茶树花发育MADS-box转录因子CsGLO1、CsGLO2与CsAG之间的互作关系研究

利用酵母双杂交方法和双分子荧光互补技术（BiFC）研究了茶树（Camellia sinensis（L.）O.Kuntze）花发育MADS-box的B类转录因子CsGLO1、CsGLO2与C类转录因子CsAG间的互作关系及其可能发生互作的亚细胞定位。通过构建5个酵母表达载体,利用酵母单杂交实验检测了3个蛋白的转录激活活性,并通过酵母双杂交实验分析了蛋白间的互作关系。结果显示,3个蛋白均无转录激活活性,且两两之间可发生相互作用。进一步构建6个BiFC表达载体,采用压力注射法瞬时浸染烟草（Nicotiana benthamiana L.）叶表皮细胞,并利用激光共聚焦显微镜观察荧光信号,结果表明茶树B类CsGLO与C类CsAG蛋白可在植物活细胞内形成同源和异源二聚体,并具有在细胞质中发生互作的特定模式。本研究可为利用分子生物学技术抑制茶树“花果同现”现象提供理论依据。     

表达GFP/mCherry白色念珠菌的构建及其在与巨噬细胞互作中的应用

白色念珠菌(Candida albicans)被巨噬细胞吞噬的效率与被吞噬后的形态观察是研究白色念珠菌与巨噬细胞互作的重要内容。【目的】以野生型菌株SC5314为母本,构建能够表达绿色荧光蛋白(green fluorescent protein, GFP)/mCherry的白色念珠菌,应用于巨噬细胞与白色念珠菌互作的研究。【方法】通过生长与形态观察、细胞活性检测及小鼠系统性感染模型确定荧光蛋白的表达对菌株生长、形态与毒力的影响;在共培养条件下,通过流式细胞术及荧光显微镜检测巨噬细胞的吞噬率及白色念珠菌的形态变化。【结果】构建的菌株在表型上与野生型菌株一致,并可用于在共培养下测定巨噬细胞吞噬率的流式细胞术以及观察白色念珠菌的形态变化。【结论】表达荧光蛋白的菌株为研究巨噬细胞与白色念珠菌的互作提供了新方法。     

甘蓝型油菜BnaCIPK15基因的表达分析与功能鉴定

CBL-CIPK是高等植物中广泛存在的一类解析Ca~(2+)信号的蛋白。该研究在前期工作基础上,对甘蓝型油菜(Brassica napus L.)的BnaCIPK15基因进行了亚细胞定位、双分子荧光互补(BiFC)、酵母双杂交和qRT-PCR检测等一系列分析,以探究BnaCIPK15蛋白在ABA激素响应中的作用。结果显示:(1)亚细胞定位发现,BnaCIPK15蛋白定位于细胞质和细胞核中; BiFC分析发现,BnaCIPK15蛋白与BnaCBL1/3/4/9蛋白之间的互作较强,与BnaCBL10仅有微弱互作。(2)qRT-PCR检测发现,BnaCIPK15基因受ABA和冷胁迫的诱导极显著上调表达,而对百草枯(Paraquat)、活性氧(H_2O_2)和热胁迫的诱导较弱,表明BnaCIPK15基因很可能参与ABA和冷胁迫的调控过程。(3)酵母滴定实验结果显示,BnaCIPK15蛋白与脱落酸(ABA)信号通路中的BnaHAB1蛋白(属于蛋白磷酸酶PP2C家族)存在明显的互作,而与BnaABFs/AREB3/ABI5转录因子无明显互作;BiFC验证显示,BnaCIPK15与BnaHAB1蛋白之间存在互作信号,而BnaCIPK15与BnaHAB2组合没有观察到信号,证明BnaCIPK15与BnaHAB1磷酸酶具有特异互作特征,推测BnaCIPK15可能参与调控ABA信号转导。研究认为,甘蓝型油菜中可能存在基于BnaCIPK15-BnaHAB1的互作模块,并参与ABA的信号转导和网络调控。     

双分子荧光互补技术

双分子荧光互补（bimolecular fluorescence complementation, BiFC）是近年发展起来的用于体内或体外检测蛋白质相互作用的一项新技术.该技术是将荧光蛋白在合适的位点切开形成不发荧光的2个片段,这2个片段借助融合于其上的目标蛋白的相互作用,彼此靠近,重新形成能具有活性的荧光蛋白.BiFC方法简单直观,既可以检测蛋白之间的相互作用,也可以定位相互作用蛋白质的位点.多色BiFC系统共用或与荧光共振能量转移（FRET）技术联用,还可以检测细胞内多个蛋白质的相互作用.     

酿酒酵母荧光定位报告菌株的开发

【目的】为了给外源蛋白在酿酒酵母细胞中的定位提供参考,构建酿酒酵母荧光定位报告菌株。【方法】运用染色体同源重组的方法,将突变的、已进行酵母表达优化的红色荧光蛋白RedStar分别整合到12个酵母细胞器标记蛋白的C端,与之进行融合表达,用特异性引物对每一个酵母荧光定位报告菌株进行PCR扩增和测序验证,用激光共聚焦显微镜进行荧光检测,对线粒体和细胞核进行特异性染料染色,用EGFP标记沙门氏菌已知定位蛋白SipA,与构建的相应荧光定位报告菌株进行共定位。【结果】构建的酿酒酵母荧光定位报告菌株可分别标示酵母细胞的肌动蛋白、晚期胞内体、细胞核、核周质、纺锤体、线粒体、过氧化物酶体、脂滴、初级内吞体、次级内吞体、高尔基体顺面及高尔基体反面。PCR扩增及测序验证、荧光检测、染料与相应报告菌株的共定位、已知定位蛋白SipA与相应报告菌株的共定位均提示报告菌株构建成功。【结论】这些报告菌株的构建,为日后在酵母中观察细胞器动态变化,以及未知蛋白在酵母中的定位提供了基础性工具。     

酿酒酵母GPCR蛋白Ste2亚细胞定位信号探索

G蛋白偶联受体(G protein-coupled receptor,GPCR)家族蛋白在细胞感受各种胞外信号过程中发挥重要作用。Ste2是酵母细胞中GPCR蛋白之一。大量文献报道了Ste2蛋白突变体对其功能和表达的影响,但关于Ste2亚细胞定位的研究相对较少。这项工作的目的在于确定Ste2亚细胞定位,探究Ste2不同跨膜域、胞内外环状结构域和N端、C端对其亚细胞定位的影响。构建了一系列结构域删除或替换突变体,通过荧光显微镜观察判断不同结构区域对Ste2亚细胞定位的影响,并通过与已知的细胞器标记蛋白共定位观察验证亚细胞定位判读结果。结果显示:野生型Ste2荧光信号出现在质膜和液泡内腔; C端缺失突变体荧光信号出现在质膜和内质网。在N端、C端、各环状结构域序列采用动物GPCR蛋白ORI7、OR17-40相应结构域替换的突变体中,C端替换导致液泡内腔信号消失,质膜信号强于野生型; N端和部分环状结构域替换不同程度减弱或消除了质膜定位,液泡腔内信号类似于野生型;部分突变体在胞内出现点状分布的荧光信号。由此推断:Ste2 N端,第一、第二胞外环状结构域和第三胞内环状结构域可能具有影响Ste2运输定位到质膜的功能;而C端则可能在Ste2离开细胞膜进入液泡的过程中发挥作用。初步确定了Ste2的不同结构区域对其定位的影响,为深入研究GPCR蛋白的亚细胞定位机制奠定基础。     

灰葡萄孢菌过氧化物酶体及细胞核的荧光蛋白标记

【目的】对灰葡萄孢菌(Botrytis cinerea)的细胞核和过氧化物酶体进行荧光蛋白标记,为研究其生长发育和侵染过程中细胞结构和细胞器动态提供基础。【方法】以绿色荧光蛋白(GFP)和红色荧光蛋白(DsRED、mCherry)为报告基因,利用根癌农杆菌介导转化(Agrobacterium tumefaciens mediated transformation,AtMT)将3种荧光蛋白标记载体分别导入灰葡萄孢菌标准菌株B05.10;通过PCR检测及荧光观察筛选和验证转化子,并进行单孢纯化;利用共聚焦显微镜记录细胞器荧光定位情况。【结果】获得了过氧化物酶体或细胞核稳定表达红、绿色荧光的重组单孢菌株,PCR验证表明标记基因成功整合入转化子基因组。在标记细胞核的菌株中,菌丝和孢子中可见多个明亮、圆形的荧光点,与DAPI染色共定位。标记过氧化物酶体的菌株中,菌丝和孢子中可见小点状绿色或红色荧光,在脂类物质诱导下荧光点数量明显增加,符合过氧化物酶体分布及动态特征。细胞壁染色结果显示,细胞壁染色产生的蓝色荧光与红、绿荧光蛋白的荧光互不干扰,标记效果良好。【结论】获得了理想的过氧化物酶体或细胞核荧光标记的灰葡萄孢菌菌株,为研究其细胞器动态以及生长发育与致病分子机制提供了参考和材料。     

食源米曲霉菌株的分离及其降解高效氯氰菊酯和3-苯氧基苯甲酸的特性研究

【目的】通过研究不同食源米曲霉菌株对高效氯氰菊酯(beta-cypermethrin,β-CP)及其必经代谢产物3-苯氧基苯甲酸(3-phenoxybenzoic acid,3-PBA)的降解特性,了解不同菌株的降解共性及差异性,为农副产品和发酵食品的农残减除提供理论基础和食品用安全微生物资源。【方法】以发酵食品为菌源,通过形态学鉴定、ITS测序和菌株产黄曲霉毒素B1 (AFB1)的测定筛选鉴定米曲霉菌株,采用高效液相色谱法(HPLC)、气相色谱-质谱法(GC-MS)、液相色谱-质谱法(LC-MS)对米曲霉模式菌株RIB40 (保藏编号:ATCC 42149)、米曲霉M4 (保藏编号:CGMCC 11645)和鉴定获得的米曲霉菌株的β-CP和3-PBA降解特性进行研究。【结果】鉴定获得15株不产AFB1的食源米曲霉,17株米曲霉在马铃薯液体培养基(PD)中振荡培养5d,对50mg/L的β-CP降解率为19.33%-50.29%不等,检测到降解产物3-PBA,对50 mg/L的3-PBA降解率为45.59%-99.67%不等;分别在添加50 mg/Lβ-CP和3-PBA的无机盐培养基(MM)中振荡培养5 d,米曲霉菌株均未生长,对β-CP和3-PBA无降解;在富集培养基(GM)中振荡培养2 d,对100 mg/L的3-PBA转化或降解率为69.28%-99.58%不等,检测到3-苯氧基苄醇(3-PBlc)和羟基-3-苯氧基苯甲酸(HO-3-PBA)。【结论】食源米曲霉具有共代谢降解β-CP和3-PBA的共性,3-PBA为β-CP降解中间产物,米曲霉对3-PBA普遍具有较高的降解率。在3-PBA降解初期,米曲霉可将其短暂还原生成毒性相对较低的3-PBlc,同时,3-PBA逐渐羟基化生成水溶性更强的HO-3-PBA参与下游降解。     

玉米酵母双杂交cDNA文库的构建及ZmCEN互作蛋白的筛选

为阐明玉米中心蛋白(ZmCEN)的生物学功能,采用酵母双杂交技术对其互作蛋白进行研究。提取玉米(Zea mays L.)自交系‘郑58’幼苗的总RNA,利用SMART技术反转录合成ds cDNA,构建以pGBKT7为载体的酵母双杂交cDNA文库;依据ZmCEN基因的CDS序列设计引物,构建重组诱饵载体(pGBKT7-ZmCEN)转化酵母菌株Y2HGold,检测诱饵载体的毒性与自激活能力后,筛选与玉米中心蛋白(ZmCEN)互作的猎物蛋白。将筛选的互作蛋白NAC67和TONNEAU1b(TON1b)再次验证相互作用,并选取互作蛋白TON1b,采用BiFC实验分别构建ZmCEN-pSPYNE和TON1b-pSPYCE BiFC半分子重组载体,转化拟南芥原生质体,进一步验证它们在细胞内的互作;并利用Uniprot和KEGG在线网站对互作蛋白进行gene ontology(GO)注释分析。结果表明:玉米全株幼苗的cDNA文库库容量达到2.56×107 CFU,文库滴度5.36×108 CFU/mL,符合建库要求。经检测诱饵载体无毒性也无自激活功能,所筛选的cDNA文库经测序和Blast比对分析以及共转验证,最终得到28个与诱饵蛋白ZmCEN互作的蛋白质。GO注释显示互作蛋白参与的生物过程有21种。BiFC结果显示,蛋白TON1b与ZmCEN在拟南芥原生质体细胞内互作而形成互补,从而产生黄色荧光,进一步证实了两者存在互作关系。酵母双杂交系统cDNA文库的成功构建与筛选,为进一步研究玉米ZmCEN及其与互作蛋白的作用机制奠定了基础。     

双分子荧光互补技术及其在蛋白质相互作用研究中的应用

双分子荧光互补(bimolecularfluorescencecomplementation,BiFC)分析技术,是由Hu等在2002年最先报道的一种直观、快速地判断目标蛋白在活细胞中的定位和相互作用的新技术.该技术巧妙地将荧光蛋白分子的两个互补片段分别与目标蛋白融合表达,如果荧光蛋白活性恢复则表明两目标蛋白发生了相互作用.其后发展出的多色荧光互补技术(multicolorBiFC),不仅能同时检测到多种蛋白质复合体的形成,还能够对不同蛋白质间产生相互作用的强弱进行比较.目前,该技术已用于转录因子,G蛋白βγ亚基的二聚体形式,不同蛋白质间产生相互作用强弱的比较以及蛋白质泛素化等方面的研究工作上.     

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.     

Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta

Bimolecular fluorescence complementation (BiFC) represents one of the most advanced and powerful tools for studying and visualizing protein-protein interactions in living cells. In this method, putative interacting protein partners are fused to complementary non-fluorescent fragments of an autofluorescent protein, such as the yellow spectral variant of the green fluorescent protein. Interaction of the test proteins may result in reconstruction of fluorescence if the two portions of yellow spectral variant of the green fluorescent protein are brought together in such a way that they can fold properly. BiFC provides an assay for detection of protein-protein interactions, and for the subcellular localization of the interacting protein partners. To facilitate the application of BiFC to plant research, we designed a series of vectors for easy construction of N-terminal and C-terminal fusions of the target protein to the yellow spectral variant of the green fluorescent protein fragments. These vectors carry constitutive expression cassettes with an expanded multi-cloning site. In addition, these vectors facilitate the assembly of BiFC expression cassettes into Agrobacterium multi-gene expression binary plasmids for co-expression of interacting partners and additional autofluorescent proteins that may serve as internal transformation controls and markers of subcellular compartments. We demonstrate the utility of these vectors for the analysis of specific protein-protein interactions in various cellular compartments, including the nucleus, plasmodesmata, and chloroplasts of different plant species and cell types.     

Visualization of cofilin-actin and Ras-Raf interactions by bimolecular fluorescence complementation assays using a new pair of split Venus fragments

The bimolecular fluorescence complementation (BiFC) assay is a method for visualizing protein-protein interactions in living cells. To visualize the cofilin-actin interaction in living cells, a series of combinations of the N- and C-terminal fragments of Venus fused upstream or downstream of cofilin and actin were screened systematically. A new pair of split Venus fragments, Venus (1-210) fused upstream of cofilin and Venus (210-238) fused downstream of actin, was the most effective combination for visualizing the specific interaction between cofilin and actin in living cells. This pair of Venus fragments was also effective for detecting the active Ras-dependent interaction between H-Ras and Raf1 and the Ca(2+)-dependent interaction between calmodulin and its target M13 peptide. In vitro BiFC assays using the pair of purified BiFC probes provided the means to detect the specific interactions between cofilin and actin and between H-Ras and Raf1. In vivo and in vitro BiFC assays using the newly identified pair of Venus fragments will serve as a useful tool for measuring protein-protein interactions with high specificity and low background fluorescence and could be applied to the screening of inhibitors that block protein-protein interactions.     

Improvement of a Venus-based bimolecular fluorescence complementation assay to visualize bFos-bJun interaction in living cells

Bimolecular fluorescence complementation (BiFC) assay makes it possible to visualize protein-protein interactions in living cells. In this assay, Venus, a bright-yellow variant of green fluorescent protein, is known to produce fluorescent backgrounds due to non-specific interactions. In this study we found that the V150A mutation increased by 8.6-fold the signal-to-noise ratio in the BiFC assay of bFos-bJun interaction.     

An improved mRFP1 adds red to bimolecular fluorescence complementation

Protein-protein interactions are fundamental to virtually every aspect of cellular functions. Blue, green and yellow bimolecular fluorescence complementation (BiFC) systems based on GFP and its variants allow the investigation of protein-protein interactions in vivo. We have developed the first red BiFC system based on an improved monomeric red fluorescent protein (mRFP1-Q66T), expanding the range of possible applications for BiFC.     

LEC–BiFC: a new method for rapid assay of protein interaction

Abstract

Protein–protein interactions play fundamental roles in most biological processes. Bimolecular fluorescence complementation (BiFC) is a promising method for its simplicity and direct visualization of protein–protein interactions in cells. This method, however, is limited by background fluorescence that appears without specific interaction between the proteins. We report here a point mutation (V150L) in one Venus BiFC fragment that efficiently decreases background fluorescence of BiFC assay. Furthermore, by combining this modified BiFC and linear expression cassette (LEC), we develop a simple and rapid method (LEC–BiFC) for protein interaction analysis that is demonstrated by a case study of the interaction between Bcl–X_L and Bak BH3 peptide. The total analysis procedure can be completed in two days for screening tens of mutants. LEC–BiFC can be applied easily in any lab equipped with a fluorescence microscope.     

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.     

Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions

Protein-protein interactions play a pivotal role in coordinating many cellular processes. Determination of subcellular localization of interacting proteins and visualization of dynamic interactions in living cells are crucial to elucidate cellular functions of proteins. Using fluorescent proteins, we previously developed a bimolecular fluorescence complementation (BiFC) assay and a multicolor BiFC assay to visualize protein-protein interactions in living cells. However, the sensitivity of chromophore maturation of enhanced yellow fluorescent protein (YFP) to higher temperatures requires preincubation at lower temperatures prior to visualizing the BiFC signal. This could potentially limit their applications for the study of many signaling molecules. Here we report the identification of new fluorescent protein fragments derived from Venus and Cerulean for BiFC and multicolor BiFC assays under physiological culture conditions. More importantly, the newly identified combinations exhibit a 13-fold higher BiFC efficiency than originally identified fragments derived from YFP. Furthermore, the use of new combinations reduces the amount of plasmid required for transfection and shortens the incubation time, leading to a 2-fold increase in specific BiFC signals. These newly identified fluorescent protein fragments will facilitate the study of protein-protein interactions in living cells and whole animals under physiological conditions.