qRT-PCR-生物分析检测技术

,单击此处编辑母版标题样式,*,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,第二节,实时荧光定量,PCR,实时荧光定量,PCR,仪、技术原理及应用,一、实时荧光定量,PCR,仪,二、实时荧光定量,PCR,的技术原理,1.,定量与常规的差别,2.,荧光扩增曲线,3.,荧光阈值和,CT,值,4.,熔解曲线,5.,标准曲线,6.,何为实时？,7. SYBR Green I,的工作原理,8. Molecular Beacons,（发夹型杂交探针）的工作原理,9. TaqMan,（水解型杂交探针）的工作原理,10.,多色多通道技术应用,基因表达分析,11.,内标技术介绍,三、 实时荧光定量,PCR,技术的应用介绍,1.,医疗方面,2.,研究方面,3.,其它方面,四、荧光定量,PCR,实验室所需仪器设备清单,五、实时定量,PCR,及应用于植物分子生物学的,研究,一、实时荧光定量,PCR,仪,热循环仪（,PCR,仪）,荧光检测系统,计算机及软件系统,不产热的光源-,发光二极管（,LED,）,灵敏度高的检测器-,光电倍增管（,PMT）,优点：,不产热,-无散热装置，全封闭,无机械转动装置-抗震性强,无需经常校准，耐用,灵敏度,高,线性范围,广,Opticon,实时荧光定量,PCR,仪,荧光检测 热循环仪,光色: 蓝色光 均一性: 0.4,C,染料:,FAM, SYBR Green I,Molecular beacons,TaqMan Probes,精确性: 0.3,C,激发光波长：,450-495,nm,发射光波长:,515-545,nm,升降温速率: 最高3,C/,秒.,灵敏度: 5,nM,的荧光素 温度梯度: 有,检测范围:,10,0,- 10,8,拷贝,容量: 96样品,反应管: 0.2,mL,反应管& 96 孔板,操作系统:,Windows NT,二、实时荧光定量,PCR,的技术原理,1.,定量与常规的差别,常规,PCR,技术：对,PCR,扩增反应的,终产物,进行定量及定性分析,定量,PCR,技术：对,PCR,扩增反应中,每一个循环的产物,进行定量及定性分析,SYBR Green I,定量原理,确定初始模板的浓度,:,初始,DNA,量越,多, 荧光达到某一值（域值）时所需要的,循环数越少,Log,浓度与循环数呈线性关系,：,根据样品扩增,达到域值的循环数,就可计算出样品中所含的模板量,实时荧光定量,PCR,技术简介,实时荧光定量,PCR,技术简介,实时荧光定量,PCR,技术简介,实时荧光定量,PCR,技术简介,实时荧光,定量,PCR,：其反应体系中，引入了,一种荧光化学物质,，随着,PCR,反应的进行，,PCR,反应产物不断累计，荧光信号强度也,等比例增加,。,每经过一个循环，收集一个荧光强度信号,，这样我们就可以通过,荧光强度变化监测产物量的变化,，从而得到一条荧光扩增曲线。,2.,荧光扩增曲线,荧光扩增曲线可以分三个阶段：荧光,背景信号阶段,，荧光信号,指数扩增阶段,和,平台期,。,荧光背景信号阶段,：扩增的荧光信号被荧光背景信号所掩盖，我们无法判断产物量的变化。,平台期,：扩增产物已,不再呈指数级的增加,。,PCR,的终产物量与起始模板量之间没有线性关系，所以根据最终的,PCR,产物量不能计算出起始,DNA,拷贝数。,只有在,荧光信号指数扩增阶段,：,PCR,产物量的对数值与起始模板量之间存在线性关系,，我们可以选择在这个阶段进行定量分析。,3.,荧光阈值和,CT,值,荧光阈值,是在荧光扩增曲线上人为设定的一个值 ，它可以设定在,荧光信号指数扩增阶段任意位置上,，但一般我们将荧光域值的缺省设置是,3-15个循环,的,荧光信号的标准偏差的10倍,。,荧光阈值线的位置,一般事实上,位于减去本底的位置上,，这时的荧光信号超过了荧光背景信号并且开始增加。,对某一样品的,C（t）,值,就定义为,荧光量与荧光阈值线交叉,时的,循环数,。即每个反应管内的荧光信号到达设定的域值时所经历的循环数被称为,CT,值,（,threshold value,）,。,定量荧光扩增曲线,是荧光量与循环数的图表。,4.,熔解曲线,在,PCR,结束后，我们可以根据,变性过程中的荧光值变化,绘出每个样品的,熔解曲线,。,绘制熔解曲线时，,Real-Time PCR,仪连续监测每个样品在从,双链完全配对,到,完全解链的升温过程中,荧光值的变化过程,。,不同的扩增产物因为其,长度,和,GC,含量,不同,而在,不一样的温度,下解链，当,产物解链时，,SYBR Green,的荧光值将降低,并被仪器监测到。由此绘制出,荧光强度随温度变化,的,负一次倒数图,。,荧光强度变化的拐点（熔点，,Tm）,即为,熔解峰值,。,熔解曲线是扩增反应的,质控途径,，当图中,没有,出现,杂峰,，也未出现,主峰的异常增宽,：表明实验中,未出现,污染,、,引物二聚体,和,非特异性扩增,。,5.,标准曲线,CT,值与起始模板的关系研究表明，,每个模板的,CT,值与该模板的起始拷贝数的对数,存在,线性关系,：,起始拷贝数越多，,CT,值越小,。,利用,已知起始拷贝数的标准品,可作出标准曲线，其中,横坐标代表,CT,值，纵坐标代表起始拷贝数的对数,（如图3所示）。,因些,只要获得未知样品的,CT,值,，即可从标准曲线上计算出,该样品的起始拷贝数,。,This technology is,very,sensitive,in that it is able to detect,single copies of genes,and,requires very little starting material,across,a wide spectrum of conditions,.,The system monitors PCR at each cycle of a reaction, and estimates the cycle when the reaction reaches,log phase increments,(when the,most useful quantitative information,about the sample is available),Quantitation can be,relative,to an,internal standard,such as a housekeeping gene, or,absolute,when compared to a,standard curve,generated from known concentrations.,6.,何为实时？,试剂方面： 如何做到,相对荧光强度反应的是,PCR,产物的相对,量；如何做到,相对荧光强度,反应的是,特定,PCR,产物,的,相对量,；,仪器方面： 如何,测定相对荧光强度,Opticon,实时荧光定量实,PCR,仪试剂,工作原理,工作原理,哪一步可以观察到荧光信号？ 变性；复性；延 伸,应用,7. SYBR Green I,的工作原理,SYBR Green I,结合到,双链,DNA,的小沟部,SYBR Green I,染料只有和,双链,DNA,结合后才发荧光。,SYBR Green,的,最大吸收波长,约为,497,nm,，,发射波长,最大约为,520,nm,。,在,PCR,反应体系中，加入过量,SYBR,荧光染料，,SYBR,荧光染料,特异性地掺入,DNA,双链,后，发射强荧光信号,;,而,不掺入,链中的,SYBR,染料分子,仅有微弱荧光信号背景,，从而保证,荧光信号的增加与,PCR,产物的增加同步,。,变性：无荧光信号，未结合,SYBR Green I Dye,通过产物的,Tm,值,来,确定产物,SYBR Green,的特点,由于它与所有的双链,DNA,相结合，不必因为模板不同而特别定制，因此,通用性好,，且,价格相对较低,。,利用,荧光染料可以指示双链,DNA,熔点,的性质，通过熔点曲线分析可以,识别扩增产物,和,引物二聚体,，因而可以,区分非特异扩增,。,此外，由于一个,PCR,产物可以与多个分子的染料结合，因此,SYBR Green,的检测,灵敏度很高,。,但是，由于,SYBR Green,与所有的双链,DNA,相结合，因此由,引物二聚体、单链二级结构,以及,错误的扩增产物,引起的,假阳性,会影响定量的精确性。,通过测量,升高温度后荧光的变化,可以,帮助降低非特异产物的影响,。,由熔解曲线来分析产物的均一性,有助于更,准确地,分析,SYBR GreenI。,CYBR GreenI,的应用范围及优缺点,起始模板浓度定量,融解曲线分析：可区分单一产物、变异产物、多种产物和（或）引物二聚体,基因型分析,使用方便：不必设计复杂的引物,没有序列特异性：可以用于不同的模板,便宜,灵敏,与,非特异性产物,结合,Molecular Beacons,（,发夹型杂交探针）的工作原理,原理：荧光谐振能量传递（,FRET）,环,与,目标序列,完全配对,茎,由,互补配对的序列,组成,变性: 产生,非特异性,的荧光,延伸:,没有,荧光,分子,Beacons,是一种在,靶,DNA,不存在,时形成,茎环结构,的,双标记寡核苷酸探针,。,在此,发夹结构,中：位于分子一端的荧光基团与分子另一端的淬灭基团紧紧,靠近,。,在此结构中，,荧光基团被激发后,不是产生光子,，而是将,能量传递给淬灭剂,，这一过程称为,荧光谐振能量传递（,FRET,）,。,分子信标的结构：是一个具有,茎环,（,loop-stem,）结构的,寡核苷酸探针,，通常有,25,35,个核苷酸，两端分别标上一个,荧光基团和淬灭基团,。,分子,Beacons,的茎环结构中，,环,的部分用于,与靶序列杂交,（与目标序列互补），通常由,15,30,个核苷酸组成，要求,与靶序列杂交后,能形成,与探针靶序列,双螺旋,有关的,反式构型,，并,使干的部分打开,，尽量得使,荧光基团和淬灭基团分开,足够的距离,。,一般,茎,（,干,的部分）一般,5-8,个核苷酸长（碱基对），并,相互配对,形成茎的结构。,荧光基团,连接在,茎臂的一端,，一般连在,5,端,；而,淬灭基团,一般连在,3,端,。,通常用,4-,（,4-,二甲基氨基偶氮苯基）苯甲酸,（,DABCYL,）作为,淬灭基团,。,分子,Beacons,必须非常仔细的设计，以致于在,复性温度,下：,模板不存在,时形成,茎环结构,；,模板存在,时则,与模板配对,。,自由状态,时，分子信标呈,发夹型结构,，荧光基团和淬灭基团靠得,很近,，二者之间发生,能量转移,（,FRET,和,直接能量转移,），荧光基团的能量被淬灭基团吸收并以,热,的形式散发，荧光几乎完全,被猝灭,。,。,当有,靶序列存在,的时候，靶序列和分子信标的探针序列杂交形成有一定刚性的,双螺旋结构,，导致分子信标的,构象改变,，,干,的部分打开，荧光基团与猝灭基团,分开,，二者之间的能量转移终止；,在有,相应的单色光激活,时，荧光基团发出荧光。荧光强度与溶液中,靶序列的多少,成正比，通过检测,荧光的强度,就可以计算出靶序列的量。,Beacons,与模板配对,后：,分子,Beacons,的构象改变,使得,荧光基团与淬灭剂分开,；当荧光基团被激发时，它发出,自身波长,的,光子,。,分子,Beacons,不同于,SYBR Green,的一个优点就是它,特异性地检测感兴趣,的,目标,DNA,。,通过,精心设计分子,Beacons,和,优化反应条件,（温度和缓冲液）后，其灵敏度非常高，可用于,单核苷酸多态性,（,SNPs,）的检测。,但是，为检测某一特定的目标,DNA,，,每一个探针都必须,单独仔细地设计,。,分子,Beacons,是美国纽约公共健康研究学会的技术专利。,Molecular Beacons,的应用范围及优缺点,定量起始模板浓度,基因型分析,鉴定产物,单核苷酸多态性（,SNP）,检测,对目标序列有,很高的特异性,，用于,SNP,检测的最灵敏的试剂之一,荧光背景,低,设计,困难,无终点分析功能,只能用于,一个特定的目标,价格较,高,9.,TaqMan（,水解型杂交探针）的工作原理,目标特异性探针,5为荧光素，3为淬灭剂,模板和探针杂交,延伸,: 聚合反应，,Taq,酶切下5端荧光 素,出现荧光,TaqMan,(,水解型杂交探针)的,应用范围及优缺点,定量起始模板浓度,基因型分析,产物鉴定,SNP,分析,对目标序列有,很高的特异性,特别适合于,SNP,检测,与,Molecular Beacons,相比，,设计相对简单,价格较,高,只适合于一个,特定的目标,不能,进行融解曲线分析,Quantitative Real-Time Polymerase Chain Reaction for the Core Facility Using TaqMan and the Perkin-Elmer/Applied Biosystems Division 7700 Sequence Detector,Deborah S. Grove,Nucleic Acid Facility, Life Science Consortium, The Pennsylvania State University, University Park, PA 16802,Probe,A probe (ie, TaqMan) is designed to anneal to the target sequence between the,traditional,forward,and,reverse,primers,.,The probe is labeled at the,5 end,with,a reporter fluorochrome,(usually,6-carboxyfluorescein,6-FAM,，,6-,羧基荧光素,) and,a quencher,fluorochrome (,6-carboxy-tetramethyl-rhodamine,TAMRA,6-,羧基,-,四甲基,-,若丹明,) added at any T position or at,the 3 end.,The probe is designed to have,a higher T,m,than,the primers, and during the,extension phase,the probe must be 100% hybridized,for success of the assay.,Principle,As long as,both fluorochromes,are on the probe, the quencher molecule,stops,all fluorescence by the reporter.,However, as,Taq,polymerase extends the primer, the,intrinsic,（内在的，固有的）,5 to 3 nuclease,activity of,Taq,degrades the probe, releasing the reporter fluorochrome.,The,amount,of fluorescence released,during the amplification cycle is,proportional to,the amount of product generated in each cycle,.,Fluorogenic 5 nuclease chemistry,. (1) Forward and reverse primers are extended with,Taq,polymerase as in a traditional PCR reaction. A probe with two fluorescent dyes attached anneals to the gene sequence between the two primers. (2) As the polymerase extends the primer, the probe is displaced. (3) An inherent nuclease activity in the polymerase cleaves the reporter dye from the probe. (4) After,release of the reporter dye from the quencher, a fluorescent signal is generated.,The sensitivity of detection allows acquisition of data when PCR amplification is still in the,exponential,phase,.,This is determined by identifying the cycle number at which,the reporter dye emission intensities rises above background noise,; this cycle number is called the threshold cycle (C,t,).,The C,t,is determined,at the most,exponential phase,of the reaction,and,is,more reliable,.,The,C,t,is inversely proportional to,the copy number of the target template; the,higher,the template concentration, the,lower,the threshold cycle measured.,One of the views available after completion of the run is an amplification window. This window shows the amount of fluorescence obtained in,each,amplification cycle,for each reaction,. The threshold cycle (C,t,) is shown,by the darker horizontal line.,Advantage,Provides,an accurate method,for determination of levels of specific DNA and RNA sequences in tissue samples.,Based on,detection of a,fluorescent signal,produced proportionally during,amplification of a PCR product,.,Turn-around time,for data acquisition and analysis,is short,Results are more reliable,than by traditional PCR methods.,Sybr greenI,detection is,less expensive,and,no sequence,specific probe is required,.,Users will also need to purchase,flat-top, optical caps,or,0.2 mm,thermal microseal,film,for their plates. All other plates and caps will not work with the new machines.,Applications,The applications for quantitative real-time PCR are,innumerable,（数不清的）,.,Detection of genomic or viral DNA,in tissues can be a,valuable diagnostic tool,.,Gene expression,can be measured after extraction of total RNA and preparation of cDNA by a reverse transcription (RT) step.,Setup and analysis are,simple,and can more easily be extended to the,clinical environment,than traditional PCR,techniques.,An,allelic discrimination assay,that can detect,single-base nucleotide mutations,and,polymorphisms,.,Allelic(,等位基因的,),discrimination assay,These assays require,two separate probes,that,differ,only by,one,base mismatch,.,One probe labeled with,6-FAM,(,6-,羧基荧光素,)represents,one allele,（等位基因）, and the other probe labeled with the,fluorochrome VIC,represents,the other allele,.,A mismatch,leads to,a less efficient amplification,.,Fluorescence spectra,are collected after the run, and using,multicomponent analysis, the software extracts the contribution of each component dye to the observed spectrum.,Homozygotes for FAM,show an increase in the,FAM signal,but,no increase in the VIC,signal, and,homozygotes for the VIC,probe show an increase in that signal.,Heterozygotes,（杂合子）,show,intermediate increases of FAM and VIC signals,.,All three groups are,clearly distinguishable, and the sensitivity is similar to that for the quantitative PCR application,10.,多色多通道技术应用,基因表达分析,PRIMER AND PROBE DESIGN,Primers and probes must be,carefully designed,because of the costs associated with producing probes with,different dyes,at 5 and 3 ends.,PE/ABD,Primer Express software,which is specifically designed to select the primers and probes. The required parameters for,well-designed primers and probe,have been well,built into the program,.,These parameters include a,T,m,for the,probe,that is,10C higher,than the primers, primer T,m,is between 58C and 60C,amplicon size,between,50 and 150 bases,absence of 5 Gs,.,Primer and probe design is,different,for,the allelic discrimination assays.,The probes designed for each allele should be,centered over,the mismatched base, and the probes,only,have to differ by that base,.,Another major difference is that the probe for these assays has a,lower Tm,requirement,than,the TaqMan PCR assays.,Prescreening assay,SYBR Green,can be used as a,probeless alternative,to the TaqMan system. Because it,binds to all double-stranded DNA, it is imperative to ensure that the PCR product being quantified is,a clean, single product, because any,primer-dimers,or,background smears,are detected. It can be used as a,prescreening assay,before ordering a probe.,If the results are satisfactory, the probe can be,ordered,and TaqMan reactions run with,higher specificity,.,11.,内标技术介绍,三、,实时荧光定量,PCR,技术的,应用介绍,荧光定量,PCR,技术的应用,对,DNA、RNA,样品进行定量和定性分析,定量分析包括,绝对定量,分析和,相对定量,分析。,前者可以得到,某个样本中基因的,拷贝数,和,浓度；,后者可以对,不同方式处理的两个样本,中的,基因表达水平,进行,比较,。,可以,不定期,对,PCR,产物或样品进行,定性分析,如：,利用,熔解曲线,分析识别,扩增产物,和,引物二聚体,，以,区分非特异扩增,；,利用,特异性探针,进行,基因型分析,及,SNP,检测,等。,目前实时荧光,PCR,技术已经被广泛应用于,基础科学研究,、,临床诊断,、,疾病研究,及,药物研发,等领域。其中最主要的应用集中在以下几个方面：,比较经过,不同处理样本,之间,特定基因,的表达差异（如,药物处理,、,物理处理,、,化学处理,等），及,特定基因,在,不同相,的表达差异；,比较,正常组织,与,病理组织,中各种,mRNA,表达量差异；,验证,基因芯片,实验结果；,验证,RNA,干扰,实验结果。,四、荧光定量,PCR,实验室,所需仪器设备清单,专用工作服和工作鞋,专用办公用品,一次性手套、一次性吸水纸,微量加样器一套（覆盖11000,ul）,以上物品各一套,。,28和-20或-80冰箱,混匀器,耐高压处理的离心管和加样器吸头,(,带滤心）,高速台式冷冻离心机,五、实时定量,PCR,及应用于,植物分子生物学的研究,Microarray,最强大的功能是,平行分析,，在单次实验中同时观察,上万种,RNA,的相对量变，也因为,平行处理,之故，无法对每一个,probe,做,最佳化、标准化,，所以目前大部份结果都会再以,Northern blot,or,Real-time quantitative PCR,再做,确认,。,虽然,Northern blot,在过去曾扮演十分称职的角色，但有時,microarray,筛选出來的基因,常有,上千个谱,，以,传统方法,来验证，仍是,耗费人力物力,的苦差事。,Real-time quantitative PCR,在近年来迅速普及，原因在其,高灵敏度,、,快速,及,精确,，,样品需求量少,也是很重要的优势，是,最适合承接,microarray,验证的工具,。,Laser Captured Microdissection (LCM),Real Time Quantitative PCR,：,量化组织中,特定基因,之表 现,共聚焦显微影像系统,(Confocal Imaging,System),流速细胞筛选分析系统,(Flow Cytometry),以上技术就,基因表现,之,各个面相,作系统探讨。,实验程序,（举例）,RNA,的定量,：,RNA,的测量仪上，以,Nono Drop,软件进行,RNA,的定量(,ng/l,)。,如浓度过高必须,稀释,后再定量，以便定量尽可能地准确。同时，可从,260/280,的比值看出,RNA,的纯度(一般在,1.9-2.1,之间)是否在要求的范围内。,RNA,的反转录,及,cDNA,模板的稀释,：用作,RNA,反转录的,RNA,量，在盐芥和拟南芥两种材料、各200,mM NaCl,处理2小时及对照的,4种,RNA,之间必须,完全准确一致,，即,Ara200、Ara0、Th200、Th0,的,RNA,量均为,2000,ng,。,以下的反转录体系中,RNA,和,RNase-free H,2,O,的总体积为,8.5 ,l,。,RNA + RNase-free H,2,O,：,8.5 l(,2000ng RNA,),Oligo-dT,18,(500ng/l),：,0.5 l,dNTP(2.5mM/each),：,4 l,计,：,13 ,l,该体系于,65-70,，,5,min,；,置于冰上冷却后，加下面反应体系,：,5,RT Buffer,：,4 l,0.1 M DTT,：,2 l,SuperScript III RT,：,1 l,总计：,20 ,l,混匀，,50,保持6小时或过夜，即得到,20,l,cDNA；,该,cDNA,稀释,20,倍,，即为,400l,稀释的,cDNA,，,此用于实时定量,PCR,作为反应模板。,DTT,即,Dithiothreitol,，中文名为二硫苏糖醇。分子式为,C,4,H,10,O,2,S,2,。是一种常用,d,的小分子,有机还原剂,，有,抗氧化,作用。,二硫苏糖醇的名字衍生自,苏糖,（一种四碳,单糖,）。,DTT,的,异构体,为,Dithioerythritol,（,DTE,），即,DTT,的氧化结构。,实时定量,PCR (Real-time PCR),SYBR Green PCR Master Mix：12.5 l,稀释的,cDNA,模板： 2 ,l,Primer-F： 1l,Primer-R： 1l,总计： 25 ,l,2,-,CT,方法与相对基因表达差异的分析,使用内标基因的目的是为了,对加入到反转录反应中的,RNA,进行均一化处理,，,标准的看家基因,一般都可被用作内标基因。,适合于实时,PCR,反应的,内标基因,包括,GAPDH,（,glyceraldehyde-3-phosphate dehydrogenase,）,，,-actin,(,细胞骨架蛋白,beta-actin,或,beta-tubulin ),、,2,-microglobulin,(,微球蛋白,),以及,rRNA,；,当然，其它的看家基因也同样能被用作内标。,我们在,应用某一基因作为内标之前,首先确证,该基因的表达,不会受实验处理的影响,。,相对定量的方法分析基因表达差异,(,i,),选择一个,内标基因,；,(,ii),确定内标的,有效性,，确保它,不会受到实验,处理的影响,；,(,iii),通过,PCR,扩增,目标基因,和,内标基因,RNA,或,cDNA,的,一系列梯度稀释模板,，确保它们的,扩增效率相同,。,(,iv),最后通过,2,-,CT,计算将,统计数据,转化成,线,性形式,而不是原始,CT,值。,CT,表示,目标基因,和,内标基因,CT,值的差异,;,2,-CT,方法,是实时定量,PCR,实验中,分析基因表 达相对变化的一种简便方法,。,Giovanna(2002),提出“,相对,C,T,”,或“,- CT”,的方法，这个方法的优点是,不需要为每次实验制作标准曲线,，只需要一个优化步骤证明,外源基因,与,内源基因,有,相同的,、至少是,相似的反应效率,即可。,比较,CT,方法的优点是,无需标准曲线,、,适合多个样品,，当然它也,消除了,创造标准曲线时,任何稀释错误的不利影响,。,相比于,标准子,(,normalizer,)，,比较,CT,法,(,CT),在对模板进行相对定量、监测基因的表达水平时，,不需标准曲线,并,增加了样品的通量,；,为了使这个方法成功应用，,目标序列,和,内参序列,的,扩增效率必须相等,。,只要,靶基因,和,标准者,具有,相似的动力学范围,，比较,CT(CT),方法就是,最具有实际应用价值的方法,。,2,-,CT,方法中,参照因子,的选择决定于,基因表达定量实验的类型,；,本实验中,参照因子,是,未经,NaCl,处理的对照样品,；,内标基因,为,标准的,看家基因,Actin,。,根据实验获得的经验值，使用公式：,1.9,-,CT,进行统计分析。,每个基因在两种处理情况下的表达水平均来自,3次,实时定量,PCR,的,平均值。,Ct-ActinCt (control),Ct-ActinCt (200mMNaCl,处理),delta Ct,Fold difference,Ave Fold Change,StDev,The,Pyruvate decarboxylase1,Gene of Arabidopsis Is Required during Anoxia,(,缺氧症,）,But Not Other Environmental Stresses,Oliver Kursteiner, Isabelle Dupuis, and Cris Kuhlemeier*,Institute of Plant Sciences, Altenbergrain 21, CH3013 Berne, Switzerland,Plant Physiology, June 2003, Vol. 132, pp. 968978, www.plantphysiol.org 2003 American Society of Plant Biologists,Fermentation has important functions in the presence of oxygen, mainly in germinating pollen and during,abiotic stress,.,Pyruvate decarboxylase,(,PDC,丙酮酸盐,(,或酯,),脱羧酶,), which catalyzes the first step in this pathway, is thought to be the main regulatory enzyme.,PDC is,encoded by four closely related genes,in Arabidopsis,.,Using real-time quantitative polymerase chain reaction, we determined the,expression levels of each individual gene,in different tissues,under,normal growth conditions, and when the plants were subjected to,anoxia,or,other environmental stress conditions,.,PDC1,is the only gene induced under oxygen limitation among the,PDC1,gene family,and that a,pdc1,null mutant is comprised in anoxia tolerance but not other environmental stresses.,Characterize,the expression of the aldehyde dehydrogenase (,ALDH,) gene family. None of the three genes is induced by anoxia,，,but,ALDH2B7,reacts strongly to,ABA application,and,dehydration, suggesting that ALDH may play a role in,aerobic detoxification of acetaldehyde.,Discuss,the possible role of ethanolic fermentation as a robust back-up energy production pathway,under adverse conditions when mitochondrial function is disturbed.,Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR.,Yang L, Ding J, Zhang C, Jia J, Weng H, Liu W, Zhang D.School of life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, Peoples Republic of China.,Plant Cell Rep. 2005 Mar;23(10-11):759-63. Epub 2004 Oct 1.,In transgenic plants, transgene copy number can greatly influence the,expression level and genetic stability of the target gene, making,estimation of transgene copy number,an important area of,genetically modified (GM),crop research.,Transgene copy numbers are currently estimated by,Southern analysis, which is,laborious,and,time-consuming, requires relatively,large amounts of plant materials,and may involve,hazardous radioisotopes,.,Report here the development of,a sensitive, high-throughput real-time (RT)-PCR technique,for estimating,transgene copy,number in,GM rice,.,Use TaqMan quantitative RT-PCR and comparison to a novel rice,endogenous reference gene,coding for,sucrose phosphate synthase (SPS),to determine the copy numbers of the,exogenous beta-glucuronidase (GUS),and,hygromycin phosphotransferase (HPT) genes,in transgenic rice.,The,copy numbers,of the,GUS,and,HPT,in,primary rice transformants (T,0,),were calculated by comparing quantitative PCR results of the GUS and HPT genes with those of the,internal standard, SPS.,With optimized PCR conditions, achieved significantly accurate estimates of,one, two, three,and,four transgene copies,in the,T,0,transformants,.,Copy number estimations of both the,GUS reporter,gene,and the,HPT selective marker gene,showed that,rearrangements of the T-DNA,occurred,more frequently,than is generally believed in transgenic rice.,An expression analysis of a gene family encoding plasma membrane aquaporins in response to abiotic stresses in,Arabidopsis thaliana,.,Jang JY, Kim DG, Kim YO, Kim JS, Kang H.Division of Applied Plant Science and Agricultural Plant Stress Research Center, College of Agriculture and Life Sciences, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, Korea.Plant Mol Biol. 2004 Mar;54(5):713-25.,Aquaporin,belongs to a,highly conserved group of membrane proteins,called major intrinsic proteins that,facilitate water transport across biological membranes,.,The genome of Arabidopsis encodes,35 aquaporin genes,with 13 homologs in the,plasma membrane intrinsic protein (PIP),subgroup.,As a step toward understanding the,aquaporin function,in plants under,various environmental stimuli,the,expressions of a gene family encoding 13 PIPs,in,Arabidopsis thaliana,under various abiotic stress conditions including,drought,cold, and,high salinity, or,abscisic acid (ABA),treatment were investigated by a quantitative real-time reverse transcription-PCR analysis.,Several PIP genes were,predominantly expressed,either in the roots or in the flowers.,The expressions of both the highly expressed aquaporins including PIP1;1, PIP1;2, and PIP2;7 and the weakly expressed aquaporins such as PIP1;4, PIP2;1, PIP2;4, and PIP2;5,were modulated by external stimuli.,The analyses of our data revealed that only the PIP2;5 was up-regulated by cold treatment, and most of the PIP genes were down-regulated by,cold stress,.,Marked up- or down-regul