Plastid biotechnology: prospects for herbicide and insect resistance, metabolic engineering and molecular farming

Transgene expression from the chloroplast (plastid) genome offers several attractions to plant biotechnologists, including high-level accumulation of foreign proteins, transgene stacking in operons and a lack of epigenetic interference with the stability of transgene expression. In addition, the technology provides an environmentally benign method of plant genetic engineering, because plastids and their genetic information are maternally inherited in most crops and thus are largely excluded from pollen transmission. During the past few years, researchers in both the public and private sectors have begun to explore possible areas of application of plastid transformation in plant biotechnology as a viable alternative to conventional nuclear transgenic technologies. Recent proof-of-concept studies highlight the potential of plastid genome engineering for the expression of resistance traits, the production of biopharmaceuticals and metabolic pathway engineering in plants.     

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.     

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。     

Genetic engineering of the chloroplast

Transformation of the plastid genome has a number of inherent advantages for the engineering of gene expression in plants. These advantages include: 10-50 times higher transgene expression levels; the absence of gene silencing and position effect variation; the ability to express polycistronic messages from a single promoter; uniparental plastid gene inheritance in most crop plants that prevents pollen transmission of foreign DNA; integration via a homologous recombination process that facilitates targeted gene replacement and precise transgene control; and sequestration of foreign proteins in the organelle which prevents adverse interactions with the cytoplasmic environment. It is now 12 years since the first conclusive demonstration of stable introduction of cloned DNA into the Chlamydomonas chloroplast by the Boynton and Gillham laboratory, and 10 years since the laboratory of Pal Maliga successfully extended these approaches to tobacco. Since then, technical developments in plastid transformation and advances in our understanding of the rules of plastid gene expression have facilitated tremendous progress towards the goal of establishing the chloroplast as a feasible platform for genetic modification of plants.     

Advances in chloroplast engineering

The chloroplast is a pivotal organelle in plant cells and eukaryotic algae to carry out photosynthesis, which provides the primary source of the world＇s food. The expression of foreign genes in chloroplasts offers several advantages over their expression in the nucleus： high-level expression, transgene stacking in operons and a lack of epigenetic interference allowing stable transgene expression. In addition, transgenic chloroplasts are generally not transmitted through pollen grains because of the cytoplasmic localization. In the past two decades, great progress in chloroplast engineering has been made. In this paper, we review and highlight recent studies of chloroplast engineering, including chloroplast transformation procedures, controlled expression of plastid transgenes in plants, the expression of foreign genes for improvement of plant traits, the production of biopharmaceuticals, metabolic pathway engineering in plants, plastid transformation to study RNA editing, and marker gene excision system.     

高等植物叶绿体基因工程

叶绿体基因工程作为一项新技术具有一系列传统核基因工程所不具备的优点,在基础性及应用性研究中极具吸引力,已经成功应用于了解质体基因组,调控植物代谢系统,农作物抗旱、抗虫、抗病、抗除草剂及以植物为生物反应器生产抗体、疫苗等方面的研究。本文主要介绍叶绿体基因工程的原理、操作体系及其在高等植物中的应用。     

高等植物叶绿体基因工程

叶绿体基因工程作为一项新技术具有一系列传统核基因工程所不具备的优点,在基础性及应用性研究中极具吸引力,已经成功应用于了解质体基因组,调控植物代谢系统,农作物抗旱、抗虫、抗病、抗除草剂及以植物为生物反应器生产抗体、疫苗等方面的研究.本文主要介绍叶绿体基因工程的原理、操作体系及其在高等植物中的应用.     

Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit

Transgenic chloroplasts offer unique advantages in plant biotechnology, including high-level foreign protein expression, absence of epigenetic effects, and gene containment due to the lack of transgene transmission through pollen. However, broad application of plastid genome engineering in biotechnology has been largely hampered by both the lack of chloroplast transformation systems for major crop plants and the usually low plastid gene expression levels in nongreen tissues such as fruits, tubers, and other storage organs. Here we describe the development of a plastid transformation system for tomato, Lycopersicon esculentum. This is the first report on the generation of fertile transplastomic plants in a food crop with an edible fruit. We show that chromoplasts in the tomato fruit express the transgene to approximately 50% of the expression levels in leaf chloroplasts. Given the generally very high foreign protein accumulation rates that can be achieved in transgenic chloroplasts (>40% of the total soluble protein), this system paves the way to efficient production of edible vaccines, pharmaceuticals, and antibodies in tomato.     

Identification of cis-elements conferring high levels of gene expression in non-green plastids

Although our knowledge about the mechanisms of gene expression in chloroplasts has increased substantially over the past decades, next to nothing is known about the signals and factors that govern expression of the plastid genome in non-green tissues. Here we report the development of a quantitative method suitable for determining the activity of cis-acting elements for gene expression in non-green plastids. The in vivo assay is based on stable transformation of the plastid genome and the discovery that root length upon seedling growth in the presence of the plastid translational inhibitor kanamycin is directly proportional to the expression strength of the resistance gene nptII in transgenic tobacco plastids. By testing various combinations of promoters and translation initiation signals, we have used this experimental system to identify cis-elements that are highly active in non-green plastids. Surprisingly, heterologous expression elements from maize plastids were significantly more efficient in conferring high expression levels in root plastids than homologous expression elements from tobacco. Our work has established a quantitative method for characterization of gene expression in non-green plastid types, and has led to identification of cis-elements for efficient plastid transgene expression in non-green tissues, which are valuable tools for future transplastomic studies in basic and applied research.     

叶绿体转化及其用于疫苗表达研究的最新进展

随着植物转基因研究的不断深入,核基因组转化的转基因沉默现象严重影响了基因工程的应用效果。植物叶绿体遗传转化以叶绿体基因组为平台对植物进行遗传操作,外源基因定点整合及母性遗传特性能较好地解决"顺式失活"和"位置效应"等类的基因沉默问题和转基因逃逸等安全问题,成为植物基因工程发展的新方向,在工业、农业及医药生物领域发挥了重要作用,也为生产廉价、安全的植物疫苗提供了新思路。本文在简要介绍叶绿体转化的原理、转化方法与优势的基础上,重点综述了近年来通过该技术表达的一些重要的病毒抗原和细菌抗原。最后,对叶绿体转化技术在表达外源基因方面存在的问题进行分析。未来随着叶绿体基因表达、调控机制研究的逐渐深入及相关技术体系的日臻完善,叶绿体转化有望成为疫苗生产的生力军。     

Synthetic biology in plastids

Plastids (chloroplasts) harbor a small gene‐dense genome that is amenable to genetic manipulation by transformation. During 1 billion years of evolution from the cyanobacterial endosymbiont to present‐day chloroplasts, the plastid genome has undergone a dramatic size reduction, mainly as a result of gene losses and the large‐scale transfer of genes to the nuclear genome. Thus the plastid genome can be regarded as a naturally evolved miniature genome, the gradual size reduction and compaction of which has provided a blueprint for the design of minimum genomes. Furthermore, because of the largely prokaryotic genome structure and gene expression machinery, the high transgene expression levels attainable in transgenic chloroplasts and the very low production costs in plant systems, the chloroplast lends itself to synthetic biology applications that are directed towards the efficient synthesis of green chemicals, biopharmaceuticals and other metabolites of commercial interest. This review describes recent progress with the engineering of plastid genomes with large constructs of foreign or synthetic DNA, and highlights the potential of the chloroplast as a model system in bottom‐up and top‐down synthetic biology approaches.     

Nuclear and plastid genetic engineering of plants: Comparison of opportunities and challenges

Plant genetic engineering is one of the key technologies for crop improvement as well as an emerging approach for producing recombinant proteins in plants. Both plant nuclear and plastid genomes can be genetically modified, yet fundamental functional differences between the eukaryotic genome of the plant cell nucleus and the prokaryotic-like genome of the plastid will have an impact on key characteristics of the resulting transgenic organism. So, which genome, nuclear or plastid, to transform for the desired transgenic phenotype? In this review we compare the advantages and drawbacks of engineering plant nuclear and plastid genomes to generate transgenic plants with the traits of interest, and evaluate the pros and cons of their use for different biotechnology and basic research applications, ranging from generation of commercial crops with valuable new phenotypes to ‘bioreactor’ plants for large-scale production of recombinant proteins to research model plants expressing various reporter proteins.     

质体基因工程中选择标记基因研究进展

与细胞核基因工程相比,质体基因工程能更安全、精确和高效地对外源基因进行表达,作为下一代转基因技术已广泛用于基础研究和生物技术应用领域。与细胞核基因工程一样,质体基因工程中也需要合适的选择标记基因用于转化子的筛选和同质化,但基于质体基因组的多拷贝性和母系遗传特点,转化子的同质化需要一个长期的筛选过程,这就决定了质体基因工程中选择标记基因的选择标准将不同于细胞核基因工程中广泛使用的现行标准。目前,质体基因工程的遗传转化操作中使用较多的是抗生素选择标记基因,出于安全性考虑,需要找到可替换、安全的选择标记基因或有效的标记基因删除方法。本文在对质体基因工程研究的相关文献分析基础之上,对主要使用的选择标记基因及其删除体系进行了综述,并对比了其优缺点,同时探讨了质体基因工程中所使用的报告基因,以期为现有选择标记基因及其删除体系的改进和开发提供一定参考,进一步推动质体基因工程,尤其是单子叶植物质体基因工程的发展。     

Efficient and Stable Transformation of Lactuca sativa L. cv. Cisco (lettuce) Plastids

Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show that lettuce leaf chloroplasts can express transgene-encoded GFP to ~36% of the total soluble protein. All transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the chloroplast genome. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.     

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.     

烟草质体分裂相关基因NtFtsZ1和NtFtsZ2对叶绿体分裂和形态的影响

质体作为植物细胞中一类重要的细胞器,控制其分裂的分子机制一直都不清楚。最近的研究表明,植物细胞中与原核细胞分裂基因fisZ类似的同源基因控制着质体的分裂过程。通过正反义转化分析了两个烟草的ftsZ基因（NtFtsZ1和NtFtsZ2)在转基因烟草中的功能。二的反义表达并未对转化烟草细胞中叶绿体的分裂和形态产生明显影响,但二过表达转化植株中叶绿体的数目和形态都发生了明显的变化,在某些转化植株的叶肉细胞中甚至只有1－2个巨大的叶绿体存在。对不同转化植株的电镜观察和叶绿素含量分析认为,NtFtsZs基因可能对叶绿体的正常发育和功能没有影响,叶绿体形态的变化是对其数目减少的一种补偿。正反义转化植株中叶绿体的不同表型暗示高等植物中同一家族的ftsZ基因可能在控制质体分裂方面具有相同的功能。同时,过表达植株中叶绿体形态的变化被认为是高等植物的FtsZ质体骨架功能的体现。     

Minimizing the unpredictability of transgene expression in plants: the role of genetic insulators

The genetic transformation of plants has become a necessary tool for fundamental plant biology research, as well as the generation of engineered plants exhibiting improved agronomic and industrial traits. However, this technology is significantly hindered by the fact that transgene expression is often highly variable amongst independent transgenic lines. Two of the major contributing factors to this type of inconsistency are inappropriate enhancer-promoter interactions and chromosomal position effects, which frequently result in mis-expression or silencing of the transgene, respectively. Since the precise, often tissue-specific, expression of the transgene(s) of interest is often a necessity for the successful generation of transgenic plants, these undesirable side effects have the potential to pose a major challenge for the genetic engineering of these organisms. In this review, we discuss strategies for improving foreign gene expression in plants via the inclusion of enhancer-blocking insulators, which function to impede enhancer-promoter communication, and barrier insulators, which block the spread of heterochromatin, in transgenic constructs. While a complete understanding of these elements remains elusive, recent studies regarding their use in genetically engineered plants indicate that they hold great promise for the improvement of transgene expression, and thus the future of plant biotechnology.     

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.     

Plastid genetic engineering in Solanaceae

Plastid genetic engineering has come of age, becoming today an attractive alternative approach for the expression of foreign genes, as it offers several advantages over nuclear transformants. Significant progress has been made in plastid genetic engineering in tobacco and other Solanaceae plants, through the use of improved regeneration procedures and transformation vectors with efficient promoters and untranslated regions. Many genes encoding for industrially important proteins and vaccines, as well as genes conferring important agronomic traits, have been stably integrated and expressed in the plastid genome. Despite these advances, it remains a challenge to achieve marked levels of plastid transgene expression in non-green tissues. In this review, we summarize the basic requirements of plastid genetic engineering and discuss the current status, limitations, and the potential of plastid transformation for expanding future studies relating to Solanaceae plants.     

Transgene-induced pleiotropic effects in transplastomic plants

Since the first demonstration of stable transgene integration in the plastid genome (plastome) of higher plants, plastid transformation has been used for a wide range of purposes, including basic studies as well as biotechnological applications, showing that transplastomic plants are an effective system to produce recombinant proteins. Compared to nuclear transformation, the main advantages of this technology are the high and stable production level of proteins as well as the natural containment of transgenes. To date, more than 100 transgenes have been successfully expressed in plant chloroplasts. In some cases, however, unintended pleiotropic effects on plant growth and physiology were shown in transplastomic plants. In this paper, we review such effects and discuss some of the technologies developed to overcome them.