影响苏云金芽孢杆菌基因在转基因植物中表达的因素

苏云金芽孢杆菌（Bacillus thuringiensis,Bt）杀虫晶体蛋白基因是植物抗虫基因工程中应用最广泛的基因资源。影响Bt基因在转基因植物中表达的因素繁多,阐明这些因素的效应对于获得Bt基因在受体植物中的稳定高效表达具有重要意义。现对Bt基因表达的主要影响因子,如Bt基因表达单元、植物发育、外部环境条件、受体植物遗传背景、整合位点及Bt基因沉默现象等进行了综述。     

转基因动物的乳腺表达

转基因动物乳腺组织特异性表达异源基因是近年来基因工程中引人注目的途径.文章介绍了这一途径有关的乳汁蛋白基因、乳汁蛋白基因与异源基因的融合方式、重组基因的必要构成以及可能影响高效表达的因素.     

Expression of tobacco ethylene receptor NTHK1 alters plant responses to salt stress

Ethylene has been regarded as a stress hormone involved in many stress responses. However, ethylene receptors have not been studied for the roles they played under salt stress condition. Previously, we characterized an ethylene receptor gene NTHK1 from tobacco, and found that NTHK1 is salt-inducible. Here, we report a further investigation towards the function of NTHK1 in response to salt stress by using a transgenic approach. We found that NTHK1 promotes leaf growth in the transgenic tobacco seedlings but affects salt sensitivity in these transgenic seedlings under salt stress condition. Differential Na+/K+ ratio was observed in the control Xanthi and NTHK1-transgenic plants after salt stress treatment. We further found that the NTHK1 transgene is also salt-inducible in the transgenic plants, and the higher NTHK1 expression results in early inductions of the ACC (1-aminocyclopropane-1-carboxylic acid) oxidase gene NtACO3 and ethylene responsive factor (ERF) genes NtERF1 and NtERF4 under salt stress. However, NTHK1 suppresses the salt-inducible expression of the ACC synthase gene NtACS1. These results indicate that NTHK1 regulates salt stress responses by affecting ion accumulation and related gene expressions, and hence have significance in elucidation of ethylene receptor functions during stress signal transduction.     

Development of the binary vector pTACAtg1 for stable gene expression in plant: Reduction of gene silencing in transgenic plants carrying the target gene with long flanking sequences

Genetic modification in plants helps us to understand molecular mechanisms underlying on plant fitness and to improve profitable crops. However, in transgenic plants, the value of gene expression often varies among plant populations of distinct lines and among generations of identical individuals. This variation is caused by several reasons, such as differences in the chromosome position, repeated sequences, and copy number of the inserted transgene. Developing a state-of-art technology to avoid the variation of gene expression levels including gene silencing has been awaited. Here, we developed a novel binary plasmid (pTACAtg1) that is based on a transformation-competent artificial chromosome (TAC) vector, harboring long genomic DNA fragments on both sides of the cloning sites. As a case study, we cloned the cauliflower mosaic virus 35S promoter:β-glucuronidase (35S:GUS) gene cassettes into the pTACAtg1, and introduced it with long flanking sequences on the pTACAtg1 into the plants. In isolated transgenic plants, the copy number was reduced and the GUS expressions were detected more stably than those in the control plants carrying the insert without flanking regions. In our result, the reduced copy number of a transgene suppressed variation and silencing of its gene expression. The pTACAtg1 vector will be suitable for the production of stable transformants and for expression analyses of a transgene.     

转基因植物表达重组蛋白的研究进展

植物表达系统的一些潜在优点 ,如重组蛋白的高积累水平 ,糖基化 ,细胞内的定位和自然储藏的稳定性是目前植物生产重组蛋白系统研究成为热点的主要原因 .在研究和选择转基因植物表达系统的过程中 ,转化 ,转化后 ,翻译 ,翻译后等环节都会影响到最终产物的数量和质量 ,因此应该了解基因表达的规律 ,以制定植物生产重组蛋白合适的策略 ,重组蛋白积累水平是关键 ,但其它因素如植物的选择 ,转基因植物的处理 ,下游加工等同样重要 .某些情形下 ,仅下游加工的成本一项就影响到特定植物表达系统的实际应用价值 .     

Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity

Expression and stability of immunoglobulins in transgenic plants have been investigated and optimized by accumulation in different cellular compartments as cytosol, apoplastic space and endoplasmic reticulum (ER) as will be discussed in this review. In several cases described the highest accumulation of complete active antibodies was achieved by targeting into the apoplastic space. High-level expression of active recombinant single-chain Fv antibodies (scFv's) was obtained by retention of these proteins in the lumen of the endoplasmic reticulum. This has been shown for leaves and seeds of transgenic tobacco as well as for potato tubers. Transgenic tobacco seeds, potato tubers and tobacco leaves can facilitate stable storage of scFv's accumulated in the ER over an extended (seeds, tubers) or a short (leaves) period of time. The expression of specific scFv's in different plant species, plant organs and cellular compartments offers the possibility of blocking regulatory factors or pathogens specifically. Examples are scFv's expressed in the cytosol and the apoplastic space of transgenic plant cells modulating the infection process of plant viruses and a cytosolically expressed scFv that influenced the activity of phytochrome A protein. The immunomodulation approach has been shown to be also applicable for investigating the action of the phyto-hormone abscisic acid (ABA). High-level accumulation of specific anti-ABA scFv's in the ER of all leaf cells has been used to block the influence of ABA on the stomatal functions. Seed-specific expression of high amounts of anti-ABA-scFv's at a defined time of seed-development induced a developmental switch from seed ripening to vegetative growth. It has been demonstrated that ER retention is essential for the accumulation of sufficient scFv to bind high concentrations of ABA in the transgenic seeds.     

转Bt基因植物中外源基因时空动态表达的研究现状

在转Bt基因植株中 ,外源基因的时空动态表达对于害虫的防治和转基因安全评价管理具有重要意义。利用生物测定法和酶联免疫吸附测定法 (ELISA) ,对植物不同组织在同一发育阶段、同一组织在不同的发育阶段以及不同转基因植株的外源基因的时空动态表达进行研究。本文综述了转基因植物中外源基因时空动态表达的研究进展和现状。     

基质结合区与转基因动物的基因表达

基质结合区(MAR)在稳定转染的细胞系中的研究结果显示,能缓冲在其侧翼的染色质某些拮抗作用.这为外源基因在染色体中随机整合的转基因动物研究提供了新的方向.文章对其在转基因动物中的探索性研究及可能的机理进行综述.指出在转基因动物中,MAR的应用能导致建立独立的基因活性结构域.它对基因高效表达无疑具有重要作用.MAR可能是一种新的顺式作用元件,与增强子、启动子协同作用调节基因的表达.     

内含子对提高转基因动物基因表达效率的影响

综述了内含子在转基因动物基因表达中的作用,对内源性内含子和异源内含子对转基因表达的影响进行分析,讨论了内含子提高转基因动物基因表达效率的三种可能机制,并指出在表达载体构建中应考虑到内含子的重要性．     

Geminivirus vectors for high-level expression of foreign proteins in plant cells

Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins.     

Expression of engineered wheat dwarf virus in seed-derived embryos

Delivery of DNA into dry and viable embryos of wheat by imbibition in DNA solution was detected by monitoring the transient expression of chimeric genes. The gene expression vectors used comprise of a neomycin phosphotransferase II (NPT II) reporter gene, under the control of various promoters or as part of a cloned plant virus genome. The genome of wheat dwarf virus (WDV), a monopartite Gemini virus of gramineae, has been used to amplify the NPT II gene in embryos of wheat, thereby leading to high levels of transient expression. Constructions were designed which permitted NPT II expression only if inter- or intramolecular recombination had occurred.     

Human placental alkaline phosphatase as a histochemical marker of gene expression in transgenic mice

The human placental alkaline phosphatase (PLAP) gene was analysed for its utility as a histochemically detectable reporter gene in transgenic mice. A reporter gene was made by linking the PLAP structural gene to an enhancerpromoter element from the human -actin gene. This gene was inserted into the mouse genome by transfection of embryonic stem cells, and by microinjection of fertilized eggs. Histochemical staining showed that the transgene was uniformly expressed in four of four stable ES cell lines, and in all ten tissues examined from adult animals from five lines of transgenic mice. Non-transgenic cells did not stain. These results suggest that the human PLAP gene will be of utility in studies requiring phenotypic marking of cells in tissues of mice.     

Role of carbon nanotubes (CNTs) in transgenic plant development

Carbon nanotubes (CNTs) are nanostructures, allotropes of carbon which are made up of graphene sheets wrapped around it forming cylindrical structures. CNTs have been regarded to have interesting and attractive physical and chemical properties and have been tremendously used in genetic engineering. Understanding the role of CNTs in development of transgenic plants, review of research papers in the field was done. CNTs are classified into two categories: the single-walled and multiwalled (MWCNTs) structures. They are valuable vectors in various biomedicine fields such as Gene delivery, Drug delivery, Immunotherapy, Tissue engineering, and Biomedical imaging and also, they deliver the DNA without damaging the cells. Based on recent studies, the functionalization of CNTs when combined with some other suitable molecules can drastically subside their toxic effects. Having unique properties such as small size, larger surface area is useful in delivering DNA into mammalian cells as well. Modifications in CNTs can make nucleic acids adhere to them even more efficiently. Also, MWCNTs are crucial in delivery DNA into the cytoplasm. Based on other methods, the CNTs-DNA are a preferred choice and the inclination toward double-stranded DNA is used over single-stranded DNA in gene delivery shows effective results. The only downside of CNTs is that they are hydrophobic and are difficult to form an aqueous solution, thus limiting their applicability. This review will aid you in comprehending useful knowledge related to a general overview of topics related to CNTs.     

Direct gene transfer to protoplasts: Fate of the transferred genes

Direct gene transfer to protoplasts is one of several methods developed for the production of transgenic plants. This method utilizes the efficient uptake of DNA from the surrounding medium by protoplasts (cell wall-less plant cells). Where a suitable protoplast system exists large numbers of transformant clones can be efficiently produced and often regenerated to normal fertile plants. This review concentrates on the fate of the DNA which is taken up into the protoplasts. Particular emphasis is given to the factors which can influence the integration and form of the transferred DNA, the expression of transferred genes, and the inheritance in further generations of those genes. The information available suggests (1) that DNA is taken up by a large proportion of the cells in a transformation mixture, (2) that this DNA forms complexes sometimes involving carrier DNA, (3) that fewer cells actually take up DNA into the nucleus, and (4) that the complex may be rearranged and/or amplified and then integrated into the genome. If the DNA is arranged in such a way that a gene can be expressed it does so in a normal manner and is stably inherited both mitotically and meiotically.     

动物转基因高效表达策略研究进展

外源基因在动物体内、细胞内表达时,常由于各种原因导致基因沉默。目前,主要有两种策略用于打破该瓶颈:一是利用友好位点;二是优化外源基因顺式作用元件。利用友好位点就是通过定点整合技术将外源基因插入至活性转录区域;优化外源基因顺式作用元件即采用合适的启动子、增强子、内含子、染色质开放元件、核基质附着区等元件,构建出高效表达载体。对相关策略进行概述,为动物基因工程育种提供参考。     

Human anti-rhesus D IgG1 antibody produced in transgenic plants

Transgenic plants represent an alternative to cell culture systems for producing cheap and safe antibodies for diagnostic and therapeutic use. To evaluate the functional properties of a plantibody, we generated transgenic Arabidopsis plants expressing full-length human IgG1 against the Rhesus D antigen, which is responsible for alloimmunization of RhD– mothers carrying an RhD+ fetus. Anti-RhD extracted from plants specifically reacted with RhD+ cells in antiglobulin technique, and elicited a respiratory burst in human peripheral blood mononuclear cells. Plant-derived antibody had equivalent properties to CHO cell-produced anti-RhD antibody, indicating its potential usefulness in diagnostic and therapeutic programs.