛋白(motor protein),它能通过水解ATP将化学能转化为机械能,推动微管产生运动.微管系统作为一种主要的细胞骨架存在于所有真核细胞中,它们对于维持细胞形态,细胞的分裂,染色体的运动及细胞内的物质运输起着重要作用.细胞质力蛋白(dynein)和动蛋白是公认的推动这类运动的运动蛋白.自从1985年Vole首次在鱿鱼大轴突(squidgiant axon)中发现动蛋白以来,人们先后在许多种动物细胞中发现有动蛋白存在,甚至在低等真核生物棘状变形虫,盘基网柄茵和高等植物烟草花粉管中发现有动蛋白的存在.研究结果表明,动蛋白参与了真核细胞中的许多重要生命活动,如细胞中的细胞器及囊泡的运动,染色体排裂和分离等运动.动蛋白很可能是普通存在于所有真核细胞中的一种运动蛋白.多头绒泡菌(Physarum poly-cephalum)属于粘菌纲(Myxomycetes)的一种低等真核生物,它表现出许多显著的细胞运动特征如原生质团迁移,细胞质的穿梭运动(shuttle streaming)等,是研究非肌细胞运动和收缩蛋白的经典材料.在多头绒泡菌胞质中也具有微管系统,它们构成其纺锤丝等,参与染色体的运动及其它胞质运动,但至今国内外尚无人证明其中有与微管作用的运动蛋白——动蛋白的存在,作者利用抗牛脑动蛋白的单克隆抗体,     

Developmental regulation and identification of an isotype encoded by altB, an alpha-tubulin locus in Physarum polycephalum.

A subcloned portion of the 5' nontranslated sequence from a Physarum alpha-tubulin cDNA is specific for a single alpha-tubulin locus, altB, of Physarum polycephalum. We find that this locus is expressed only in the plasmodium and encodes at least an alpha 1-tubulin isotype, which we have designated alpha 1B. Hybridization patterns of other subclones of this cDNA reveal two sequences for alpha-tubulin at the altB locus.     

Sounds Synthesis with Slime Mould of Physarum Polycephalum

This paper introduces a novel application of bionic engineering: a bionic musical instrument using Physarum polycephalum. Physarum polycephalum is a huge single cell with thousands of nuclei, which behaves like a giant amoeba. During its foraging behavior this plasmodium produces electrical activity corresponding to different physiological states. We developed a method to render sounds from such electrical activity and thus represent spatio-temporal behavior of slime mould in a form apprehended auditorily. The electrical activity is captured by various electrodes placed on a Petri dish containing the cultured slime mold. Sounds are synthesized by a bank of parallel sinusoidal oscillators connected to the electrodes. Each electrode is responsible for one partial of the spectrum of the resulting sound. The behavior of the slime mould can be controlled to produce different timbres.     

Electron microscopy of the slime moldPhysarum polycephalum

Protoplasma - Electron micrographs of fixed sections of the plasmodium of the classic protoplasmic material,Physarum polycephalum, are presented. Numerous round nuclei having well-defined membranes...     

Towards Physarum Robots： Computing and Manipulating on Water Surface

Plasmodium of Physarum polycephalum is an ideal biological substrate for implementing concurrent and parallel com-putation, including combinatorial geometry and optimization on graphs. The scoping experiments on Physarum computing in conditions of minimal friction, on the water surface were performed. The laboratory and computer experimental results show that plasmodium of Physarum is capable of computing a basic spanning tree and manipulating of light-weight objects. We speculate that our results pave the pathways towards the design and implementation of amorphous biological robots.     

Acetylated alpha-tubulin in Physarum: immunological characterization of the isotype and its usage in particular microtubular organelles

We have used monoclonal antibodies specific for acetylated and unacetylated alpha-tubulin to characterize the acetylated alpha-tubulin isotype of Physarum polycephalum, its expression in the life cycle, and its localization in particular microtubular organelles. We have used the monoclonal antibody 6-11B-1 (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094) as the probe for acetylated alpha-tubulin and have provided a biochemical characterization of the monoclonal antibody KMP-1 as a probe for unacetylated tubulin in Physarum. Concomitant use of these two probes has allowed us to characterize the acetylated alpha-tubulin of Physarum as the alpha 3 isotype. We have detected this acetylated alpha 3 tubulin isotype in both the flagellate and in the myxameba, but not in the plasmodium. In the flagellate, acetylated tubulin is present in both the flagellar axonemes and in an extensive array of cytoplasmic microtubules. The extensive arrangement of acetylated cytoplasmic microtubules and the flagellar axonemes are elaborated during the myxameba-flagellate transformation. In the myxameba, acetylated tubulin is not present in the cytoplasmic microtubules nor in the mitotic spindle microtubules, but is associated with the two centrioles of this cell. These findings, taken together with the apparent absence of acetylated alpha-tubulin in the ephemeral microtubules of the plasmodium suggest a natural correspondence between the presence of acetylated alpha-tubulin and microtubule organelles that are intrinsically stable or cross-linked.     

Emergence of morphological order in the network formation of Physarum polycephalum

Emergence in a system appears through the interaction of its components, giving rise to higher order or complexity in the system. We tested for the presence of emergent properties in a biological system using the simplest biological entity of a unicellular organism; the plasmodium of Physarum polycephalum, a giant unicellular amoeboid organism that forms a network-like tubular structure connecting its food sources. We let two plasmodium networks within a single cell interact with each other, and observed how the intracellular interaction affected the morphologenesis of the plasmodium networks. We found that the two networks developed homologous morphology. We further discuss the presence of autonomous and emergent properties in homologous network formation.     

Path finding by tube morphogenesis in an amoeboid organism

We have studied how the plasmodium of Physarum polycephalum, a large amoeboid cell, is able to track the shortest path between two selected points in a labyrinth. When nutrients are supplied at these points to a sheet-like plasmodium extended fully in a maze, the organism forms a single tube which connects the two sites via the shortest route. During the path finding, plasmodial parts in dead ends of the maze shrink and finally the tube with the minimum-length is selected from the existing possibilities. A simple cellular mechanism based on interacting cellular rhythms may describe the experimental observations.