植物基因工程中化学诱导表达系统研究近况

利用可诱导表达系统可使目的基因在特定的时间内表达，从而有利于我们研究其基因产物的作用，本文着重介绍了几种利用植物远缘物种的调控元件构建而成的化学诱导系统，及它们在植物基因工程中的应用。     

烟草化学诱导表达系统的建立

化学诱导表达系统对植物功能基因的研究及植物基因工程应用有重要意义。XVE是以雌激素为基础的用于诱导转基因植物中目的基因表达的一种系统,能够在可调控方式下诱导基因的表达。目前,以烟草为遗传转化材料进行XVE系统研究的详细报道还未见发表。本文以烟草作为研究对象,利用PCR技术从本实验保存的质粒pROKII-GFP中扩增出GFP基因。构建雌激素诱导型植物表达载体(p ER8-GFP)并利用农杆菌介导的叶盘法将外源基因导入野生型烟草中;经潮霉素抗性筛选出转化植株后,用PCR鉴定出阳性转化植株,将阳性转化植株利用不同浓度和不同时间的雌激素进行处理,并结合定量PCR和Night SHADE植物活体成像系统对转基因植株的表达水平进行检测。结果表明诱导p ER8-GFP载体在烟草中表达的最适雌激素浓度为25μmol·L~(-1),最适时间为48 h;同时在烟草胚轴与根尖细胞中检测到荧光信号,表明XVE化学诱导系统在烟草中也可以高效、严格的依赖雌激素的诱导来控制目的基因表达。本研究将为烟草相关的化学诱导表达研究提供技术支持与解决方案。     

植物广谱抗病基因工程策略与研究进展

系统获得性抗性（SAP）是植物防御病原微生物侵染的一条有效途径。利用基因工程技术改造其表达特性可以提高植物的抗病性,从活性氧的代谢,抗病基因的利用、过敏反应的诱导和SAR的组成性表达等方面论述了植物广谱抗病基因工程的研究策略。已取得的成就及今后的研究方向。     

转基因植物高效删除标记基因的实用型双元转化载体

利用雌激素诱导的Cre/loxPDNA重组系统,构建用于转基因植物删除标记基因的实用新型载体,将所有非目的基因放于两同向loxP位点间,多克隆位点位于两loxP位点外,可插入目的基因,载体设计非常实用。在插入目的基因GUS后转化烟草,雌激素诱导,分析诱导前后DNA的重组情况、不同时间基因转录水平,以及目的基因在诱导前后的表达。结果显示,诱导后染色体DNA实现了高效重组,非目的基因片段消失,目的基因GUS在诱导后表达正常。     

基因AtHsp17.6-C2启动子的克隆及其热诱导动力学研究(英文)

介绍一个植物表达系统,由热激蛋白基因(AtHsp17.6-C2)的启动子来驱动GUS基因的表达。在22℃生长条件下,稳定遗传的转基因植株中几乎检测不到GUS的活性。但是当温度升至34～37℃时,GUS的活性迅速升高。37℃是该植物表达系统最适诱导温度。转基因植株经37℃热诱导2小时后再返回22℃培育2小时,GUS的活性增加80多倍。多次热诱导实验表明这个表达系统是能够被重复多次热诱导的。实验结果表明这个植物诱导表达系统能够适用于多种目的需要。     

不结球白菜BcDF1.2基因克隆与诱导表达

利用RACE技术,获得了不结球白菜防卫素基因全长序列,命名为BcDF1.2(DDBJ登录号AB302891).序列分析发现,BcDF1.2全长532 bp包含1个长度为96 bp的内含子区域,该基因编码79个氨基酸残基组成的蛋白质,与其他植物的防卫素基因和抗真菌蛋白基因有较高的同源性.系统进化树分析表明,该基因在不同植物间具有高度保守性.基因组DNA杂交表明BcDF1.2属于较小的多基因家族.水杨酸和霜霉病原菌诱导后,该mRNA不仅在诱导的叶片中转录表达增加,而且在未诱导的系统叶片中表达增加.BcDF1.2在不结球白菜叶片中的转录表达特点表明可能参与对霜霉病的抗性反应.     

拟南芥FT基因原核表达载体的构建、表达和蛋白纯化

FT基因是植物成花素,在植物的开花调控中起着重要作用。构建了用于原核表达的FT-eGFP表达载体,并在大肠杆菌BL21中进行重组蛋白的诱导表达。从IPTG诱导浓度、诱导时间和诱导温度等方面进行了细致的分析,最终建立了FT-eGFP融合蛋白诱导表达的优化体系:当菌液OD600=0.6-1.0时,采用IPTG1.0mol/L,在28℃诱导表达6h。摸索和建立了利用HisTrapKit标签,经过镍柱纯化,纯化目的蛋白的技术体系,为进一步研究FT基因在植物开花调控中的应用奠定了基础。     

植物低温诱导转录因子CBF研究进展

植物在低温驯化过程中能诱导许多基因的表达。其中CBF转录因子是目前植物抗寒分子生物学领域研究的热点之一。它通过与低温诱导基因启动子区域中的CRT/DRE调控元件结合,调控一系列低温诱导基因的表达,从而提高植物的抗寒性。对植物低温诱导CBF转录因子的结构、功能及其表达调控等方面的研究进展进行了综述。     

草甘膦诱导表达的大豆与棉花EST序列的分离

利用差异显示PCR方法分离了63个草甘膦诱导后在大豆和棉花中差异表达的片段,测序分析结果表明属于33个草甘膦诱导的大豆和棉花EST序列。通过在GenBank中进一步比时研究发现:约85％的EST序列与水杨酸、冷、创伤、氧化等非生物胁迫诱导后表达库中的EST序列有高达95％以上的同源性,由此可推测这些基因参与了植物对非生物胁迫的反应过程。草甘膦诱导后高表达EST、序列的获得将有利于进一步分离相关非生物胁迫诱导表达基因及启动子,研究其转录调控的机理,有望建立草甘膦诱导系统。从而解决组成型表达造成外源基因在植物体所有发育阶段和所有组织部位表达,造成植物体能量浪费。     

植物基因的人工调控表达

通过增加或减少特定诱导物,可精确调节目的基因上游构建的“基因开关”,实现外源目的基因的准确调控,使外源基因在植物体内适时、适量、有效地表达。因此构建一个拥有“基因开关”的人工调控表达系统是势在必行的,该系统的成功构建也将有利于提高转基因植物广泛应用的可能性 。     

An Inducible DamID System for Profiling Interactions of Nuclear Lamina Protein Component Lamin B1 with Chromosomes in Mouse Cells

At the level of DNA organization into chromatin, there are mechanisms that define gene expression profiles in specialized cell types. Genes within chromatin regions that are located at the nuclear periphery are generally expressed at lower levels; however, the nature of this phenomenon remains unclear. These parts of chromatin interact with nuclear lamina proteins like Lamin B1 and, therefore, can be identified in a given cell type by chromatin profiling of these proteins. In this study, we created and tested a Dam Identification (DamID) system induced by Cre recombinase using Lamin B1 and mouse embryonic fibroblasts. This inducible system will help to generate genome-wide profiles of chromatin proteins in given cell types and tissues with no need to dissect tissues from organs or separate cells from tissues, which is achieved by using specific regulatory DNA elements and due to the high sensitivity of the method.     

Reversible, cell-heritable changes during the development of tobacco pith tissues

Cytokinin requiring cells of Nicotiana tabacum L. cv "Havana 425" can be induced in culture to become cytokinin autotrophic. This process is known as cytokinin habituation. Earlier we showed that pith parenchyma tissue consists of inducible cells, which habituate at high rates when treated with cytokinin, and noninducible cells, which remain cytokinin requiring under these conditions. The inducible and noninducible phenotypes are determined states that arise during the development of the tobacco plant and are inherited by individual cells. Here we show that pith tissue of plants regenerated from cloned lines of noninducible cells exhibits the inducible phenotype indicating that noninducible cells, or their descendants, can become inducible. This change in competence for habituation appears to have an epigenetic basis; it is reversible, occurs at high rates, and depends on the developmental state of the cells. The habituated state occurs in two forms that can be distinguished by their difference in developmental potential. Habituated cells derived from inducible pith cells give rise to normal plants whose leaf and pith tissues require cytokinin for growth in culture. In contrast, habituated cells obtained by transferring noninducible cells on media with progressively lower cytokinin concentrations give rise to plants whose leaf and pith tissues exhibit a cytokinin-habituated phenotype in culture.     

Recent advances in inducible expression in transgenic mice

In order to accurately analyze gene function in transgenic mice, as well as to generate credible murine models of human diseases, the ability to regulate temporal- and spatial-specific expression of target genes is absolutely critical. Pioneering work in inducible transgenics, begun in the 1980s and continuing to the present, has led to the development of a variety of different inducible systems dedicated to this goal, the shared basis of which is the accurate conditional expression of a given transgene. Recent advances in inducible transgene expression in mice are discussed.     

Larval feeding structure plasticity during pre-feeding stages of echinoids: Not all species respond to the same cues

Much of the work on phenotypic plasticity has focused on inducible defenses. As a result, little is known about induced phenotypes that improve the acquisition of resources (i.e. inducible offenses). Feeding larvae of several marine invertebrate species, gastropods and echinoderms, have inducible offenses, and produce larger feeding structures when given less food. To better understand inducible offenses, I investigated when in development sea urchin and sand dollar larvae can first alter their feeding morphology in response to different concentrations of food. Food induced feeding structure changes in both sea urchin and sand dollar larvae before larvae were able to ingest food. This suggests that the nervous system and a regulator gene, orthopedia, play a mechanistic role. In addition, larvae of the two species, Strongylocentrotus purpuratus and Dendraster excentricus, responded to different cues. Pre-feeding larvae of both species developed relatively shorter arms when given algal cells (i.e. chemical and physical stimuli), whereas only pre-feeding larvae of D. excentricus developed shorter arms when exposed to algal exudates (i.e. chemical stimuli). Larvae of neither species responded morphologically to the presence of polystyrene beads (i.e. physical stimuli).     

Design of a Lentiviral Vector for the Inducible Expression of <Emphasis Type="Italic">MYC</Emphasis>: A New Strategy for Construction Approach

Lentiviral vectors are powerful tools for gene expression studies. Here we report the construction of pTIJ, a vector for inducible gene expression. pTIJ was generated from pTRIPZ backbone, which is designed for the inducible expression of shRNA sequences, by the introducing of a multiple cloning site upstream of the Tet promoter and the removal of miR30 flanking sequences. To evaluate pTIJ as a tool for the inducible expression of genes of interest, we introduced MYC cDNA into pTIJ and infected two small cell lung cancer cell lines, H209 and H345. Induction of MYC expression by doxycycline was detectable in both cell lines by real-time PCR and western blot analysis. This study highlights the relevance of pTIJ vector to allow the inducible expression of any gene of interest. In our belief, pTIJ will be an extremely useful tool to simplify the generation of genetically engineered cell lines for the inducible expression of cDNA sequences in biological studies. Furthermore, we report the generation of a pTIJ-MYC vector for the inducible expression of the oncogene MYC.     

Arginine catabolism in Agrobacterium strains: role of the Ti plasmid.

We present a study of the enzymatic activities involved in the pathway for arginine catabolism by Agrobacterium tumefaciens. Nitrogen from arginine is recovered through the arginase-urease pathway; the genes for these two activities are probably chromosomally born. Arginase was found to be inducible during growth in the presence of arginine or ornithine. Urease was constitutively expressed. Ornithine, resulting from the action of arginase on arginine, could be used as a nitrogen source via transamination to delta 1-pyrroline-5-carboxylate and reduction of the latter compound to proline by a reductase (both enzymatic activities are probably chromosomally encoded). Ornithine could also be used as a carbon source. Thus, we identified an ornithine cyclase activity that was responsible for direct conversion of ornithine to proline. This activity was found to be Ti plasmid encoded and inducible by growth in medium containing octopine or nopaline. The same activity was also chromosomally encoded in some Agrobacterium strains. In such strains, this activity was inducible during growth in arginine-containing medium.     

Determinants of resistance to chlortetracycline and other antibiotics in chlortetracycline-producing strain of Streptomyces aureofaciens

Data are presented on resistance of Streptomyces aureofaciens strain TB-633 FU--the producer of chlortetracycline (CTC) to autogenous antibiotics and a number of other antibiotics. It is demonstrated that resistance to CTC is specified by ctr genes of constitutive expression as well as by inducible genes. CTC and ethidium bromide may serve as efficient inductors of inducible ctr genes. The induction process is accompanied by increase in antibiotic biosynthesis level. Genes responsible for strain resistance to a number of macrolide antibiotics and thiostrepton are inducible and only function in the presence of appropriate antibiotics in the medium. The action of inducible mtr gene(s) is described in detail. The gene(s) simultaneously ensure increase in resistance to CTC and a number of macrolide antibiotics in the presence of exogenous inductors in media, such as both CTC and macrolide antibiotics. Mutants have been isolated which provide constitutive level of resistance to these antibiotics. A series of ctr and mtr mutants have increased CTC biosynthesis as compared to the initial level. Data on comparative analysis of the results obtained from hybridization of fragments of S. aureofaciens and S. rimosus DNAs to actI and actIII genes, responsible for polyketide synthases' synthesis, demonstrate that genes for CTC and OTC biosynthesis are situated on DNA fragments of similar size. This determines the strategy for cloning ctr and mtr genes as well as genes for CTC biosynthesis from S. aureofaciens.