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。     

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。     

丝状真菌基因工程研究进展

本文评述了丝状真菌基因工程的研究进展,内容涉及已被转化成功的９０余种丝状真中类及其所利用的选择标记,比较了几种外源ＤＮＡ进入丝状真菌受体的方法,并较为详细地评述了丝状真菌复制型与整合型转化及其转化子的有性生殖与无性生殖的遗传稳定性,最后,展望了丝状真菌基因工程在农业,工业和医药方面的应用。表明了丝状真菌基因工程具有广阔的应用前景。     

海洋微藻基因工程的选择标记

６种海洋微藻新月菱形藻（Ｎｉｔｚｓｃｈｉａ ｃｌｏｓｔｅｒｉｕｍ Ｅｈｒ．）、牟勒氏角刺藻（Ｃｈａｅｔｏｃｄｒｏｓ ｍｕｅｌｌｅｒｉ Ｌｅｍｍ．）、三角褐指灌（Ｐｈａｅｏｄａｃｔｙｌｕｍ ｔｒｉｃｏｒｎｕｔｕｍ Ｂｏｈｌ．）绿光等鞭金藻８７０１（Ｉｓｏｃｈｒｙｓｉｓ ｇａｌｂａｎａ Ｐａｒｋｅ ８７０１）、亚心形扁藻（Ｐｌａｔｙｍｏｎａｓ ｓｕｂｃｏｒｄｉ－ｆｏｒｍｉｓ（Ｗｉｌｌｅ）Ｈａｚｅｎ）和     

叶绿体遗传转化研究中的选择标记

叶绿体遗传转化研究需要有合适的选择标记作为辅助手段,多种选择标记已经在叶绿体转化中得到应用。本文综述了国内外叶绿体转化研究中主要使用的选择标记,并着重介绍了非抗生素选择标记－甜菜碱醛脱氢酶和选择标记的删除体系。     

杜氏盐藻基因工程选择标记的研究

研究了杜氏盐藻对链霉素,卡那霉素,潮霉素,G418和氯霉素5种常用抗生素的敏感性。结果表明,经4周培养,盐灌对链霉素,卡那霉素,潮霉素和G418不敏感,浓度高达600ug/ml时,也不能抑制盐藻生长,对氯霉素很敏感,60ug/ml即可完全抑制固体和液体培养中盐藻的生长,氯霉素合作为盐藻基因工程的筛选抗生素,CAT基因在其阳性筛选标记基因。     

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.     

无选择标记基因植物转化系统研究进展

在转基因植物中,将选择标记基因去掉,将提高转基因植物的食用安全性和对环境的安全性,更易为广大消费者所接受,也有利于对同一个植物品种进行多次转基因操作。科学工作者已经在建立无选择标记基因转化系统方面作了大量尝试,获得了无标记基因的转基因植物（MFTPs：Marker-Free Transgenic Plants)。本文将这方面的研究进展介绍给大家,以推动植物生物技术产业化进程。     

葡萄基因工程研究进展

植物基因工程技术为培育优良葡萄品种开辟了一条全新而有效的途径。葡萄基因转化受体系统的建立主要包括器官发生途径和胚状体发生途径,建立良好的受体系统是葡萄基因转化成功的关键,遗传转化途径主要有根癌农杆菌介导的遗传转化和基因枪法。概述了迄今国内外葡萄基因工程的研究进展,着重对葡萄基因转化受体系统的建立、转化的方法、转化植株的筛选和检测、影响葡萄基因转化的主要因素等进行了综述,并展望了葡萄基因工程的发展前景。     

丝状真菌基因工程研究进展

本文评述了丝状真菌基因工程的研究进展,内容涉及已被转化成功的９０余种丝状真菌种类及其所利用的选择标记,比较了几种外源ＤＮＡ进入丝状真菌受体的方法,并较为详细地评述了丝状真菌复制型与整合型转化及其转化子的有性生殖与无性生殖的遗传稳定性,最后,展望了丝状真菌基因工程在农业、工业和医药方面的应用。表明了丝状真菌基因工程具有广阔的应用前景。     

Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system

Incorporation of a selectable marker gene during transformation is essential to obtain transformed plastids. However, once transformation is accomplished, having the marker gene becomes undesirable. Here we report on adapting the P1 bacteriophage CRE-lox site-specific recombination system for the elimination of marker genes from the plastid genome. The system was tested by the elimination of a negative selectable marker, codA, which is flanked by two directly oriented lox sites (>codA>). Highly efficient elimination of >codA> was triggered by introduction of a nuclear-encoded plastid-targeted CRE by Agrobacterium transformation or via pollen. Excision of >codA> in tissue culture cells was frequently accompanied by a large deletion of a plastid genome segment which includes the tRNA-ValUAC gene. However, the large deletions were absent when cre was introduced by pollination. Thus pollination is our preferred protocol for the introduction of cre. Removal of the >codA> coding region occurred at a dramatic speed, in striking contrast to the slow and gradual build-up of transgenic copies during plastid transformation. The nuclear cre gene could subsequently be removed by segregation in the seed progeny. The modified CRE-lox system described here will be a highly efficient tool to obtain marker-free transplastomic plants.     

运动发酵单胞菌ZM4、CP4菌株基因工程选择标记的研究

尽管质粒和选择标记的使用作为基因工程最基本的一环而为人们所熟知,但对一些特殊菌种（菌株）或研究很少的菌种（菌株）的基因工程操作来说,质粒和选择标记可能仍然是一个并未完全解决的问题,因而需要不断提高认识、不断改进。运动发酵单胞菌Zymomonasmobilis具有突出的产醇性能,但其多种内源质粒和多种抗性的特点,增加了其基因工程操作时质粒和选择标记选用的难度。本研究在测定四个抗生素即Ap、Cm、Te、Km对典型菌株ZM4、CP4的最低生长抑制浓度的基础上,初步确定了这两个菌株基因工程操作时的四个抗生素使用浓度依次分别为300、100、25、350μg／mL（ZM4）和500、100、25、250μg]mL（CP4）;并进一步通过穿梭载体pZB21、宽宿主载体pBBR1MCS-2和整合载体pBR328-ldhR—cml—ldhL的转化,初步分析和证明了这些选择标记和在相应抗生素浓度下的效果：首先,对每一个选择标记基因来说,前述抗生素浓度是适于携带此选择标记基因的质粒的转化筛选和相应转化子培养的;其次,在前述抗生素浓度下,综合筛选平板阳性率和转化效率、培养物菌体形态异常程度等指标,四个选择标记基因中,以Cm和Tc抗性标记基因效果最好,Km抗性标记基因居中,Ap抗性标记基因最差。这些结果为ZM4、CP4基因工程遗传改造用抗性标记基因、质粒、抗生素的选择及转化系统的完善奠定了基础。     

Generation and characterization of soybean and marker-free tobacco plastid transformants over-expressing a bacterial 4-hydroxyphenylpyruvate dioxygenase which provides strong herbicide tolerance

Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is part of the biosynthetic pathway leading to plastoquinone and vitamin E. This enzyme is also the molecular target of various new bleaching herbicides for which genetically engineered tolerant crops are being developed. We have expressed a sensitive bacterial hppd gene from Pseudomonas fluorescens in plastid transformants of tobacco and soybean and characterized in detail the recombinant lines. HPPD accumulates to approximately 5% of total soluble protein in transgenic chloroplasts of both species. As a result, the soybean and tobacco plastid transformants acquire a strong herbicide tolerance, performing better than nuclear transformants. In contrast, the over-expression of HPPD has no significant impact on the vitamin E content of leaves or seeds, quantitatively or qualitatively. A new strategy is presented and exemplified in tobacco which allows the rapid generation of antibiotic marker-free plastid transformants containing the herbicide tolerance gene only. This work reports, for the first time, the plastome engineering for herbicide tolerance in a major agronomic crop, and a technology leading to marker-free lines for this trait.     

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.     

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.     

Progress in cellular engineering of plants: biochemical and genetic assessment of selectable markers from cultured cells

Summary Recent availability of stable and well characterized selectable markers and ability to combine alien genomes parasexually have contributed to the development of molecular biology in higher plants, including gene expression and genetic manipulation.Several types of biochemical mutants (resistant to inhibitory concentrations of aminoacid(s) or aminoacid analogs as well as deficient for enzyme activity) have recently been isolated and characterized biochemically and genetically. Among them, mutants with alterations in the nitrogen and aminoacid metabolism, or in the activity of alcohol dehydrogenases are being used in the development of more efficient techniques of gene transfer.The manipulation of whole genomes by sexual or somatic cell fusion offers new potential in this field, but refinement of transfer techniques is desirable. The new set of selectable markers obtained through advanced cellular technology, as well as our ability to regenerate plants from manipulated cell lines are expected to play a major role in cellular engineering.     

An efficient and novel plant selectable marker based on organomercurial resistance

A selectable marker gene facilitates the detection of genetically modified plant cells during transformation experiments. So far, these marker genes are almost exclusively of two types, conferring either antibiotic resistance or herbicide tolerance. However, more selectable markers must be developed as additional transgenic traits continue to be incorporated into transgenic plants. Here, we used mercury resistance, conferred by the organomercurial lyase gene, as a selectable marker for transformation. The merB gene fromStreptococcus aureus was modified for plant expression and transferred to a hybrid poplar(Populus alba xPopulus glandulosa), using the stem segment-agrobacteria co-cultivation method. The transformed cells were selected on a callus-inducing medium containing as little as 1 μM methylmercury. Subsequent plant regeneration was done in the presence of methylmercury. Resistance to Hg was stably maintained in mature plants after two years of growth in the nursery. We suggest that this gene could serve as an excellent selectable marker for plant transformation.     

Molecular analysis of the acetolactate synthase gene of Chlamydomonas reinhardtii and development of a genetically engineered gene as a dominant selectable marker for genetic transformation

Genomic and cDNA clones of the acetolactate synthase (ALS) gene of Chlamydomonas reinhardtii have been isolated from a mutant, c85-20 (Hartnett et al., 1987), that is resistant to high concentrations of sulfometuron methyl (SMM) and related sulfonylurea herbicides. Comparison of the ALS gene sequences from the wild-type and the SMM resistant (SMMr) strains revealed two amino acid differences in the mature enzyme, a lysine to threonine change at position 257 (K257T) and a leucine to valine change at position 294 (L294V). Transformation of wild-type C. reinhardtii with the mutant ALS gene produced no transformants with ability to grow in the presence of a minimum toxic concentration of SMM (3 microm). Substitution of the ALS promoter with the promoter of the C. reinhardtii Rubisco small subunit gene (RbcS2) permitted recovery of SMMr colonies. In vitro mutagenesis of the wild-type ALS gene to produce various combinations of mutations (K257T, L294V and W580L) indicated that the K257T mutation was necessary and sufficient to confer the SMMr phenotype. Optimum transformation rates were obtained with two constructs (pJK7 and pRP-ALS) in which all introns in the coding region were present. Rates of transformation with construct pJK7 were approximately 2.5 x 10-4 transformants/cell (i.e. one transformant for each of 4000 initial cells) using electroporation and 8.5 x 10-6 transformants/cell using the glass bead vortexing method. These results suggest that pJK7 and pRP-ALS can serve as important additional dominant selectable markers for the genetic transformation of C. reinhardtii.     

Characterization of a higher plant herbicide-resistant phytoene desaturase and its use as a selectable marker

Three natural somatic mutations at codon 304 of the phytoene desaturase gene (pds) of Hydrilla verticillata (L. f. Royle) have been reported to provide resistance to the herbicide fluridone. We substituted the arginine 304 present in the wild-type H. verticillata phytoene desaturase (PDS) with all 19 other natural amino acids and tested PDS against fluridone. In in vitro assays, the threonine (Thr), cysteine (Cys), alanine (Ala) and glutamine (Gln) mutations imparted the highest resistance to fluridone. Thr, the three natural mutations [Cys, serine (Ser), histidine (His)] and the wild-type PDS protein were tested in vitro against seven inhibitors of PDS representing several classes of herbicides. These mutations conferred cross-resistance to norflurazon and overall negative cross-resistance to beflubutamid, picolinafen and diflufenican. The T3 generation of transgenic Arabidopsis thaliana plants harbouring the four selected mutations and wild-type pds had similar patterns of cross-resistance to the herbicides as observed in the in vitro assays. The Thr304 Hydrilla pds mutant proved to be an excellent marker for the selection of transgenic plants. Seedlings harbouring Thr304 pds had a maximum resistance to sensitivity (R/S) ratio of 57 and 14 times higher than that of the wild-type for treatments with norflurazon and fluridone, respectively. These plants exhibited normal growth and development, even after long-term exposure to herbicide. As Thr304 pds is of plant origin, it could become more acceptable than other selectable markers for use in genetically modified food.