gus基因检测

报告基因●定义报告基因 (reporter gene)是一种编码可被检测的蛋白质或酶的基因，也就是说，是一个其表达产物非常容易被鉴定的基因。

把它的编码序列和基因表达调节序列相融合形成嵌合基因，或与其它目的基因相融合，在调控序列控制下进行表达，从而利用它的表达产物来标定目的基因的表达调控，筛选得到转化体。

特征作为报告基因，在遗传选择和筛选检测方面必须具有以下几个条件：(1)已被克隆和全序列已测定；(2)表达产物在受体细胞中本不存在，即无背景，在被转染的细胞中无相似的内源性表达产物；(3)其表达产物能进行定量测定。

●应用在植物基因工程研究领域，已使用的报告基因有以下几种：胭脂碱合成酶基因(nos)、章鱼碱合成酶基因(ocs)、新霉素磷酸转移酶基因(nptⅡ)、氯霉素乙酰转移酶基因(cat)、庆大霉素转移酶基因、葡萄糖苷酶基因、荧光酶基因等。

nos、ocs这两个基因是致瘤土壤农杆菌(Agrobacterium tumfaciens)的Ti质粒特有的，对Ti质粒进行改造，用相应的致瘤农杆菌转化植物体时，如果外源基因转入植物体中，则这两种报告基因在植物根茎叶中均能表达，不受发育调控，检测时直接用转化体提取液进行纸电泳，染色后在紫外光下观察荧光即可。

nptⅡ、cat及庆大霉素转移酶基因，均为抗生素筛选基因，相关的酶可以对底物进行修饰(磷酸化、乙酰化等)，从而使这些抗生素失去对植物生长的抑制作用，使得含有这些抗性基因的转化体能在含这些抗生素的筛选培养基上正常生长，也可以用转化体提取液体，外用同位素标记，放射自显影筛选转化体。

目前常用的一种报告基因是β-D-葡萄糖苷酶基因，该酶催化底物形成β-D-葡萄糖苷酸，它在植物体中几乎无背景，组织化学检测很稳定，可用分光光谱、荧光等进行检测。

荧光酶基因(luc)是1985年从北美荧火虫和叩头虫cDNA 文库中克隆出来的，该酶在有ATP、Mg2＋、O2和荧光素存在下发出荧光，这样就可用转基因植物整株或部分直接用X-光片或专门仪器进行检测。

GUS报告基因范文GUS报告基因是一种用于筛选转基因植物的报告基因。

它在植物细胞内表达的酵素β-葡萄糖苷酶（β-Glucuronidase，GUS），能够将葡萄糖醛酸（X-Gluc）转化为蓝色产物。

通过观察和分析植物组织的GUS活性，可以判断是否发生了基因转化。

下面将详细介绍GUS报告基因的特点、应用以及实验方法。

1.GUS报告基因的特点（1）GUS基因来自于大肠杆菌，它很少在真核生物中表达，因此不会对植物正常生长发育产生影响。

（2）GUS基因编码的酵素活性能够方便、快速地用染色剂标记出来，实验结果直观可见。

（3）转GUS基因的步骤相对简单，转化率较高，且不需要使用昂贵的设备。

2.GUS报告基因的应用（1）植物转基因筛选：通过观察和分析转基因植物的GUS活性，可以确定哪些植株成功地转化了外源基因。

（2）基因调控研究：GUS报告基因可以用来研究目的基因的表达调控机制，例如在转基因植物中瞬时表达GUS基因，观察其在各种组织和发育阶段的表达情况，可以推测目的基因的启动子活性。

（3）信号传导途径研究：通过构建GUS基因的操纵，可以研究植物信号传导途径中特定基因的表达情况，进而了解信号传导途径的效率和调节机制。

3.实验方法以下是GUS报告基因实验的一般步骤：（1）构建GUS载体：将GUS基因与适合的植物表达载体进行连接，形成GUS转化载体。

（2）遗传转化：将GUS转化载体导入要进行转基因植物研究的植物细胞中，使用适当的生物技术方法（如冲击法、农杆菌介导法）实现遗传转化。

（3）植物筛选：选择经过转化的植株进行分析，通常可以通过PCR、Southern blot、Western blot等技术检测GUS基因的存在。

（4）组织切片染色：收集不同部位的植物组织，例如叶片、根、花等，制作切片。

使用X-Gluc作为底物加入切片中，观察蓝色染色产物的形成。

（5）定量分析：通过测定GUS活性，使用亲合素、含有底物的液体培养基等方法，可以 quantitatively 地测定GUS酶活性。

gus报告基因随着基因科技的不断发展，越来越多的人开始关注自己的基因信息。

而近年来，Gus报告基因成为了热门话题之一。

那么什么是Gus报告基因呢？Gus报告基因是由Gus Health公司提供的基因检测服务，旨在通过对个体DNA样本的分析，为客户提供关于健康、运动、营养、皮肤护理、基因亲属关系等方面的个性化建议和指导。

近年来，越来越多的人通过Gus报告基因获取自己的基因信息，并通过这些信息来优化自己的生活方式。

Gus报告基因的检测流程十分简单，客户只需要在网上下单，收到检测包裹后依照说明书采集口腔唾液后将样本寄回公司。

约两周后，客户可以获得自己的基因检测结果，并获得个性化的建议和指导。

那么Gus报告基因检测的结果都包括哪些内容呢？首先是健康方面。

Gus报告基因可以检测出客户是否携带有害突变基因，以及是否存在易感基因，提前了解个体健康的风险，有利于尽早采取措施进行预防和治疗。

其次是运动方面。

据研究，每个人的身体对不同类型的锻炼有不同的反应。

Gus报告基因检测结果可以显示客户是否适合进行高强度训练、有氧运动还是力量训练等不同类型的锻炼。

再者是营养方面。

Gus报告基因可以检测出客户是否存在乳糖不耐受、咖啡因代谢能力等基因变异，为客户提供更加个性化的饮食建议。

此外，Gus报告基因还可以检测出客户皮肤老化的风险，提供关于皮肤护理的建议；并且可以通过DNA亲属检测，了解亲属间的遗传关系。

但是需要注意的是，Gus报告基因并不是一份“命运报告单”，不能确定一个人未来发生某种疾病的概率。

每个人的基因信息都受到许多因素的影响，因此仅仅基于基因信息来进行健康管理是远远不够的。

另外，Gus报告基因也不应被视为100%准确的检测解决方案。

每种基因检测技术都有其局限性和误差率。

因此，必须对检测结果不以基因为唯一依据，而是作为综合评估的参考之一。

总的来说，Gus报告基因是一项非常有用的检测服务，可以为人们提供个性化的健康管理指导。

gus报告基因GUS报告基因。

GUS报告基因是一种用于植物基因表达研究的重要工具。

它是由β-葡萄糖苷酶（β-glucuronidase）基因构建而成，可以被广泛地应用于植物遗传转化和表达分析中。

GUS报告基因的研究对于理解植物基因表达及其调控机制具有重要意义。

首先，GUS报告基因可以用于植物遗传转化的研究。

通过将GUS报告基因导入植物细胞，可以观察到该基因在植物体内的表达情况。

这对于研究外源基因在植物中的表达特点以及转基因植物的遗传稳定性具有重要意义。

通过对GUS报告基因的研究，可以更好地了解植物转基因技术的应用前景及其潜在风险。

其次，GUS报告基因也可以用于植物基因表达分析。

通过将GUS报告基因与目标基因进行融合，可以观察到目标基因在植物体内的表达情况。

这对于研究目标基因在不同组织和发育阶段的表达模式，以及其受到外界环境因素影响的调控机制具有重要意义。

通过对GUS报告基因的研究，可以更好地了解植物基因表达调控的分子机制及其在植物生长发育过程中的作用。

此外，GUS报告基因还可以用于植物转基因技术的研究与应用。

通过对GUS报告基因的表达情况进行定量和定位分析，可以更好地评估转基因植物的表达效率和稳定性。

这对于改良转基因植物的遗传背景和表达性能具有重要意义。

通过对GUS报告基因的研究，可以更好地指导植物转基因技术的开发和应用，为农业生产和生物技术研究提供有力支持。

综上所述，GUS报告基因在植物基因表达研究中具有重要的应用价值。

通过对GUS报告基因的研究，可以更好地理解植物基因表达及其调控机制，促进植物遗传转化和基因功能研究的进展，为植物生物技术的发展和应用提供重要支持。

因此，对GUS报告基因的深入研究具有重要的理论和实际意义，值得进一步深入探讨和应用。

gus报告基因作用原理
Gus报告基因作用原理
基因是生命的基本单位，它们控制着生物体的生长、发育和功能。

基因作用原理是指基因在生物体内的作用方式和机制。

Gus报告基因是一种常用的基因标记技术，它可以用来研究基因的表达和调控。

Gus报告基因是一种β-葡萄糖苷酶（β-glucuronidase）基因，它可以将X-葡萄糖苷（X-Gluc）转化为蓝色产物。

Gus报告基因可以被插入到其他基因的上游或下游区域，从而形成Gus报告基因转录本。

当这些转录本被转录和翻译时，Gus报告基因就会被表达出来，从而产生蓝色染色。

Gus报告基因的作用原理是基于基因表达的调控机制。

基因表达是指基因转录和翻译的过程，它受到许多因素的调控，包括转录因子、启动子、增强子、剪接和RNA降解等。

Gus报告基因可以被插入到这些调控元件的上游或下游区域，从而研究它们对基因表达的影响。

Gus报告基因可以用来研究基因的表达模式、组织特异性和响应信号等。

例如，可以将Gus报告基因插入到一个特定的基因的上游或下游区域，从而研究它在不同组织和发育阶段的表达模式。

此外，还可以将Gus报告基因插入到一个响应信号的基因的上游或下游区域，从而研究它对该信号的响应机制。

Gus报告基因是一种常用的基因标记技术，它可以用来研究基因的
表达和调控。

它的作用原理是基于基因表达的调控机制，通过插入到调控元件的上游或下游区域，从而研究它们对基因表达的影响。

Gus报告基因的应用可以帮助我们更好地理解基因的功能和调控机制，为生命科学研究提供重要的工具和方法。

14Histochemical and FluorometricAssays for uidA(GUS) Gene DetectionMagdalena CerveraSummaryTransgenic plant production has been intimately connected to the β-glucuronidase (uidA or GUS) gene used as a reporter marker gene. The enzyme stability and the high sensitivity and amenability of the GUS assay to qualitative (histochemical assay) and to quantitative (fluorometric or spectrophotometric assay) detection are some of the rea-sons that explain the extensive use of uidA gene in plant genetic transformation. Meth-ods for uidA gene detection have been thoroughly described in the literature. The aim of this chapter is to describe the basic protocols needed for GUS detection in a plant genetic transformation laboratory.Key Words: Fluorometric GUS detection; β-glucuronidase; GUS; histochemical GUS detection; reporter marker genes; uidA gene.1. IntroductionA reporter gene codes for an enzyme or other protein that can be detected directly or indirectly using a biochemical assay. The establishment of genetic transformation procedures has relied on, among other factors, the use of effi-cient reporter marker genes, which easily allows the detection of transgenic events after a transformation experiment, in either a transient or stable expres-sion assay. The production of transgenic plants in many cases depends also on the use of reporter genes, as it facilitates the identification of stably transformed individuals once they have undergone a selection process based on the select-able marker gene used in the same experiment. It should also be mentioned that gene reporter systems have played a key role in many gene expression and regulation studies, in which expression of a reporter gene under, for instance, From:Methods in Molecular Biology, vol. 286: Transgenic Plants: Methods and ProtocolsEdited by: L. Peña © Humana Press Inc., Totowa, NJ203the direction of different promoters or the presence of different transcription factors may be investigated.Since the β-glucuronidase (GUS) gene (gus,gusA, or uidA) was first isolated from Escherichia coli (1), many efforts have been made to develop the E. coli uidA gene as a reporter system for plant transformation(2,3). Indeed, it has be-come the most widely used marker system, mainly because of the enzyme stabil-ity and the high sensitivity and amenability of the assay to detection by fluorometric, spectrophotometric, or histochemical techniques. In addition, there is little or no detectable GUS activity in almost any higher plant tissues (2), with some exceptions (4–7). These compile a great part of the attributes a reporter marker gene must account for.E. coli GUS has a monomeric molecular weight of 68,200 and appears to function as a tetramer (8). This enzyme hydrolyzes β-glucuronides as substrates and the detection method will vary depending on the specific substrate and prod-uct formed after the reaction. In plants it works as a fusion gene, where a pro-moter coming from a different organism directs the transcription of the uidA coding sequence, specifically regulating gene expression in time, quantity, and cell or tissue location. Some potential limitations have been reported in the use and subsequent detection of GUS activity in transformed plant tissues: back-ground activity (4,9), normally because of diffusion of the reaction product or to endogenous activity; autofluorescence (9); quenching or inhibitors (9,10); or microbial contamination (11,12). In an Agrobacterium-mediated transformation system, it is adequate to work with a reporter gene modified by the presence of an intron, which will impede gene expression in bacteria and thus interferences in the detection assay (12).One disadvantage of the uidA gene as a reporter marker is that commonly used GUS assays involve destruction of plant material. However, it is pos-sible to detect glucuronidase activity in a nondestructive manner. Exposure of plant material to 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) or 4-methyl-umbelliferyl-β-D-glucuronide (MUG) for short periods of time reduces toxicity of these substrates as to allow later rescue of plant material (13,14). Nevertheless, in the past few years, other markers have been devel-oped, for example, that encoding the green fluorescent protein (GFP) of Aequorea victoria (15), which has proved to be more useful for some appli-cations than the uidA gene, as it can be visualized without demanding the destruction of plant material.GUS assays can be performed in a wide variety of tissues, even in proto-plasts, taking into account that differences in the tissue structure might require slight changes in the detection protocols. Besides, it is important to know that reproductive tissues may exhibit endogenous GUS activity (4)and that plant development may also affect gus gene expression (10).Although there are othersubstrates available, two of them are the most currently used, X-Gluc for GUS histochemical localization and MUG for GUS fluorometric quantitation. This chapter focuses our attention on describing histochemical and fluorometric detection assays involving these substrates.2. Materials2.1. Histochemical GUS Detection Assay2.1.1. Stock Solutions1. 1 M Tris-HCl buffer, pH 7.0.2.0.1 M Tris-HCl buffer, pH 7.0.3. 5 M NaCl.4.1% (v/v) Triton X-100.5. 5 m M Potassium ferricyanide, pH 7.0.6. 5 m M Potassium ferrocyanide, pH7.0.7.1% (v/v) Glutaraldehyde in 0.1 M Tris-HCl buffer, pH 7.0.2.1.2. Substrate Solution (X-Gluc) (see Note 1)1.100 m M Tris-HCl buffer, pH 7.0 (see Note 2), 50 m M NaCl, 0.01% Triton X-100, 0.5 m M potassium ferricyanide, pH 7.0 (see Note 3), 0.5 m M potassium ferrocyanide, pH 7.0, 2 m M X-Gluc (Duchefa Biochemie BV; Haarlem, The Netherlands) (powder; for 10 mL of reagent mix dissolve 10.41 mg of X-Gluc in0.4 mL of N,N-dimethylformamide [DMF] before mixing with the other compo-nents) (see Note 4).2.Although there are other ways, we recommend keeping separate stock solutionsof each component, storing them in the refrigerator. We do not keep a substrate stock solution, but weigh the required amount for each assay. Once substrate is dissolved in DMF, reagent mix is prepared fresh from stocks and adjusted to the final volume. If GUS assays are not often performed in the laboratory, we also prefer not to store a potassium ferrocyanide stock, as it oxidizes quickly (it does not last more than 2 mo in the refrigerator). It is better to weigh the amount required for each assay.2.2. Fluorometric GUS Detection Assay2.2.1. Stock Solutions1. 1 M Sodium phosphate buffer, pH 7.0.2.0.25 M Na2EDTA, pH 8.0.3.0.2 M Na2CO3, pH 9.5.4.1% (v/v) Triton X-100.5. 1 mg/mL of bovine serum albumin (BSA).6.Bradford solution for protein determination (Bio-Rad, Hercules, CA).7.Diluted Bradford solution (1:5) in sterile water (prepare just before use).8.Extraction buffer: 50 m M Na phosphate buffer, pH 7.0, 10 m M Na2ethylenedi-aminetetraacetic acid (EDTA), pH 8.0, 0.1% Triton X-100, 10 m Mβ-mercapto-ethanol.2.2.2. Substrate and Product Solutions1.Substrate solution: 2 m M4-MUG (Sigma, St. Louis, MO) (3.5 mg of 4-MUG in5 mL of extraction buffer).2.Product solution: 1 m M4-methylumbelliferone (MU) (Sigma) (1.98 mg of MUin 10 mL of 0.2 M Na2CO3, pH 9.5). This is the stock solution, from which work-ing solution will be prepared.2.2.3. EquipmentFluorometer (DNA fluorometer model TKO 100, Bio-Rad).3. MethodsThe methods described here are based on the protocols by Jefferson et al.(2)and Gallagher (16), and some slight modifications have been introduced on our experience working with transgenic plant material. They are indicated and explained in the Notes.3.1. Histochemical GUS Detection AssayIn the histochemical assay, hydrolysis of X-Gluc by GUS gives an insoluble and highly colored indigo dye, visualized as a blue precipitate at the site of enzyme activity that is easily detectable (Fig. 1).3.1.1. Staining Assay1.Cut plant sections to be assayed and place them inside testing tubes, Eppendorftubes, small beakers, or multiple-well plates.2.Add a generous volume of substrate solution to plant sections in the wells tocover them completely.3.If necessary, infiltrate tubes or plates under vacuum for about 1–2 min to enhancepenetration of substrate.4.Cover tubes or plates with Parafilm or similar to avoid evaporation during incu-bation.5.Incubate at 37°C a minimum of 3–4 h to a maximum of 14–18 h (see Note 5).6.Uncover plates and remove carefully substrate solution using a Pasteur pipet.Wash tissues three times with 0.1 M Tris-HCl, pH 7.0.7.Fix tissues, if necessary, with 1% glutaraldehyde in 0.1 M Tris-HCl, pH 7.0.Incubate at 15°C for 2–3 h (see Note 6).8.Wash three times with 0.1 M Tris-HCl, pH 7.0.9.Proceed to destaining of tissues (see Note 7) by following a series of increasingethanol mixtures (30, 50, 70, 90, and 100%; 5 min each) to achieve total dehydra-tion of tissues. Keep tissues with pure ethanol for about 1 h. This will remove the chlorophyll and will allow an easier detection of GUS-positive events.10.Proceed to rehydrate tissues by submerging them in decreasing ethanol mixtures(70, 50, and 30%; 5 min each) and diluted Tris-HCl buffer in the last step. Tissue samples are now ready to be observed under a stereomicroscope (see Note 8). If desired, samples can be mounted for microscopy. Figure 2shows the aspect of different transgenic citrus tissues after GUS staining assays; all were performed in our laboratory.3.2. Fluorometric GUS Quantitation AssayUnlike the histochemical detection, fluorometric analysis allows quanti-tation of GUS activity. In the presence of GUS, MUG is hydrolyzed to a fluo-rescent product, 4-methylumbelliferone (MU) (Fig. 3). After the reaction, total fluorescence is measured and product concentration is calculated based on a previous MU standardization curve. The fluorometric assay is highly reliable and simple to use. However, precautions must be taken to perform the analysis in steady conditions and achieve maximum assay repeatability.Fig. 1. Reaction taking place in the histochemical GUS assay (from Guivarc’h et al.[20]). 5-Bromo-4-chloro-3-indolyl-β-D -glucuronide (X-Gluc) is used as the substrate for GUS, and cleavage of X-Gluc leads to precipitation of a blue product (diXH-indigoor ClBr-indigo) at the site of enzyme activity.Fig. 3. Reaction taking place in the fluorometric GUS assay. 4-Methylumbelliferyl-β-D -glucuronide (MUG) is used as the substrate for GUS in a GUS fluorometric quantitation. Cleavage of MUG leads to the formation of fluorescent 4-methylumbelliferone (MU). At pH > 8.2 the phenoxide form is predominant and fluo-rescence at 460 nm is measured to quantitate GUS activity.Fig. 2. Transgenic citrus tissues showing GUS staining after histochemical uidA gene detection: (A)leaf pieces showing different levels of GUS expression (a control from a nontransformed plant is shown on the left). (B)Flower from an adult sweet orange tree (a control from a non-transformed plant is shown on the left). (C)Embryos from transgenic seeds. (D)Transverse section of a fruit from an adult tree (a control from a non-transformed plant is shown on the left). (E)cut end from a stem segment showing GUS+ (dark blue) transformation events. All tissues came from Agrobacterium -mediated transformation and regeneration experiments, where A.tumefaciensEHA105 p35SGUSINT was used as the vector system.3.2.1. Fluorometric Assay3.2.1.1. E XTRACTI O N M ETH O De fresh material or material frozen in liquid nitrogen.2.Extract 20–50 mg of tissue in an Eppendorf tube in 400 μL of extraction bufferand homogenize using a driller. Work on ice.3.Centrifuge 10 min at 4°C at 13,000g to remove unlysed cells and debris (seeNote 9).4.Transfer the supernatant to a fresh tube and centrifuge again 10 min at 4°C at13,000g.5.Samples may be stored at –80°C, but it is not convenient to thaw and freeze themmore than twice, as activity is partially lost.3.2.1.2. B RADF O RD A SSA Y (P R O TEIN C O NCENTRATI O N)1.Prepare diluted Bradford solution and distribute 1 mL in Eppendorf tubes.2.Make dilutions of the BSA stock solution for calibration (in triplicate). Use thisrange: 0, 1.0, 2.0, 4.0, 6.0, 10.0, and 20.0 μg of BSA (see Note 10). Add appro-priate volumes of BSA stock to the Bradford solution (for a more accurate cali-bration, previously remove the same volume of Bradford solution from the Eppendorf tubes) and mix well.3.Prepare samples by adding 2–10 μL of protein extracts to 1 mL of dilutedBradford solution. Make sure that colors of solutions are in the range of BSA standards.4.Determine the absorbance at 600 nm in a spectrophotometer (see Note 11).5.Calculate the total protein concentration in the samples using the BSA standard-ization curve.3.2.1.3. M U G A SSA Y (E NZ Y ME A CTIVIT Y)1.Keep a bath at 37°C and prepare three Eppendorf tubes per sample with 900 μLof 0.2 M Na2CO3 to stop the reaction at different times.2.Prepare the substrate solution to a final concentration of 2 m M4-MUG (3.5 mgof 4-MUG in 5 mL of extraction buffer).3.Mix protein extracts with substrate in a 1:1 proportion to a total volume of 300μL. Incubate at 37°C.4.Take 100 μL from mixtures at three different times (e.g., 10, 20, and 30 min) (seeNote 12) and mix well with 900 μL of Na2CO3(ready in Eppendorf tubes) (see Note 13). Keep in dark until measurement is performed.5.Prepare stock product solution to a concentration of 1 m M MU (1.98 mg of MUin 10 mL of 0.2 M Na2CO3). Prepare a working dilution of 0.1 μM MU making an intermediate 10 μM dilution from the 1 m M stock. Keep both refrigerated in the dark.6.Calibrate the fluorometer using the diluted MU solution as standard and measureMU fluorescence in the samples (see Note 14). Calculate GUS activities as pmol of MU/min/μg of total protein.4. Notes1.We usually use in the laboratory for GUS assays Tris-HCl buffer (+ NaCl) insteadof phosphate buffer, as described originally by Jefferson et al. (2),but both have given good results in our case. At any rate, as substrate solution in phosphate buffer is obviously the most used reagent mixture, it is probably convenient to mention it here: 0.1 M Na phosphate buffer pH 7.0, 10 m M EDTA (see Note 3), 0.5 m M potassium ferricyanide, 0.5 m M potassium ferrocyanide, 1 m M X-Gluc, 0.1% Tri-ton X-100 (final concentrations) (17).2.Apparently some buffer substances might have effects on certain promoters (14),if fixation is not performed before staining, leading to equivocal results. This is not a substantial issue for the most usual promoters, and Tris-HCl, phosphate, or other buffers at pH 7.0 give good results. pH is another important factor in the case of the GUS staining assay, as most endogenous plant GUS activities exhibit at pH 5.0. However, it can occur at higher pH; for instance, we found endogenous GUS activity in citrus seeds at pH 7.0. This can be normally avoided by increas-ing working pH to pH 8.0 or even pH 9.0 (14),adding methanol (5,14,18),or polyvinylpolypyrrolidone (PVPP) (10).3.X-Gluc hydrolysis is usually restricted to the site of GUS activity. However, atsites presenting local oxidative processes, such as high peroxidase activity, pre-cipitation of diX-indigo may occur. A slow oxidation step (and following dimer-ization into insoluble indigo) allows the soluble intermediate to diffuse away from the site of reaction, making an accurate localization of GUS activity (back-ground activity) difficult. Addition of a mixture of 0.5 M potassium ferrocya-nide, and 0.5 M potassium ferricyanide (6)in the substrate solution accelerates oxidation of the reaction intermediates to diXH-indigo. It is convenient to note that a high concentration of potassium ferricyanide may inhibit GUS activity (6), so this should be taken into account when the uidA gene is driven by a weak promoter. EDTA is added to mitigate the partial inhibition of the enzyme by the oxidation catalyst.4.Higher substrate concentrations may be used to increase signal in case of weakstaining. Increasing potassium ferrocyanide/potassium ferricyanide concentra-tions or the incubation periods may also be useful in these cases.5.Substrate penetration may be a problem in the case of some tissues, such as leafpieces. Leaf cuticle may difficult the penetration of X-Gluc in this tissue, so normally it is better to punch the leaf with a punctilious object, add Triton X-100 to the substrate mixture and infiltrate tissue in the substrate solution by using a vacuum pump. Calluses, stem or young root pieces do not usually present this problem. Results can be visible after 1–2 h of incubation, but normally it is better to let the reaction finish. Infiltration problems or low expression could lead to errors in the final reading of results. Nevertheless, owing to the stability of the enzyme and extreme sensitivity of the assay, the product is accumulated during the entire period and relative quantification could lead as well to overinterpretation of the data.6.Fixation can be performed before or after staining. If done before staining, fixa-tion time must be short so as not to lose the activity of the glucuronidase (14).Fixation after staining can be performed as explained in the text or with other typical fixatives, such as ethanol.7.The process of destaining is not normally necessary in the case of calluses orother tissues with low chlorophyll content, but it is highly recommended in leaf or stem tissues, where GUS-positive events may appear masked by chlorophyll.8.Photographic records should be kept from these assays, so they can be useful forlater comparisons. Stained samples can be stored in ethanol in well-sealed con-tainers.9.When working with protoplasts, collect them by centrifugation for 5 min at 85–90g(three or four times, 1.5 mL of protoplast suspension) in an Eppendorf tube.Discard supernatant each time. Add 0.4 mL of extraction buffer to the pellet and homogenize gently by ultrasound (10–20 s).10.Values included in the BSA curve can be modified depending on the samples weare working with and their expression. The colors of the extract mixtures give an initial clue, they should fall among the colors of BSA standards.11.If a reader of multiwell plates is available, the BSA standard curve and extractscan be prepared and read in the same plate, in volumes of 200 μL. This facilitates and even homogenizes readings. If it is not available in the laboratory, standards and samples must be measured one by one in the spectrophotometer.12.Incubation times and extract:substrate proportion may vary depending on thesamples. Longer incubation and higher extract volumes are probably needed when working with samples with low expression or in transient expression assays. 13.Fluorescent properties of phenolic and phenoxide forms of 4-MU are different:phenolic form (excitation 323, emission 386 nm) and phenoxide form (excitation 363, emission 447 nm). Owing to the equilibrium at physiological pH between phenolic and phenoxide forms of 4-MU (Fig. 3), the fluorescence value at the reaction pH is relatively low. Treatment of samples after incubation with 0.2 M Na2CO3buffer, pH 9.5 stops the enzyme reaction and raises the final pH above the p K a of 4-MU (p K a8.2), shifting the reaction to produce the maximum amount of fluorescence at 447 nm (for more details, see ref.19).14.At the end of the assay, there should be three fluorescence values per sample,corresponding to different reaction times. Final data are expressed as the slope of the amount of product formed in the reaction (starting from an extract with a determined concentration of total protein) vs time (pmol of MU/min/μg of total protein). In our laboratory, we use the Bio-Rad DNA fluorometer model TKO 100. It is a filter fluorescence photometer with a fixed excitation bandpass source (365 nm) and an emission (460 nm) bandpass filter. Calibration and measure-ments are easy to perform and sensitivity is acceptable. AcknowledgmentsThe author thanks A. Navarro for his excellent help in many GUS assays. Part of this work was supported by grants from the Generalitat Valenciana No.CTIDIA/2002/89, from the Instituto Nacional de Investigaciones Agrarias No.RTA-01-120 and from CICYT AGL2003-01644.References 1.Novel, G. and Novel, M. (1973) Mutants d’Escherichia coli affectés pour leur croissance sur methyl β-glucuronide: localisation du gene de structure de la β-glucuronidase (uidA ).Mol. Gen. Genet.120, 319–335.2.Jefferson, R. A., Kavanagh, T. A., and Bevan, M. W. (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.Embo J.6,3901–3907.3.Jefferson, R. A., Bevan, M., and Kavanagh, T. (1987) The use of the Escherichia coli beta-glucuronidase as a gene fusion marker for studies of gene expression in higher plants. Biochem. Soc. Trans.15, 17–18.4.Hu, C.-Y., Chee, P. P., Chesney, R. H., Zhou, J. H., and Miller, P. D. (1990)Intrinsic GUS-like activities in seed plants. Plant Cell Rep.9,1–5.5.Kosugi, S., Ohashi, Y., Nakajima, K., and Arai, Y. (1990) An improved assay for β-glucuronidase in transformed cells: methanol almost completely suppresses a putative endogenous β-glucuronidase activity. Plant Sci.70, 133–140.6.Mascarenhas, J. P. and Hamilton, D. A. (1992) Artifacts in the localization of GUS activity in anthers of petunia transformed with a CaMV 35S-GUS construct.Plant J.2, 405–408.7.Muhitch, M. J. (1998) Characterization of pedicel β-glucuronidase activity in developing maize (Zea mays ) kernels. Physiol. Plant.104,423–430.8.Jefferson, R. A., Burgess, S. M., and Hirsh, D. (1986) β-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc. Natl. Acad. Sci. USA 83,8447–84519.Thomasset, B., Ménard, M., Boetti, H., Denmat, L. A., Inzé, D., and Thomas, D.(1996)β-Glucuronidase activity in transgenic and non-transgenic tobacco cells:specific elimination of plant inhibitors and minimization of endogenous GUS background.Plant Sci.113,209–219.10.Serres, R., McCown, B., and Zeldin, E. (1997) Detectable β-glucuronidase activ-ity in transgenic cranberry is affected by endogenous inhibitors and plant devel-opment.Plant Cell Rep.16, 641–646.11.Tör, M., Mantell, S. H., and Ainsworth, C. (1992) Endophytic bacteria expressingβ-glucuronidase cause false positives in transformation of Dioscorea species.Plant Cell Rep.11,452–456.12.Vancanneyt, G., Schmidt, R., O’Connor-Sanchez, A., Willmitzer, L., and Rocha-Sosa, M. (1990) Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium -mediated plant transformation. Mol. Gen. Genet.220, 245–250.13.Kirchner, G., Kinslow, C. J., Bloom, G. C., and Taylor, D. W. (1993) Non-lethalassay system of β-glucuronidase activity in transgenic tobacco roots. Plant Mol.Biol. Rep. 11, 320–325.14.Martin, T., Wöhner, R.-V., Hummel, S., Willmitzer, L., and Frommer, W. B.(1992) The GUS reporter system as a tool to study plant gene expression, in GUS 12357891012Assays for uidA (G U S) Gene Detection 213Protocols: Using the GUS Gene as a Reporter of Gene Expression (Gallagher, S.R., ed.), Academic Press, San Diego, CA, pp. 23–43.15.Stewart, C. N., Jr. (2001) The utility of green fluorescent protein in transgenic plants.Plant Cell Rep.20, 376–382.16.Gallagher, S. R., ed. (1992) GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression , Academic Press, San Diego, CA.17.Stomp, A.-M. (1992) Histochemical localization of β-glucuronidase, in GUS Pro-tocols: Using the GUS Gene as a Reporter of Gene Expression (Gallagher, S. R.,ed.), Academic Press, San Diego, CA, pp. 103–113.18.Wilkinson, J. E., Twell, D., and Lindsey, K. (1994) Methanol does not specifi-cally inhibit endogenous β-glucuronidase (GUS) activity. Plant Sci.97, 61–67.19.Naleway, J. J. (1992) Histochemical, spectrophotometric, and fluorometric GUS substrates, in GUS Protocols: Using the GUS Gene as a Reporter of Gene Expres-sion (Gallagher, S. R., ed.), Academic Press, San Diego, CA, pp. 61–76.20.Guivarc’h, A., Caissard, J. C., Azmi, A., Elmayan, T., Chriqui, D., and Tepfer, M.(1996)In situ detection of expression of the gus reporter gene in transgenic plants:ten years of blue genes. Transgen. Res.5, 281–288.1518。

1．GUS报告基因的定性检测GUS（β- 葡萄糖苷酸酶）能与显色底物X-gluc 反应，显现蓝色，因而可以通过组织化学染色定性研究GUS的表达水平和表达模式。

GUS 染色的稳定性好，组织定位精确，成为在植物中运用很广的报道基因。

gus基因是目前常用的一种报告基因，是β－D－葡萄糖苷酸酶(gus)基因，其表达产物β-葡萄糖苷酸酶(GUS)能催化裂解一系列的β－葡萄糖苷酸，它可以将5-溴-4-氯-3-吲哚-β-葡萄糖苷酸酯（X－Gluc）分解为蓝色的物质,也可以将4-甲基-伞形花酮-β-D-葡萄糖苷酯(4-MUG)分解为蓝色物质。

其检测方法简单、快速、灵敏、稳定，且背景活性低。

1.1 GUS染色底物的配制* 0.5M Na2HPO4配制方法：将17.907g Na2HPO4溶于水，定容至100ml。

0.5M NaH2PO4的配制方法：将7.800g NaH2PO4溶于水，定容至100ml。

实验步骤：1.将准备好的拟南芥植株放入小EP管中，加入染色液浸没试材，封好盖子；可用抽真空，5min，200mbr。

2.37℃培养箱中温育12h，或有蓝色出现，水中洗涤一次；3.将浸染过的试材转入70%（或95%乙醇）中脱色2-3次（除去叶绿素），每隔1小时更换一次脱色液，至阴性对照材料呈白色为止。

（漂洗：先后用50%，70%，100%的乙醇漂洗样品，每次浸泡5分钟。

脱色：加入100%乙醇浸泡直至完全脱色。

）4.立体显微镜观察拍照。

2．GUS报导基因的定量检测GUS 能与底物MUG（分子量352.3，4-甲基伞型酮-β-葡萄糖醛酸苷，4-methylumbelliferylβ-D-glucuronide）反应产生荧光物质MU（分子量198.2，4-甲基伞型酮，4-methylumbelliferone）。

MU的激发波长为365nm，发射波长为456nm，其含量可由荧光分光光度计测出。

因此，我们可以根据单位质量的植物总蛋白在单位时间内产生的荧光物质的多少来定量的检测GUS含量。

转基因植物中报告基因表达检测-gus活性检测的思考题1、对实验结果进⾏⽂字描述。

2、根据你的实习谈谈gus活性检测时应注意哪些问题。

注意事项：⽤于染⾊的植物材料的制备⽅法要因涉及的特定组织和器官的不同⽽异。

例如，拟南芥的根、花和叶⽚以及烟草幼苗的根就可以不作任何预处理⽽直接染⾊。

但是像烟草和马铃薯这些植物的茎和叶就必须在染⾊前切成薄⽚（1-3mm）。

当操作⼤的组织和样品时，可以选⽤真空渗⼊法来帮助底物和酶渗⼊细胞。

3、gus基因为什么被称之为报告基因？在转基因应⽤中有哪些优缺点。

报告基因是⼀种编码可被检测的蛋⽩质或酶的基因，也就是⼀个其表达产物⾮常容易被鉴定的基因。

把它的编码序列和基因表达调节序列相融合形成嵌合基因，或与其它⽬的基因相融合，在调控序列控制下进⾏表达，从⽽利⽤它的表达产物来标定⽬的基因的表达调控，筛选得到转化体。

报告基因必须具备的条件：(1)已被克隆和全序列已测定; (2)表达产物在受体细胞中不存在，即⽆背景，在被转染的细胞中⽆相似的内源性表达产物; (3) 报告基因编码的产物的检测应该快速、简便、灵敏度⾼⽽且重现性好，表达产物能进⾏定量测定。

(4) 细胞内其它的基因产物不会⼲扰报告基因产物的检测。

GUS活性检测优点（1）在⼤多数植物组织中GUS活性的本底低；（2）反应产物基本不扩散，在植物细胞内积累；（3）通过简单的扩散或真空渗⼊，底物易被植物细胞吸收。

GUS基因作为报告基因的优势：1GUS蛋⽩在植物细胞中稳定存在，对较⾼的温度和去污剂有⼀定的耐受性；2检测⽅法简单，可定性或定量：使⽤X-Gal为底物，通过显⾊反应来观察；使⽤4-MU为底物，反应以后⽣成荧光物质，通过相应的荧光检测仪来进⾏经确定量；3多数植物组织中GUS背景表达低，样品表达能够很好区分。

缺点（1）不适⽤于活体组织的观察；（2）在GUS表达⽔平很⾼的组织中会发⽣⽆⾊反应产物渗漏的现象，但是可以通过在底物溶液中加⼊氧化剂氰化钾或亚铁氰化钾或者⼆者都加（终浓度为5mmol/L），来使这种渗漏减⾄最少。