拟南芥CHS基因表达的实时荧光定量PCR检测

在简要介绍实时荧光定量PCR反应和定量原理的基础上,采用TaqMan荧光定量PCR技术,研究了UV-B辐射对拟南芥(Arabidopsis thaliana)CHS(查耳酮合成酶基因)表达的诱导,获得了与传统Northern杂交一致的结果.实时荧光定量PCR用于基因表达的定量检测,具有特异性强、自动化程度高、高效快捷,避免使用放射性同位素,能同时对多个样品中的起始模板进行准确定量等特点,因此该方法已逐渐被广泛用于基因表达的定量分析.     

实时荧光定量PCR技术及其应用

实时荧光定量PCR技术是一种多色荧光检测核酸定量技术,该简要介绍实时荧光定量PCR技术的原理及其应用。     

实时荧光定量PCR技术综述

实时荧光定量PCR技术是在PCR技术基础上发展起来的一种高度灵敏的核酸定量技术。与传统PCR相比,实时定量PCR能够更加快速、灵敏,并有效地对核酸进行定量检测。     

实时荧光定量PCR的发展和数据分析

实时荧光定量PCR技术是基因时代一项用于检测mRNA的常用技术,是临床检测和基础研究中不可缺少的重要研究方法,包括绝对定量PCR和相对定量PCR。该技术的特点是可以减少PCR后操作,在比较不同浓度的mRNA方面具有非常宽的动力学范围。我们就目前实时荧光定量PCR的发展及数据的分析进行综述。     

实时荧光定量PCR 检测人肿瘤细胞NKG2D 配体基因表达

目的:建立人肿瘤细胞NKG2D配体基因(MICA、MICB、ULBP1、ULBP2、ULBP3)表达的实时荧光定量PCR(real-timefluorescence quantitative PCR)检测方法。方法:根据NCBI基因库中NKG2D配体基因序列,设计合成引物。用Trizol法从培养的肿瘤细胞(BEC-7402、HeLa、MDA-MB-435、XWLC-05)中提取总RNA,逆转录成cDNA,建立实时荧光定量PCR检测NKG2D配体基因表达的方法,并检测NKG2D配体在肿瘤细胞株中的表达。结果:经过琼脂糖凝胶电泳、熔解曲线和标准曲线分析,用所设计的引物和SYBR Green I能够特异扩增和定量检测NKG2D配体基因的表达。该方法成功检测4种肿瘤细胞NKG2D配体基因的表达。结论:建立了人NKG2D配体基因表达的实时荧光定量PCR检测方法,为进一步研究人NKG2D配体在肿瘤免疫中的作用提供了有效手段。     

实时荧光定量PCR检测人肿瘤细胞NKG2D配体基因表达

目的：建立人肿瘤细胞NKG2D配体基因（MICA、MICB、ULBP1、ULBP2、ULBP3）表达的实时荧光定量PCR（real-time fluorescence quantitativePCR）检测方法。方法：根据NCBI基因库中NKG2D配体基因序列,设计合成引物。用Trizo1法从培养的肿瘤细胞（BEC-7402、HeLa、MDA-MB-435、XWLC-05）中提取总RNA,逆转录成eDNA,建立实时荧光定量PCR检测NKG2D配体基因表达的方法,并检测NKG2D配体在肿瘤细胞株中的表达。结果：经过琼脂糖凝胶电泳、熔解曲线和标准曲线分析,用所设计的引物和SYBR GreenI能够特异扩增和定量检测NKG2D配体基因的表达。该方法成功检测4种肿瘤细胞NKG2D配体基因的表达。结论：建立了人NKG2D配体基因表达的实时荧光定量PCR检测方法,为进一步研究人NKG2D配体在肿瘤免疫中的作用提供了有效手段。     

利用实时荧光定量PCR法检测转基因水稻外源基因拷贝数的研究

转基因植物中外源基因拷贝数是影响目的基因表达水平和遗传稳定性的重要因素,因此外源基因拷贝数的检测成为转基因研究的关键．利用高通量、快速、灵敏的SYBR Green Ⅰ荧光定量实时PCR法,检测了转大麦烟酰胺合成酶基因（NASl）水稻中外源基因拷贝数．以蔗糖磷酸合成酶基因（SPS）作为水稻的内源参照基因．通过梯度稀释法．分别获得了NAS1和SPS基因的Ct值与起始模板数的相关性标准曲线,相关系数分别为0．99976和0．99571,相关性高．通过目的基因NAS1和水稻内源参照基因SPS起始模板数的比较,获得了目的基因在转基因水稻中的拷贝数。在8株转基因株系中,1株为假阳性,1株拷贝数为1,3株拷贝数为2,其余3株拷贝数分别为3、4和7．而阴性对照拷贝数为0．这种方法快速、简便、准确,可以满足转基因育种工作中对后代优良株系的选择．     

实时荧光PCR技术定量检测转Bt基因水稻的研究

以转Bt基因的"克螟稻"为研究材料,通过使用特异的引物和荧光标记探针,以已知转基因成份含量的水稻样品为模板建立标准曲线,对转基因水稻的NOS和Bt外源基因进行了荧光定量检测分析.初步建立了转基因水稻定量检测的技术方法.     

茶树实时荧光定量PCR分析中内参基因的选择

选择合适的内参基因是提高实时荧光定量PCR分析(qRT-PCR)准确性的先决条件。该文以茶树(Camellia sinensis) 芽、叶、幼根、嫩茎、花瓣、种子和愈伤组织为材料, 应用实时荧光定量PCR技术, 分析了18S rRNA、GAPDH、β-actin和α-tubulin 4个常用内参基因在茶树不同器官组织中的表达情况。经GeNorm和NormFinder软件分析发现, 当利用荧光定量PCR分析比较茶树不同器官组织中的基因表达差异时, 可选择β-actin作为校正内参基因; 而比较不同成熟度的叶片和愈伤组织时, 可以选择GAPDH作为校正内参基因。     

茶树实时荧光定量PCR分析中内参基因的选择

选择合适的内参基因是提高实时荧光定量PCR分析（qRT-PCR）准确性的先决条件。该文以茶树（Camellia sinensis）芽、叶、幼根、嫩茎、花瓣、种子和愈伤组织为材料,应用实时荧光定量PCR技术,分析了18S rRNA、GAPDH、β-actin和α-tubulin4个常用内参基因在茶树不同器官组织中的表达情况。经GeNorm和NormFinder软件分析发现,当利用荧光定量PCR分析比较茶树不同器官组织中的基因表达差异时,可选择β-actin作为校正内参基因;而比较不同成熟度的叶片和愈伤组织时,可以选择GAPDH作为校正内参基因。     

Internet Resources for Gene Expression Analysis in Arabidopsis thaliana

The number of online databases and web-tools for gene expression analysis in Arabidopsis thaliana has increased tremendously during the last years. These resources permit the database-assisted identification of putative cis-regulatory DNA sequences, their binding proteins, and the determination of common cis-regulatory motifs in coregulated genes. DNA binding proteins may be predicted by the type of cis-regulatory motif. Further questions of combinatorial control based on the interaction of DNA binding proteins and the colocalization of cis-regulatory motifs can be addressed. The database-assisted spatial and temporal expression analysis of DNA binding proteins and their target genes may help to further refine experimental approaches. Signal transduction pathways upstream of regulated genes are not yet fully accessible in databases mainly because they need to be manually annotated. This review focuses on the use of the AthaMap and PathoPlant^® databases for gene expression regulation analysis and discusses similar and complementary online databases and web-tools. Online databases are helpful for the development of working hypothesis and for designing subsequent experiments.     

Herbicide Safener-Inducible Gene Expression in Arabidopsis thaliana

The potential use of a new chemical-inducibie gene expressionsystem in Arabidopsis thaliana has been examined. The systemis based on the maize In2-2 promoter which is activated by benzenesulfonamideherbicide safe-ners. Plants transformed with the ß-glucuronidase(gus) reporter gene under the control of the In2-2 promoterwere grown in the presence of different safeners and the inducedGUS activity pattern was studied histochemically. In the absenceof safeners, the In2-2 promoter was not active. Applicationof different safeners induced distinct gus expression patterns,including expression in the root, hy-dathodes, and the shootapical meristem. Plants maintained continuously on inducingconcentrations of the safeners were retarded in growth. Thegrowth inhibition effects of the Sa5 safener could be overcomein a sul-fonylurea-resistant background. In2-2 promoter activitycould also be induced by the sulfonylurea herbicide chlor-sulfuron.In the sulfonylurea-resistant background, which derives fromherbicide-resistant acetolactate synthase activity, inductionof the In2-2 promoter by chlorsulfuron was lower. Furthermore,branched-chain amino acids, known to inhibit acetolactate synthaseactivity, also induced In2-2 promoter activity. Our data suggesta strong correlation between In2-2 expression and inhibitionof the acetolactate synthase activity. (Received November 12, 1996; Accepted February 21, 1997)     

拟南芥AHAl基因启动子的表达特性分析

从拟南芥中分离到编码质膜H^＋-ATPase的AHA1基因启动子序列813bp,并构建了此种启动子与GUS嵌合的重组载体,通过农杆菌介导转化拟南芥,得到转基因拟南芥。用组织化学方法分析AHA1基因启动子驱动GUS转基因拟南芥的结果表明,GUS基因在转基因的拟南芥根、茎、叶、花和荚的维管组织中均有表达,且不同发育时间内GUS基因表达也不同。以上研究表明拟南芥AHA1基因可能参与植物的生长发育与抗逆胁迫反应。     

番木瓜果实扩展蛋白Cp-EXP1基因表达的荧光定量PCR分析

目的：对番木瓜果实扩展蛋白Cp-EXP1的基因进行荧光定量PCR分析,以便对其功能做进一步研究。方法：以不同成熟阶段的番木瓜果实总RNA为模板,设计引物进行荧光定量PCR。结果：Cp-EXP1基因在不同成熟阶段的番木瓜果实中的表达是有差异的,其表达水平受乙烯调控。结论：Cp-EXP1基因的表达与番木瓜的成熟软化密切相关,其表达分析为研究番木瓜的生长发育及其品种改良打下了良好的基础。     

Evolution of Stress-Regulated Gene Expression in Duplicate Genes of Arabidopsis thaliana

Due to the selection pressure imposed by highly variable environmental conditions, stress sensing and regulatory response mechanisms in plants are expected to evolve rapidly. One potential source of innovation in plant stress response mechanisms is gene duplication. In this study, we examined the evolution of stress-regulated gene expression among duplicated genes in the model plant Arabidopsis thaliana. Key to this analysis was reconstructing the putative ancestral stress regulation pattern. By comparing the expression patterns of duplicated genes with the patterns of their ancestors, duplicated genes likely lost and gained stress responses at a rapid rate initially, but the rate is close to zero when the synonymous substitution rate (a proxy for time) is >~0.8. When considering duplicated gene pairs, we found that partitioning of putative ancestral stress responses occurred more frequently compared to cases of parallel retention and loss. Furthermore, the pattern of stress response partitioning was extremely asymmetric. An analysis of putative cis-acting DNA regulatory elements in the promoters of the duplicated stress-regulated genes indicated that the asymmetric partitioning of ancestral stress responses are likely due, at least in part, to differential loss of DNA regulatory elements; the duplicated genes losing most of their stress responses were those that had lost more of the putative cis-acting elements. Finally, duplicate genes that lost most or all of the ancestral responses are more likely to have gained responses to other stresses. Therefore, the retention of duplicates that inherit few or no functions seems to be coupled to neofunctionalization. Taken together, our findings provide new insight into the patterns of evolutionary changes in gene stress responses after duplication and lay the foundation for testing the adaptive significance of stress regulatory changes under highly variable biotic and abiotic environments.