通过荧光差异显示PCR法分离水稻中由ABA调节的基因

脱落酸(ABA)在植物种子发育以及植物细胞对外源环境因子(如逆境、胁迫等)反应过程中起着重要的作用,对其调节基因的分离将有助于了解其相关信号传导途径及作用机制.通过荧光差异显示PCR法我们由水稻中分离了部分ABA调节的cDNA片段,在所分离克隆的17个片段中,有14个片段被ABA诱导(2、4、8、12h),有3个片段被ABA抑制(8h).测序及序列分析表明这些片段可能分别编码与植物光合作用(7)、信号传导(1)、转录调控(2)、代谢(6)相关的蛋白或未知蛋白(1)而且其表达可能受到ABA的调节或ABA参与了其作用,对其中可能编码α/β水解酶折叠蛋白、酪氨酸磷酸酶、液泡H+-ATPase的mRNA进行的RT-PCR和Northern blot分析,确证了ABA对它们表达的调节作用.在此基础上对FDD-PCR技术及ABA作用的可能机制进行了讨论.     

差异显示法分离水稻抗稻瘟病相关基因

采用mRNA差异显示技术,分析水稻稻瘟病抗源材料“地谷”叶片受稻瘟病菌侵染前后的基因的表达差异,获得87个差异片段。对这87个差异片段进行了回收、重扩增与克隆,并对其中的81个片段进行了杂交鉴定。斑点杂交结果证实其中6个片段受稻瘟病菌诱导表达。进一步克隆测序并进行数据库比对分析表明其中一个与水稻4号染色体中一推测的苹果酸合成酶高度同源,一个与水稻11号染色体上的RPR1基因高度同源,RPR1基因具有保守的NBS-LRR结构,并与水稻防卫反应的信号传导有关;另一个与水稻第6号染色体上一推测的硫氧还蛋白高度同源,其余3个为新的cDNA片段。     

荧光差异显示PCR克隆参与胃腺癌转移的基因wcl1

使用来源于同一个胃腺癌病人的原发灶RF 1(ATCC编号CRL 186 4 )和转移灶RF 4 8(ATCC编号CRL 186 3)作为研究肿瘤转移的模型 .通过荧光差异显示PCR(FDD PCR)技术 ,克隆了 4 5个涉及胃腺癌转移相关基因 .和原发灶CRL 186 4相比 ,发现在转移灶CRL 186 3细胞中有 38个基因被显著上调 ,7个基因被显著下调 ,包括未被发现的基因 3个 .利用生物信息学技术对其中 1个在RF 4 8中高度上调的wcl1进行克隆和鉴定 ,发现wcl1的cDNA全长为 6 6 4bp ,含有 1个 2 4 0bp的完整阅读框 .用RT PCR和Northern印迹证实 ,结果与克隆拼接的大小完全一致 (NCBI数据库收录号AF36 4 86 3) .wcl1编码肽位于胞浆内含 79个氨基酸 ,理论上的pI Mr:5 2 8 896 1 72 .氨基酸序列分析发现 ,其N端 4～ 5 5氨基酸处为未知功能区UPF0 0 16蛋白 ,5 7～ 78处有一段亮氨酸拉链结构域 ,未发现与已知的蛋白质有高度的同源性 .该基因定位于 11q14.组织分布表明 ,wcl1除在分化差的胃腺癌中的表达要高于相应的正常胃组织 ,在微血管内皮中表达也明显高于乳腺管癌、脑纤维状星形细胞瘤 ,未检测到在其它组织有分布 .研究提示 ,Wcl1可能为胃腺癌转移过程中参与核内基因转录的相关蛋白质 .     

荧光差异显示技术在分离细菌体内诱导表达基因中的应用

面对复杂的宿主环境,细菌是通过调节表达不同的毒力基因实施其感染过程。目前已建立起多种分析鉴定体内特异表达的细菌毒力基因的方法;其中荧光差异显示技术的应用为鉴定细菌的毒力基因以及揭示细菌致病机理开辟了新途径。本对此作一扼要综述。     

运用多种策略改良差异显示PCR

目的：改进差异显示PCR技术,提高其在筛选差异表达基因方面的效率。方法：①采用单碱基金铆钉引物;②增加引物长度;③提高PCR反应的严谨性;④应用同步重复测序电泳。结果：减少经典方法的工作量,降低了非特异性和假阳性率。用改良技术研究热适应大鼠下丘脑基因的差异表达,发现了一个差异表达基因片段,斑点杂交证实为阳性片段。结论：改进后的差异显示PCR技术是一种研究基因差异表达从而发现新基因或基因新功能的有效方法。     

用基因芯片结合荧光差异显示—PCR筛选和识别胃腺癌转移相关的基因

使用来源于同一个胃腺癌病人的原发灶RF－1（ATCC编号：CRL－1864）和转移灶RF－48细胞系（ATCC编号,CRL－1863）作为研究肿瘤转移分子机制的模型,RF－1（实验组）和RF－48（对照组）的mRNA通过逆转录方法,将Cy3和Cy5两种荧光染料分别标记到两种细胞的cDNA上,制备成cDNA探针,并与表达谱芯片（双点4096条基因）进行杂交与扫描,重复2次实验,利用计算机数据处理判断基因是否在上述两种细胞中有表达差异,共筛选出差异表达的基因共138条,其中81条在RF－48细胞中表达明显上调,57条在RF－48细胞中表达显著下调,同时也通过荧光差异显示－PCR（FDD－PCR）技术,克服了45个涉及胃腺癌转移相关基因,包括未被发现的基因3个,在两种筛选方法中都存在差异表达的基因共有7条,对部分可能与肿瘤志移机制有关的差异表达基因的作用进行了分析和讨论,基因芯片技术可高通量,大规模地研究基因表达水平,FDD－PCR技术可克隆出未发现的新基因,二者结合,初步筛选出与转移相关的基因,有助于揭示胃腺癌转移的分子机制。     

差异显示法分离赤霉素调控的水稻（Oryza sativa）cDNA

赤霉素是植物生长发育过程中一类重要的调节激素。本文运用反转录和聚合酶链式反应建立了一套旨在分离差异表达ｃＤＮＡ的差异显示方法。ＤＤＦ１所对应的基因是一个单拷贝基因,可被高浓度的ＧＡ３诱导并获得高水平表达。     

快速、有效筛选新的功能基因——荧光差异显示技术

控制生物性状的基本单位是基因,研究细胞的基因表达一直是分子生物学的重要课题之一。不同细胞间基因表达的差异决定了生命活动的多样性,如发育与分化、内环境稳定、细胞周期调节等。近年来,随着DNA重组技术的发展,已有数万个人类基因被克隆分析,要从如此众多的表达基因中找出引起细胞生理或病理变化的基因并非易事。应用荧光标记的mRNA差异显示技术将有可能在一定程度上起到关键的作用。以总RNA为模板,采用荧光标记的锚定引物,通过逆转录、差异显示PCR反应,经5.6％变性聚丙烯酰胺凝胶电泳分离差异条带,条带清晰、明亮、背景低,各样本间的差异不仅呈有无的变化,亦表现出很多强弱改变;本实验室经过多年的坚持摸索、研究,现已能成功的应用荧光标记差异显示技术,可快速（2d）、敏感、经济有效的筛选各种未知的表达基因。     

利用mRNA荧光差异显示技术规模化筛选棉纤维特异表达基因

以陆地棉（Gossypium hirsutum L.）品种TM-1开花后9d、21d、27d三个不同发育时期的棉花纤维为材料,利用mRNA荧光差异显示 (FDD) 技术,筛选到109个差异显示的cDNA片段。在此基础上,结合两轮反Northern杂交筛选和Northern杂交分析,获得了多个仅在棉花纤维细胞中特异表达或在纤维中优先表达基因的cDNA片段,序列测定和数据库搜索分析表明,这些cDNA片段中的多数还未有报道。本工作为克隆上述基因的全长cDNA,并进一步研究它们在棉纤维发育中的功能奠定了基础。     

Isolation of rice genes possibly involved in the photoperiodic control of flowering by a fluorescent differential display method

To better understand the molecular mechanisms of the photoperiodic regulation of rice, a short-day plant, we isolated 27 cDNAs that were differentially expressed in the photoperiod-insensitive se5 mutant from approximately 8,400 independent mRNA species by the use of a fluorescent differential display (FDD). For this screening, we isolated mRNAs at five different time points during the night and compared their expression patterns between se5 and the wild type. Of 27 cDNAs isolated, 12 showed diurnal expression patterns often associated with genes involved in the determination of the flowering time. In se5, expression of nine cDNAs was increased. Five of these cDNAs were up-regulated under SD, suggesting that they may promote flowering under SD. They included genes encoding a cDNA containing a putative NAC domain, the fructose-bisphosphate aldolase, and a protease inhibitor. Expression of three cDNAs was decreased in se5 but not photoperiodically regulated. These cDNAs included a rice homolog of Arabidopsis GIGANTEA (GI), lir1, and a gene for myo-inositol 1-phosphate synthase, all of which were previously shown to be under the control of circadian clocks. The expression patterns of the rice homolog of GI, OsGI, were similar to those of the Arabidopsis GI, suggesting the conservation of some mechanisms for the photoperiodic regulation of flowering between these two species.     

Male specific genes from dioecious white campion identified by fluorescent differential display

Fluorescent differential display (FDD) has been used to screen for cDNAs that are differentially up-regulated in male flowers of the dioecious plant Silene latifolia in which an X/Y chromosome system of sex determination operates. To adapt FDD to the cloning of large numbers of differential cDNAs, a novel method of confirming the differential expression of these has been devised. FDD gels were Southern electro-blotted and probed with mixtures of individual cDNA clones derived from different FDD product ligation reactions. These Southern blots were then stripped and re-probed with further mixtures of individual cloned FDD products to identify the maximum number of recombinant clones carrying the true differential amplification products. Of 135 differential bands identified by FDD, 56 differential amplification products were confirmed; these represent 23 unique differentially expressed genes as determined by virtual Northern analysis and two genes expressed at or below the level of detection by virtual Northern analysis. These two low expressed genes show bands of hybridization on genomic Southern blots that are specific to male plants, indicating that they are derived from, or closely related to, Y chromosome genes.     

Isolation of developmentally regulated genes by differential display screening of cDNA libraries.

mRNA differential display RT-PCR has been extensively used for the isolation of genes differentially expressed between RNA populations. We have assessed its utility for the identification of developmentally regulated genes in plasmid cDNA libraries derived from individual tissues dissected from early mouse embryos. Using plasmid Southern blot hybridisation as a secondary screen, we are able to identify such genes and show by whole-mount in situ hybridisation that their expression pattern is that expected from the differential display profile.