高等植物叶绿体基因组转化的应用

叶绿体基因组转化技术由于其独特的优越性,现已成为植物基因工程的研究热点。本文简单介绍了叶绿体基因组转化技术的原理和方法;并重点综述了该技术在基础研究和实践中的应用。这些应用主要包括利用叶绿体基因组转化技术进行Rubisco的组装,叶绿体基因结构、转录、翻译和RNA编辑等研究;利用叶绿体作为生物反应器生产人生长激素、霍乱毒素抗体、聚羟基丁酸脂和生物弹性蛋白等;获得抗虫、抗病、抗除草剂和耐旱的转基因植物;以及降低转基因植物的外源基因扩散等。     

疫苗生产的新途径——转基因植物

与发酵生产方式相比,转基因植物疫苗生产技术具有高效、经济和简便等特点。植物表达系统生产外源蛋白一般采用两种方式：（１）编码外源抗原基因与植物基因组稳定整合;（２）利用植物病毒载体,使外源蛋白在植物细胞中瞬时表达。植物系统生产的抗原疫苗可保持自然免疫原性质,口服后能够诱发体液和粘膜免疫反应。     

高等植物叶绿体表达重组蛋白研究进展

近年来,基因工程技术发展迅速,许多重组蛋白得以表达。其中利用植物生物反应器表达特异药物蛋白为人类一些重要疾病的预防和治疗提供了新途径。植物叶绿体遗传转化和表达系统成为目前植物生物反应器的研究热点。因结构和遗传上的特殊性,高等植物叶绿体在重组蛋白表达方面具有独特优势,外源基因表达量高、定点整合,而且叶绿体母系遗传特性保证了生物安全性。很多重要药用蛋白质在植物叶绿体中表达成功。烟草作为高等植物叶绿体转化模式植物,在疫苗抗原、抗体等药物蛋白和其他重要重组蛋白表达方面取得显著进展。高等植物叶绿体遗传转化也为叶绿体基因的表达和调控机制的研究提供新的技术和方法。文中从叶绿体遗传转化原理、载体构建、重组蛋白和重要药物蛋白在叶绿体中的表达以及重组蛋白表达对植物代谢和性状影响等多个角度,对高等植物叶绿体遗传转化体系研究的新进展进行了综述,以期为叶绿体表达平台的开发和重要药用蛋白质的表达提供新思路。     

转基因苜蓿口服疫苗的研究和应用

随着植物转基因技术的成熟,利用转基因牧草生产人或动物可食性疫苗更成为当今研究的热点.苜蓿以其营养价值高,粗蛋白、维生素和矿物质含量丰富,氨基酸平衡、适口性好等优点,成为研制植物口服疫苗最为理想的生物反应器.该文分别从植物口服疫苗的优点、苜蓿的遗传转化技术、外源基因在转基因苜蓿中的表达水平及免疫效果等,介绍国内外以苜蓿作为受体生产植物可食疫苗的研究进展,指出研究中存在的问题,并展望其广阔的应用前景.     

转基因植物中T-DNA整合的分子特征及表达

植物中不同转基因方法转化外源基因的T-DNA整合特征既具有共性,又具有特性,使得转基因的遗传在各独立转化体间呈现多样性,另外多种遗传因子和限制因素使受体植物中外源基因的表达存在下降,甚至出现基因沉默等复杂现象。本文主要对农杆菌介导及裸露DNA直接转化转基因植物中T-DNA的分子特征和转基因表达的影响因子进行了介绍和概述。转化体中转基因的遗传稳定性和表达主要取决于转基因在植物基因组中的整合位置、拷贝数及组成结构。因而,通过对具有表达水平各异的转化体进行深入的遗传分析和分子生物学研究以及转化体之间进行的比较研究,将对转基因技术自身的完善、定点整合以及更有效的利用转基因技术都具有十分重要的意义。     

植物转基因沉默研究进展、对策及应用

转基因沉默 (ｔｒａｎｓｇｅｎｅｓｉｌｅｎｃｉｎｇ)是导入并整合进受体基因组中的外源基因在当代转化体或其后代中表达受到抑制的现象。自Ｐｅｅｒｂｏｌｔｅ在 1986年首次报道发现转基因沉默现象以来 ,相关报道不断发表。如今 ,转基因沉默现象已成为遗传转化技术实用化 ,商品化过程中的巨大障碍。从九十年代开始 ,人们就致力于转基因沉默方面的研究。随着研究的不断深入 ,转基因沉默的各种机理不断被揭示 ,并研究出相应的对策。1.植物转基因沉默研究进展转基因沉默的各种机理都涉及到各种核酸之间的相互作用 ,包括转录水平的基因沉默 (ｔ…     

叶绿体基因工程研究进展

叶绿体是植物细胞和真核藻类执行光合作用的重要细胞器,在叶绿体中表达外源基因比在细胞核中表达具有一些独特优势。叶绿体基因工程涉及叶绿体的基因组特征、转化系统的优点、转化过程及方法等方面,叶绿体基因工程在提高植物光合效率、改良植物特性、生产生物药物及改善植物代谢途径等方面已得到应用。尽管叶绿体基因工程还存在同质化难度高、标记基因转化效率较低、宿主种类偏少等问题,但作为外源基因在高等植物中表达的良好平台其仍然具有广阔的发展和应用前景。     

根癌农杆菌介导的转新城疫病毒融合蛋白基因水稻植株的获得

利用转基因植物作为生物反应器可以表达重组蛋白、生产外源蛋白质,也可以成为动物疫苗的廉价生产系统。以编码新城疫病毒融合蛋白(NDV-F)的基因为外源基因,以玉米泛素蛋白(Ubi)启动子为启动子,以潮霉素磷酸转移酶(HPT)基因作为选择标记基因,β-半乳糖苷酸酶(GUS)基因作为报告基因构建了适宜于农杆菌介导转化水稻的表达质粒pUNDV,并通过农杆菌介导转化水稻,获得了多株转基因植株。通过PCR分析和GUS活性检测,证实含有NDV-F基因的T-DNA已整合到水稻核基因组中,为研制廉价安全的转基因水稻新城疫基因工程疫苗奠定了基础。     

苏云金芽孢杆菌杀虫蛋白基因克隆及油菜叶绿体遗传转化研究

向细胞核导入外源基因的核转化技术是植物基因工程的主要方法。然而,外源基因表达效率低,表达不稳定,基因易失活和因随机插入而造成的位置效应等是该方法不足之处。而且,由于外源基因可随花粉扩散,细胞核转化体的生物安全性问题已在全球范围内引起世人的关注和担忧。将外源基因导入叶绿体基因组有望克服细胞核转化中存在的某些弊端。油菜作为世界上重要的油料作物,其叶绿体遗传转化研究还未见报道。本研究从苏云金芽孢杆菌克隆得到野生型杀虫晶体蛋白基因,构建了用于油菜叶绿体定点转化的植物表达载体,并用基因枪法将杀虫蛋白基因导入油菜,…     

转基因植物与食用疫苗

转基因植物与食用疫苗宋长征,韩金祥,王美玲（山东省医学科学院生物技术中心,济南）转基因植物技术是随着ＤＮＡＩ组、基因的遗传转化、植物的组织培养、目的基因的整合与表达的检测等技术手段的建立而发展起来的生物技术。利用转基因植物作为生物反应器,把外源基因引...     

OBPC Symposium: Maize 2004 &; beyond—Recent advances in chloroplast genetic engineering

Summary The chloroplast genetic engineering approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. Thus far, more than 40 transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer several agronomic traits and produce vaccine antigens, industrially valuable enzymes, biomaterials, and amino acids. Functionality of chloroplastderived of vaccine antigens has been facilitated by hyperexpression in transgenic chloroplasts (leaves) or non-green plastids (carrols) and the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Despite such significant progress in chloroplast transformation, this technology has not been extended to major crops. This obstacle emphasizes the need for plastid genome sequencing to increase the efficiency of transformation and conduct basic research in plastid biogenesis and function. However, highly efficient soybean, carrot, and cotton plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors. Recent advancements facilitate our understanding of plastid biochemistry and molecular biology. This review focuses on exciting recent developments in this field and offers directions for further research and development.     

Breakthrough in chloroplast genetic engineering of agronomically important crops

Chloroplast genetic engineering offers several unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event and transgene containment by maternal inheritance, as well as a lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. More than 40 transgenes have been stably integrated and expressed using the tobacco chloroplast genome to confer desired agronomic traits or express high levels of vaccine antigens and biopharmaceuticals. Despite such significant progress, this technology has not been extended to major crops. However, highly efficient soybean, carrot and cotton plastid transformation has recently been accomplished through somatic embryogenesis using species-specific chloroplast vectors. This review focuses on recent exciting developments in this field and offers directions for further research and development.     

叶绿体基因工程：一种植物生物技术的新方法

以叶绿体转化为主的叶绿体基因工程,与传统的基因工程技术细胞核转化相比,在外源基因表达水平和转基因植物安全性等方面有明显的优势, 尤其是在控制转基因沉默和遗传稳定性方面,可以互补核转化带来的局限性。因此,叶绿体基因工程是一种很具有发展前景的植物转基因技术,并在未来工农业生物技术领域发挥重要作用。本文着重在叶绿体转化技术主要特点,应用领域及其未来的发展前景等方面进行了简单评述。     

莱茵衣藻叶绿体生物反应器研究进展

莱茵衣藻(Chlamydomonas reinharditi)是一种遗传机制已研究比较清楚的模式植物。近年来,生物反应器是当今世界上各国生物技术研究的一个热点,随着生物技术的发展,已成功实现衣藻作为生物反应器生产重组蛋白及抗体,生产的部分产品已经实现了商品化,与其他生物反应器相比,其在外源基因表达水平和转基因植物安全性等方面有明显的优势,尤其是在控制转基因沉默和遗传稳定性方面展示了极大的优越性。因此,莱茵衣藻是一种具有很好发展前景的生物反应器,必将在未来的药用蛋白生物技术领域发挥重要作用。主要对提高基因在莱茵衣藻叶绿体中表达的策略,转化技术的特点及其未来的发展前景等方面进行了简单评述。     

Chloroplast Genetic Engineering: Recent Advances and Future Perspectives

Chloroplast genetic engineering offers a number of unique advantages, including a high-level of transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects, and undesirable foreign DNA. Thus far, over forty transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer important agronomic traits, as well as express industrially valuable biomaterials and therapeutic proteins. The hyperexpression of recombinant proteins within plastid engineered systems offers a cost effective solution for using plants as bioreactors. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Oral delivery of vaccine antigens against cholera, tetanus, anthrax, plague, and canine parvovirus are made possible because of the high expression levels and antibiotic-free selection systems available in plastid transformation systems. Plastid genetic engineering also has become a powerful tool for basic research in plastid biogenesis and function. This approach has helped to unveil a wealth of information about plastid DNA replication origins, intron maturases, translation elements and proteolysis, import of proteins and several other processes. Although many successful examples of plastid engineering have set a foundation for various future applications, this technology has not been extended to many of the major crops. Highly efficient plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors in soybean, carrot, and cotton. Transgenic carrots were able to withstand salt concentrations that only halophytes could tolerate; more than twice the effectiveness of other engineering attempts. Recent advances in plastid engineering provide an efficient platform for the production of therapeutic proteins, vaccines, and biomaterials using an environmentally friendly approach. This review takes an in-depth look into the state of the art in plastid engineering and offers directions for further research and development.     

植物基因工程新途径：叶绿体转化

在植物基因工程研究中,叶绿体是继核转化之后又一新的遗传转化和表达受体,叶绿体转化体系具有可同时进行多基因转化,表达原核性,超量表达,后代遗传稳定,定点整合,不会产生基因沉默及母性遗传和安全性好等特点,本文着重介绍叶绿体转化体系的特点,国内外研究动态,存在的问题及未来发展方向。     

用于叶绿体遗传转化的表达载体

叶绿体遗传转化是植物基因工程的新方向。本文简要介绍用于叶绿体遗传转化的表达载体的构建方法,涉及同源重组片段、叶绿体特异的启动子和终止子、筛选标记基因,以及目前在叶绿体中已实现表达的外源基因等内容。 Abstract:Chloroplast genetic transformation is a new way of plant genetic engineering.This paper reviews the construction methods of expression vector used in chloroplast genetic transformation.It contains the homologous recombinant fragments,the chloroplast specific promoter and terminater,the selectable marker genes and the interest genes whose expression in chloroplast have been achieved.