差异表达基因分离技术的研究进展

分离并克隆差异表达基因是生命科学的研究热点.近年来,以差示筛选、扣除杂交等基本方法为基础,先后出现了抑制差减杂交,微阵列技术等多种分析差异表达基因的技术, 使差异表达基因分离方法不断完善.对这些方法的优缺点、发展趋势及应用前景进行了简要综述.     

水稻穗瘟防卫反应相关基因的分离和鉴定

以遗传背景相近、对叶瘟抗性相同但对穗瘟抗性不同的两个水稻株系为材料,利用抑制消减杂交（SSH）技术构建穗瘟抗／感消减cDNA文库,经差异筛选及序列分析,共获得90个独立的差异表达cDNA克隆,根据与它们刚源的基因功能推测,这些克隆可能参与了对病原菌的防卫反应、信号传导和转录等一些重要的生物学过程。利ⅢRT-PCR分析了26个所筛选到的cDNA克隆在抗／感植株接种后的表达,17个基因的表达差异得到验证。对这螳差异表达基因在抗感株系接种后不同时间点的表达谱也进行了RT-PCR的分析。文章首次报道了什关水稻对穗瘟抗性在mRNA水平进行研究,为深入研究水稻对穗瘟抗性的遗传机理打下了基础。     

银鲫原肠胚差异表达基因的筛选

胚胎发育是基因组中各个基因在时间和空间上选择性表达的结果。为了鉴定参与鱼类早期胚胎发育和胚层分化的调控因了,分别构建了银鲫(Carassiusauratusgibelio)原肠胚和成熟卵子的SMARTcDNA文库,并采用差异筛选的方法,从银鲫原肠胚SMARTcDNA文库中筛选不同于成熟卵子的差异表达基因。通过菌斑和PCR产物的两轮斑点杂交,从大约1500个克隆中筛选出131个阳性克隆,从中选择58个克隆测序并将测得的序列进行了数据库比对分析,结果显示这些差异表达基因大部分为参与转录和翻译的调控因子和核糖体蛋白以及一些在胚胎早期大量表达的参与脂类代谢的脂蛋白和一些未知的新基因。接着采用RTPCR技术,对其中6个基因在胚胎发育不同阶段的表达特征进行了分析,进一步证实这些基因在胚胎发育过程中存在表达差异;研究还从分析的基因中揭示出4类不同的表达模式。通过本研究,已筛选出一批在银鲫胚胎发育早期开始表达的调控基因,为开展鱼类胚胎发育早期表达基因的功能研究奠定了前期工作基础。       

鉴别差异表达基因的新方法──抑制消减杂交法(SSH)

抑制消减杂交法（SuppressicSubtractiveHybridization,SSH）是最近（1996）报道的能有效鉴别两个不同细胞群差异表达基因的新方法［1,2］。该方法是以抑制PCR和消减杂交法为基础,在一轮反应中实现mRNA的均等化和消减步骤。本文对SSH法的原理、优缺点作了介绍,并与其它检测差异表达基因的方法,如mRNA差别显示法（DDRT－PCR）、代表性序列差异分析（RDA）比较,认为SSH方法具有高效快速、高敏感性和假阳性低等优点。同时,625个克隆中有很大一部分是转录物丰度很低的基因,40％的克隆可能为新的基因。因此,SSH可作为鉴别差异表达基因和克隆新基因的有效方法。     

CHO/dhfr-细胞定点整合高效表达系统的建立

为了克服随机整合建立高表达细胞株时“位置效应”所带来的不可预知的后果,我们尝试建立基于定点整合的CHO高效表达系统。首先设计一个新的高效筛选载体pMCEscan。该载体含有报告基因(k2tPA)、扩增基因(dhfr)、重组酶识别序列(FRT)及筛选基因(neo),且neo基因的表达经过系统的弱化,确保能够对基因组中的整合位点进行大规模的高效筛选。然后利用该载体转染CHO/dhfr-细胞并进行大规模筛选以获得足够多的阳性克隆,并对阳性克隆进行系统分析,筛选出报告基因表达水平高、单拷贝且扩增效果好的克隆,此克隆被认为筛选载体整合入CHO细胞基因组中转录热点(Hotspot)区域,从而获得了能够实现外源基因在基因组中定点整合和有效表达的CHO/dhfr-细胞系。随后利用位点特异性重组系统(FLP-FRT)将外源基因定点整合到Hotspot区域,以实现外源基因在CHO细胞基因组中的定点整合及高效表达。并利用该细胞系实现了k2tPA的高表达,表达量达到17.1μg/106cell.24h。该研究致力于CHO细胞基因组中高表达位点的寻找和确认,建立基于定点整合的哺乳动物细胞高效表达系统。     

玉米耐渍功能基因组分析及相关基因Sicyp51的鉴定与克隆

选用耐渍性有明显差异的玉米自交系Mo17和Hz32, 对二叶一心期的幼苗进行淹水处理, 构建了这两个材料各自正向和反向的SSH(suppression subtractive hybridization)文库. 从SSH文库中筛选到105个阳性克隆; 利用处理及对照mRNA与Mo17全生育期cDNA芯片分别杂交, 得到57个3倍以上表达差异的克隆. 对所有162个克隆进行序列分析, 结果表明: 所有差异表达的克隆代表85个TUGs(tentative unique genes), 这些TUGs主要参与厌氧代谢、逆境自我保护、信号传导、细胞结构和能量代谢等生理生化途径, 同时还包括41个未知功能的cDNA, 说明玉米根系对淹水胁迫响应涉及到复杂的代谢反应. 对从Mo17和Hz32中筛选到的差异表达克隆进行比较分析发现, 在淹水条件下, 耐渍性不同的材料具有不同的耐渍相关基因的表达模式. 利用Northern及RT-PCR检测了部分阳性克隆的表达谱, 并克隆到一个与耐渍相关的新基因(Sicyp51).     

以木糖异构酶基因xylA为选择标记的Gateway系统植物表达载体的构建

目的:构建以木糖异构酶基因xylA为筛选标记的无抗生素标记Gateway系统植物表达载体。方法:克隆大肠杆菌木糖异构酶基因xylA并用其替换植物表达载体pCAMBIA1301中的hpt基因,利用载体中的多克隆位点将Gateway Binary Vector(pH7WG2D)中酶切位点XbaⅠ和HindⅢ之间包括P35S、T35S、attR1、attR2和CmR-ccdB的片段重组入表达载体pCAMBIA1301中,构建表达载体pCAMBIA1301-xylA-GW,利用含有津田芜菁HY5基因片段的BP反应产物与载体进行LR反应,获得含有目的基因的植物表达载体pCAMBIA1301-xylA-HY5,并导入根癌农杆菌LBA4404中。结果:抗生素筛选及酶切和PCR鉴定表明成功构建了以xylA为筛选标记的无抗生素标记植物表达载体pCAMBIA1301-xylA-HY5。结论:利用木糖异构酶基因xylA结合Gateway克隆技术构建无抗生素标记植物表达载体,可简化、方便植物转基因表达载体构建。     

CHO/dhfr-细胞定点整合高效表达系统的建立

为了克服随机整合建立高表达细胞株时“位置效应”所带来的不可预知的后果,我们尝试建立基于定点整合的CHO高效表达系统。首先设计一个新的高效筛选载体pMCEscan。该载体含有报告基因（k2tPA）、扩增基因（dhfr）、重组酶识别序列（FRT）及筛选基因（neo）,且neo基因的表达经过系统的弱化,确保能够对基因组中的整合位点进行大规模的高效筛选。然后利用该载体转染CHO／dhfr^-细胞并进行大规模筛选以获得足够多的阳性克隆,并对阳性克隆进行系统分析,筛选出报告基因表达水平高、单拷贝且扩增效果好的克隆,此克隆被认为筛选载体整合入CHO细胞基因组中转录热点（Hotspot）区域,从而获得了能够实现外源基因在基因组中定点整合和有效表达的CHO／dhfr-细胞系。随后利用位点特异性重组系统（FLP-FRT）将外源基因定点整合到Hotspot区域,以实现外源基因在CHO细胞基因组中的定点整合及高效表达。并利用该细胞系实现了k2tPA的高表达,表达量达到17．1μg／10^6cell·24h。该研究致力于CHO细胞基因组中高表达位点的寻找和确认,建立基于定点整合的哺乳动物细胞高效表达系统。     

Advances in maize genomics: the emergence of positional cloning

Positional cloning has been and remains a powerful method for gene identification in Arabidopsis. With the completion of the rice genome sequence, positional cloning in rice also took off, including the cloning of several quantitative trait loci. Positional cloning in cereals such as maize whose genomes are much larger than that of rice was considered near impossible because of the vast amounts of repetitive DNA. However, conservation of synteny across the cereal genomes, in combination with new maize resources, has now made positional cloning in maize feasible. In fact, a chromosomal walk is usually much faster than the more traditional method of gene isolation in maize by transposon tagging.     

Construction and characterization of new cloning vehicles. VII. Construction of plasmid pBR327par, a completely sequenced, stable derivative of pBR327 containing the par locus of pSC101

In vitro recombinant DNA experiments, using plasmid pBR327 and a DNA fragment derived from plasmid pSC101 containing the par region, resulted in the construction of plasmid pBR327par. This new cloning vehicle has all the cloning properties of the parental plasmid, and is more stable than pBR327. Since the nucleotide sequence of the par region has been determined, this new vector is completely characterized. Some features of the sequence with possible functional significance are discussed.     

Simple Method for Markerless Gene Deletion in Multidrug-Resistant Acinetobacter baumannii

The traditional markerless gene deletion technique based on overlap extension PCR has been used for generating gene deletions in multidrug-resistant Acinetobacter baumannii. However, the method is time-consuming because it requires restriction digestion of the PCR products in DNA cloning and the construction of new vectors containing a suitable antibiotic resistance cassette for the selection of A. baumannii merodiploids. Moreover, the availability of restriction sites and the selection of recombinant bacteria harboring the desired chimeric plasmid are limited, making the construction of a chimeric plasmid more difficult. We describe a rapid and easy cloning method for markerless gene deletion in A. baumannii, which has no limitation in the availability of restriction sites and allows for easy selection of the clones carrying the desired chimeric plasmid. Notably, it is not necessary to construct new vectors in our method. This method utilizes direct cloning of blunt-end DNA fragments, in which upstream and downstream regions of the target gene are fused with an antibiotic resistance cassette via overlap extension PCR and are inserted into a blunt-end suicide vector developed for blunt-end cloning. Importantly, the antibiotic resistance cassette is placed outside the downstream region in order to enable easy selection of the recombinants carrying the desired plasmid, to eliminate the antibiotic resistance cassette via homologous recombination, and to avoid the necessity of constructing new vectors. This strategy was successfully applied to functional analysis of the genes associated with iron acquisition by A. baumannii ATCC 19606 and to ompA gene deletion in other A. baumannii strains. Consequently, the proposed method is invaluable for markerless gene deletion in multidrug-resistant A. baumannii.     

A general method for inserting specific DNA sequences into cloning vehicles

A general method has been developed to introduce any double-stranded DNA molecule into cloning vehicles at different restriction endonuclease sites. In this method a chemically synthesized decadeoxyribonucleotide duplex, containing a specific restriction endonuclease sequence, is joinlex DNA is cut by the same restriction endonuclease to generate the cohesive ends. It is then inserted into the restriction endonuclease cleavage site of the cloning vehicle. To demonstrate the feasibility of this new method, we have inserted separately the synthetic lac operator DNA at the Bam I and HindIII cleavage sites of the plasmid pMB9 DNA.     

Convenient modification of the method for oligonucleotide-directed in vitro mutagenesis of cloned DNA

A new modification of the oligonucleotide-mediated mutagenesis technique has been developed. The proposed methodology has been used to produce specific base changes in the double-stranded plasmid DNA. For this purpose, special cloning vectors have been constructed using the synthetic oligodeoxyribonucleotides. The developed method allows the production of mutant DNA from those of the wild-type with a yield of 10-20%.     

利用表达序列标签电子克隆cDNA全序列的策略

基因组计划的进展及表达序列标签数据的迅速扩增使得电子克隆方法孕育而生,为进行基因克隆开辟了一条新的路径。介绍了表达序列标签和电子克隆的原理及过程,重点分析电子克隆过程中遇到的问题及解决方法,展望其在新基因功能研究中的作用。     

哺乳动物手工克隆的研究进展

体细胞核移植技术(Somatic cell nuclear transfer,SCNT)基本上分为传统克隆(Traditional cloning,TC)和手工克隆(Handmade cloning,HMC)。虽然随着SCNT的推进,科研人员已经利用TC成功克隆出多种动物,但这种方法需要使用昂贵的显微操作仪,并且克隆效率较低。因此,TC逐渐被新方法HMC替代,HMC是一种通过手工操作将体细胞与两个去核的半卵母细胞融合、激活,并在培养系统中培养后产生胚胎的方法。HMC不仅操作程序简单、不需要显微操作仪、成本较低,还可以提高囊胚率,使克隆技术向生产应用又迈进了一大步。主要对HMC的操作程序、表观遗传重编程异常和HMC重编程异常修复进行了综述,并对HMC进行了展望,以期能够更好地将其应用到生产中去。     

肿瘤的细胞免疫治疗研究进展

随着抗原递呈机制与细胞对特异性抗原识别机制的揭示、众多重要的人细胞因子及其功能的发现、分子克隆与细胞克隆技术的发展,细胞免疫治疗日益受到重视,并成为肿瘤等疾病临床治疗新思路、新方法的研究热点,肿瘤的细胞免疫治疗技术也在近20年内得到了广泛深入的研究和临床实践。在此,我们简要综述近年来肿瘤的细胞免疫治疗研究进展。     

林木基因克隆研究进展

林木种质资源丰富, 种质间遗传差异大, 控制林木重要性状的基因克隆及转化对培育优良林木新品种具有很强的实用价值, 但许多具有潜在应用价值的林木基因未得到充分发掘和有效分离。近年来, 随着各种不同林木cDNA文库的建立, 大规模随机EST测序技术的运用以及克隆技术的不断完善, 特别是毛果杨(Populus trichocarpa)基因组测序计划的完成, 大量与林木重要性状相关的基因被分离和鉴定。这些重要基因的获得为利用转基因技术培育高产、优质、抗逆、抗病虫害的林木新品种奠定了一定的基础。该文综述了20多年来国内外林木基因克隆的研究进展, 对基因克隆及其应用过程中亟待解决的问题进行了讨论, 并对其发展趋势进行展望。     

Highly efficient yeast-based in vivo DNA cloning of multiple DNA fragments and the simultaneous construction of yeast/ Escherichia coli shuttle vectors

In vivo recombinational cloning in yeast is a very efficient method. Until now, this method has been limited to experiments with yeast vectors because most animal, insect, and bacterial vectors lack yeast replication origins. We developed a new system to apply yeast-based in vivo cloning to vectors lacking yeast replication origins. Many cloning vectors are derived from the plasmid pBR322 and have a similar backbone that contains the ampicillin resistance gene and pBR322-derived replication origin for Escherichia coli. We constructed a helper plasmid pSUO that allows the in vivo conversion of a pBR322-derived vector to a yeast/E. coli shuttle vector through the use of this backbone sequence. The DNA fragment to be cloned is PCR-amplified with the addition of 40 bp of homology to a pBR322-derived vector. Cotransformation of linearized pSU0, the pBR322-derived vector, and a PCR-amplified DNA fragment, results in the conversion of the pBR322-derived vector into a yeast/E. coli shuttle vector carrying the DNA fragment of interest. Furthermore, this method is applicable to multifragment cloning, which is useful for the creation of fusion genes. Our method provides an alternative to traditional cloning methods.