植物质体基因工程:新的优化策略及应用

植物质体转化技术通过同源重组实现定点整合,与细胞核基因工程相比,使外源基因表达更为精确、安全和高效。该技术在基础研究中为叶绿体功能基因组研究提供了有效手段,同时在应用方面为外源基因表达提供了理想的平台,已成为植物遗传育种的一种新策略。本文总结了近年来质体基因工程在转化体系的建立和优化上的新思路,着重阐述了利用质体转化技术在遗传育种中提高作物抗性、改良品质等应用领域的最新研究进展。克服质体转化技术在作物遗传育种中面临的难题,必将为作物育种的发展带来新的绿色革命。     

质体

质体是植物细胞特有的一类细胞器,藻类的质体类型比较单一,一般只有叶绿体一种,到了被子植物质体的类型才增多,而且被子植物细胞的发育,很大程度表现在质体的分化上。自从本世纪60年代确证质体含有自己的DNA以来,有人将质体看作是细胞内的细胞,这对理解质体有一定意义。在分生组织中质体呈未分化状态,叫做原质体。随着细胞的分化,原质体的结构和功能都发生深刻的变化,并形成各种质体。按照惠特利(Whatley 1978)的意见,质体大体可分     

高等植物的质体基因转化

质体基因转化技术由于其独特的优越性,已开始成为植物基因工程中新的研究热点。本文对质体基因转化技术所面临的几个关键性问题,包括外源基因导入方法、外源基因的整合及表达、质体转化体的有效筛选标记等进行了讨论,同时对质体基因转化的优越性及其应用与发展前景进行了综合评述。     

高等植物的质体基因转化

质体基因转化技术由于其独特的优越性,已开始成为植物基因工程中新的研究热点。本文对质体基因转化技术所面临的几个关键性问题,包括外源基因导入方法、外源基因的整合及表达、质体转化体的有效筛选标记等进行了讨论,同时对质体基因转化的优越性及其应用与发展前景进行了综合评述。     

国外大刊重要论文简介

改变腺苷酸库的结果导致转基因植物 ①淀粉含量和产量增加RegiererB ,FernieAR ,SpringerF ,Perez_MelisA ,LeisseA ,KoehlK ,WillmitzerL ,GeigenbergerP ,KossmannJNatureBiotechnology ,2 0 0 2 ,2 0 :1 2 5 6～ 1 2 60淀粉及其精细加工产品作为人类日常饮食的重要来源。植物合成淀粉主要在质体中进行。研究发现 :增加质体腺苷酸的供应可以有效提高淀粉含量。腺苷酸激酶 (EC2 .7.4.3)主要催化ATP和AMP向ADP的转化 ,因此可以有效地调控质体腺苷酸的水平。采用反义RNA对该酶基因的下游调控对质体各种腺苷酸的量均有影响。借…     

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。     

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ftsZ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.二者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,但二者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FtsZ质体骨架功能的体现.     

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。     

利用植物质体转基因技术高效表达抗人源白介素6单链抗体

白介素6 (interleukin-6, IL-6)是与包括癌症在内的多种疾病相关的一种多功能细胞因子,免疫疗法利用抗人源IL-6抗体来治疗相关疾病取得了很好的治疗效果。植物作为生物反应器可以有效降低药用蛋白的生产成本,同时积累功能蛋白。通过基因枪轰击和再生筛选,得到了2个在烟草质体中表达了鼠源抗人源IL-6单链抗体(single chain variable fragment, scFv)的独立株系,并用Southern blotting鉴定了质体转化烟草的同质化状态。抗人源IL-6 scFv基因在质体转基因烟草中成功转录和翻译,功能性抗人源IL-6 scFv在质体转基因烟草叶片中的含量占到总可溶性蛋白的1%,达到41 mg/kg鲜重。另外,质体转基因烟草的表型与野生型烟草相比并没有显著差异,它们具有相似的生长速率、成熟植株的株高以及果荚数目。抗人源IL-6 scFv的高表达量也表明了利用质体转基因植物低成本生产scFv的潜力。     

叶绿体基因表达调控的研究进展

在植物生长和发育过程中,叶绿体基因表达包括两个方面：一方面在光照条件下质体转化成为叶绿体,一系列质体基因激活,表达叶绿体所必需的基因产物;另一方面叶绿体中由于环境条件变化引起基因表达的改变。叶绿体基因表达调控的机制主要包括转录水平、转录后的mRNA加工、mRNA稳定性和翻译水平的调节,并且在各个步骤中多种核编码蛋白因子的参与也起着重要的作用。     

Production of foreign proteins using plastid transformation

In the past decades, the progress made in plant biotechnology has made possible the use of plants as a novel production platform for a wide range of molecules. In this context, the transformation of the plastid genome has given a huge boost to prove that plants are a promising system to produce recombinant proteins. In this review, we provide a background on plastid genetics and on the principles of this technology in higher plants. Further, we discuss the most recent biotechnological applications of plastid transformation for the production of enzymes, therapeutic proteins, antibiotics, and proteins with immunological properties. We also discuss the potential of plastid biotechnology and the novel tools developed to overcome some limitations of chloroplast transformation.     

Novel approach in plastid transformation

Engineering the nuclear genome of plants is perceived to be associated with problems regarding biosafety and the stability of expression of the transgene. Alternative transformation strategies using the genomic outfit of the plastid promise to be more successful in this respect. Over the past few years progress has been made in screening procedures, and plastid transformation technology has allowed function to be assigned to open reading frames, massive expression of insecticidal agents and proteins involved in herbicide resistance, and the accumulation of biopolymers. Recently, the design of a novel femtoinjection technique that allows injection into chloroplasts has provided the opportunity to further manipulate and understand chloroplastic gene expression.     

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.     

Protein expression in plastids

The genome of the plastid has generated much interest as a target for plant transformation. The characteristics of plastid transgenes both reflect the prokaryotic origin of plastid organelles and provide a unique set of features that are currently lacking in genes introduced into the plant nucleus. Recent progress has been made in understanding plastid expression of recombinant proteins.     

Plant plastid engineering

Genetic material in plants is distributed into nucleus, plastids and mitochondria. Plastid has a central role of carrying out photosynthesis in plant cells. Plastid transformation is becoming more popular and an alternative to nuclear gene transformation because of various advantages like high protein levels, the feasibility of expressing multiple proteins from polycistronic mRNAs, and gene containment through the lack of pollen transmission. Recently, much progress in plastid engineering has been made. In addition to model plant tobacco, many transplastomic crop plants have been generated which possess higher resistance to biotic and abiotic stresses and molecular pharming. In this mini review, we will discuss the features of the plastid DNA and advantages of plastid transformation. We will also present some examples of transplastomic plants developed so far through plastid engineering, and the various applications of plastid transformation.     

Plastid transformation in the monocotyledonous cereal crop, rice (Oryza sativa) and transmission of transgenes to their progeny

The plastid transformation approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering, transgene containment, and a lack of gene silencing and position effects. The extension of plastid transformation technology to monocotyledonous cereal crops, including rice, bears great promise for the improvement of agronomic traits, and the efficient production of pharmaceutical or nutritional enhancement. Here, we report a promising step towards stable plastid transformation in rice. We produced fertile transplastomic rice plants and demonstrated transmission of the plastid-expressed green fluorescent protein (GFP) and aminoglycoside 3'-adenylyltransferase genes to the progeny of these plants. Transgenic chloroplasts were determined to have stably expressed the GFP, which was confirmed by both confocal microscopy and Western blot analyses. Although the produced rice plastid transformants were found to be heteroplastomic, and the transformation efficiency requires further improvement, this study has established a variety of parameters for the use of plastid transformation technology in cereal crops.     

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.     

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.