转基因技术在兰花上的应用(综述)

本文综述兰花转基因技术的应用、转入的目的基因和转基因组织筛选与检测的研究进展。     

兰花的功能基因

对兰花的花发育相关基因、花色、激素及代谢途径相关功能基因的研究进展和前景作了介绍和讨论。     

重要观赏兰科植物的分子生物学研究进展

兰科植物是开花植物中最大的家族之一,分子标记技术应用于兰科植物的分类鉴定和品种鉴别,为兰花的分类提供了分子水平的证据,也为兰花保护策略和措施的制定提供了理论基础。兰科植物表现有高度特异的形态、结构和生理特性,是研究花着色机理和子房发育的理想对象。兰花离体培养开花系统的建立,可以用来探明兰花从营养生长向生殖生长的转变机制,是研究花的分化和发育的理想材料。兰花具有特异的查尔酮合成酶（CHS）基因和二氢叶酸还原酶（DFR）基因等控制花色素的合成,DOH1基因控制石斛兰花芽的形成和提早开花,PHAL.039基因和ACC合成酶基因在蝴蝶兰授粉后的子房发育中起着重要的调控作用,这些特异基因的分离和克隆为兰花花的分化、发育及着色机制提供了分子基础。蝴蝶兰属、大花蕙兰(Cymbidium hybridium)、石斛兰属、文心兰属、五唇兰属和万代兰属等兰科植物都有转基因的研究报道,主要以原球茎为材料采用基因枪或农杆菌法转化,部分研究获得了转化植株。     

重要观赏兰科植物的分子生物学研究进展

兰科植物是开花植物中最大的家族之一,分子标记技术应用于兰科植物的分类鉴定和品种鉴别,为兰花的分类提供了分子水平的证据,也为兰花保护策略和措施的制定提供了理论基础.兰科植物表现有高度特异的形态、结构和生理特性,是研究花着色机理和子房发育的理想对象.兰花离体培养开花系统的建立,可以用来探明兰花从营养生长向生殖生长的转变机制,是研究花的分化和发育的理想材料.兰花具有特异的查尔酮合成酶(CHS)基因和二氢叶酸还原酶(DFR)基因等控制花色素的合成,DOHI基因控制石斛兰花芽的形成和提早开花,PHAL039基因和ACC合成酶基因在蝴蝶兰授粉后的子房发育中起着重要的调控作用,这些特异基因的分离和克隆为兰花花的分化、发育及着色机制提供了分子基础.蝴蝶兰属、大花蕙兰(Cymbidium hybrdium)、石斛兰属、文心兰属、五唇兰属和万代兰属等兰科植物都有转基因的研究报道,主要以原球茎为材料采用基因枪或农杆菌法转化,部分研究获得了转化植株.     

转基因抗虫烟草研究进展

烟草为模式植物,也是外源杀虫基因最早转化成功的植物。文章从转Bt内毒素基因,植物凝集素GNA,Plec,AHA基因,蛋白酶抑制剂PIⅠ,PIⅡ,MTI,SKTI基因,昆虫特异性神经毒素基因,几丁质酶基因,畸形细胞分泌蛋白基因以及双抗虫基因等方面综述了转基因抗虫烟草的抗虫性、转基因抗虫烟草的经济性状等,展望了转基因抗虫烟草的研究和应用前景,以期对烟草害虫的治理尤其是对其他转基因抗虫作物的培育和研究有借鉴作用。     

外源基因在转基因植物中的表达与稳定性

外源基因能否在转基因植物中稳定表达对转基因植物的应用前景有重要的影响。影响外源基因稳定表达的因素有多种,其中包括遗传和环境因素。在某些转基因植物中,外源基因表达是受发育调控,本文主要讨论了转基因沉默及发育时期和环境条件对源基因的表达及稳定性的影响,并进一步探讨了对策。     

代谢转基因植物的研究现状与展望

代谢转基因是通过基因工程技术对细胞内的代谢途径进行遗传修饰,进而完成细胞特性改造。代谢修饰转基因植物是一个极具商业前景的领域,在医药、环境、农业等方面已有许多成功应用的实例。综合调控代谢的基因工程策略,讨论了代谢转基因植物的研究现状,我国农业生产中存在的主要问题和代谢转基因技术对我国农业发展的意义和前景。     

转基因动物的检测方法

转基因动物的研究目前尚处于实验室研究阶段,获得转基因动物难度大,检测转基因比较困难,从染色体和基因水平、转录、翻译、整体表型等不同角度介绍了转基因动物的不同检测手段和方法,并对各水平存在的问题及应用前景作了阐述。     

兰花的基因操作技术

美国洛克菲勒大学植物分子生物学研究所教授Nam-Hai Chua等培育出转基因兰花,这在世界上尚属首例.在京都召开的日本农艺化学会的特别讲演中公布了这项成果.在兰花中导入荧光素酶基因作为标记,用光检测装置确认了表达.启动子使用了花菜花叶病毒35S. 同时在新加坡的一家研究所任职的Chua与Wang G.Y.共同开发在体外培养兰花的技术.150天后90%的植株开花.常规培育的兰花从出苗到开花需要几个月～3年的时间.用生物技术培育的兰花大幅度缩短了开花时间.     

抗虫转基因植物的研究进展及前景

虫害对农业生产的危害日益严重。目前对害虫的防治主要依赖于化学药物,但化学药物的副作用不容忽视。利用植物基因工程获得抗虫转基因植物是更具前景的途径。目前主要利用的抗虫基因是苏云金杆菌的δ－内毒素基因和植物来源的抗虫基因（如蛋白酶抑制剂基因、淀粉酶抑制剂基因、凝集素基因等）,各种抗虫基因在转基因应用中各有其优缺点,如苏云金杆菌δ－内毒素基因在植物中表达水平低。随着抗虫转基因植物在大田中的应用,昆虫的抗性或适应性问题也随之产生,这将是转基因植物发展道路上又一挑战。     

The firefly luciferase gene as a non-invasive reporter for Dendrobium transformation

Here a screening method is described for transformed tissues and transgenic plants of Dendrobium (Orchidaceae) using the firefly luciferase gene ( luc ) as a combined marker/reporter gene. Protocorm-likebodies (PLB) were bombarded with tungsten particles (1.3 µm) coated with plasmids carrying a 35S-luc chimeric gene. Three weeks after bombardment 1 mM luciferin was added to the tissues and transformed cells were identified by virtue of their bioluminescence as monitored by low-light video microscopy in combination with a real-time photon imaging technique. Transformed tissues were excised, allowed to proliferate, and then subjected to a second round of screening. After three rounds of growth and screening, transformed Dendrobium tissues expressing luciferase were used to generate transgenic plants. Southern blot analysis of several transgenic lines confirmed the integration of the luciferase gene into the orchid genome. It is thought that this procedure can be used for transformation of not only orchids but other species as well.     

Patterns of reproductive isolation in Mediterranean deceptive orchids

The evolution of reproductive isolation is of central interest in evolutionary biology. In plants, this is typically achieved by a combination of pre- and postpollination mechanisms that prevent, or limit, the amount of interspecific gene flow. Here, we investigated and compared two ecologically defined groups of Mediterranean orchids that differ in pollination biology and pollinator specificity: sexually deceptive orchids versus food-deceptive orchids. We used experimental crosses to assess the strength of postmating prezygotic, and postzygotic reproductive isolation, and a phylogenetic framework to determine their relative rates of evolution. We found quantitative and qualitative differences between the two groups. Food-deceptive orchids have weak premating isolation but strong postmating isolation, whereas the converse situation characterizes sexually deceptive orchids. Only postzygotic reproductive isolation among food-deceptive orchids was found to evolve in a clock-like manner. Comparison of evolutionary rates, within a common interval of genetic distance, showed that the contribution of postmating barriers was more relevant in the food-deceptive species than in the sexually deceptive species. Asymmetry in prezygotic isolation was found among food-deceptive species. Our results indicate that postmating barriers are most important for reproductive isolation in food-deceptive orchids, whereas premating barriers are most important in sexually deceptive orchids. The different rate of evolution of reproductive isolation and the relative strength of pre- and postmating barriers may have implication for speciation processes in the two orchid groups.     

Low population genetic differentiation in the Orchidaceae: implications for the diversification of the family

A leading hypothesis for the immense diversity of the Orchidaceae is that skewed mating success and small, disjunct populations lead to strong genetic drift and switches between adaptive peaks. This mechanism is only possible under conditions of low gene flow that lead to high genetic differentiation among populations. We tested whether orchids typically exhibit high levels of population genetic differentiation by conducting a meta‐analysis to compare mean levels of population genetic differentiation (F_ST) between orchids and other diverse families and between rare and common orchids. Compared with other families, the Orchidaceae is typically characterized by relatively low genetic differentiation among populations (mean F_ST = 0.146) at allozyme loci. Rare terrestrial orchids showed higher population genetic differentiation than common orchids, although this value was still lower than the mean for most plant families. All lines of evidence suggest that orchids are typically characterized by low levels of population genetic differentiation, even in species with naturally disjunct populations. As such, we found no strong evidence that genetic drift in isolated populations has played a major role in the diversification of the Orchidaceae. Further research into the diversification of the family needs to unravel the relative roles of biotic and environmental selective pressures in the speciation of orchids.     

Further advances in orchid mycorrhizal research

Orchid mycorrhizas are mutualistic interactions between fungi and members of the Orchidaceae, the world’s largest plant family. The majority of the world’s orchids are photosynthetic, a small number of species are myco-heterotrophic throughout their lifetime, and recent research indicates a third mode (mixotrophy) whereby green orchids supplement their photosynthetically fixed carbon with carbon derived from their mycorrhizal fungus. Molecular identification studies of orchid-associated fungi indicate a wide range of fungi might be orchid mycobionts, show common fungal taxa across the globe and support the view that some orchids have specific fungal interactions. Confirmation of mycorrhizal status requires isolation of the fungi and restoration of functional mycorrhizas. New methods may now be used to store orchid-associated fungi and store and germinate seed, leading to more efficient culture of orchid species. However, many orchid mycorrhizas must be synthesised before conservation of these associations can be attempted in the field. Further gene expression studies of orchid mycorrhizas are needed to better understand the establishment and maintenance of the interaction. These data will add to efforts to conserve this diverse and valuable association.     

Transformation of Dendrobium orchid using particle bombardment of protocorms

Transformed dendrobium orchids (Dendrobium x Jaquelyn Thomas hybrids) were recovered from protocorms bombarded by particles coated with the plasmid pGA482GG/cpPRV4, which contains the plant expressible Nos-NPT II and papaya ringspot virus (PRV) coat protein (CP) genes. Approximately 280 protocorms from four crosses were bombarded and potentially transformed tissues were identified by growth and green color on half-strength Murashige and Skoog medium supplemented with 2% sucrose and 50–100 mg 1^–1 kanamycin sulfate. Kanamycin concentrations that prevented growth of nontransformed tissues could not be used for long-term selection because such levels suppressed the regeneration of potentially transformed tissues. PCR and restriction analysis 21 months after treatment found 13 of 13 plants from two crosses, which appeared kanamycin-tolerant, to contain the Nos-NPT II gene, while only one of these plants carried the vector-linked PRV CP-gene. These results support use of particle bombardment for transformation of this important ornamental monocot.     

兰科植物内生细菌物种多样性及其促生机理研究进展

内生细菌影响兰科植物菌根形成和共生关系的稳定性,在兰科植物的生活史中起着重要作用。内生细菌通过分泌植物激素、采用光合作用、生物固氮或促进矿质营养的循环以及产生铁载体、合成其他活性物质等途径来促进兰科植物生长发育。综述了兰科植物内生细菌物种多样性的研究方法及其对兰科植物的促生机理,基于兰科植物与共生微生物的密切关系,认为内生细菌间、内生细菌与兰科植物菌根真菌间的互作是揭示兰科植物与内生细菌互作机理的重要方向。     

On the value of nuclear and mitochondrial gene sequences for reconstructing the phylogeny of vanilloid orchids (Vanilloideae, Orchidaceae)

Background and Aims

Most molecular phylogenetic studies of Orchidaceae have relied heavily on DNA sequences from the plastid genome. Nuclear and mitochondrial loci have only been superficially examined for their systematic value. Since 40% of the genera within Vanilloideae are achlorophyllous mycoheterotrophs, this is an ideal group of orchids in which to evaluate non-plastid gene sequences.

Methods

Phylogenetic reconstructions for Vanilloideae were produced using independent and combined data from the nuclear 18S, 5·8S and 26S rDNA genes and the mitochondrial atpA gene and nad1b-c intron.

Key Results

These new data indicate placements for genera such as Lecanorchis and Galeola, for which plastid gene sequences have been mostly unavailable. Nuclear and mitochondrial parsimony jackknife trees are congruent with each other and previously published trees based solely on plastid data. Because of high rates of sequence divergence among vanilloid orchids, even the short 5·8S rDNA gene provides impressive levels of resolution and support.

Conclusions

Orchid systematists are encouraged to sequence nuclear and mitochondrial gene regions along with the growing number of plastid loci available.Key words: 26S rDNA, 18S rDNA, 5·8S rDNA, atpA, nad1, orchids, plastid, Vanilla, vanilloid orchids, Vanilloideae