激光微束在植物外源基因转化上的应用

本文概述了近年来激光微束在植物外源基因转化上的最新进展,激光微束转化法的关键技术及应用。并对其今后的应用前景作了展望。     

Infrared picosecond laser for perforation of single plant cells

The functional analysis of plant cells at the cellular and subcellular levels requires novel technologies for the directed manipulation of individual cells. In this report, we demonstrate the use of an infrared (1,064 nm) picosecond laser for the perforation of tobacco cell protoplasts. A single pulse was sufficient to perforate the plasma membrane enabling the uptake of dye from the surrounding medium into the cytosol. Moreover, the procedure was shown to be suitable for the efficient delivery of DNA expression constructs to the nucleus, as demonstrated by the subsequent expression and correct targeting of a recombinant fluorescent protein. Single cell perforation using this novel optoporation method shows that isolated plant cells can be permeabilized without direct manipulation. This is a valuable procedure for cell-specific applications, particularly where the import of specific molecules into plant cells is required for functional analysis.     

Gene transfer by electroporation

Electroporation of cells in the presence of DNA is widely used for the introduction of transgenes either stably or transiently into bacterial, fungal, animal, and plant cells. A review of the literature shows that electroporation parameters are often reported in an incomplete or incorrect manner, forcing researchers to rely too much on a purely empirical trial and error approach. The goal of this article is to provide the reader with an understanding of electrical circuits used in electroporation experiments as well as physical and biological aspects of the electroporation process itself. Further, a simple paradigm is provided which unites all electroporation parameters. This article should be particularly useful to those new to the technique.     

Mitotic reversion in prophase of PTK1 cells induced by argon laser microirradiation

In this paper, we report the effects of laser microirradiation of prophase nucleoli and mitotic chromosomes in cells of female rat kangaroo kidney epithelial cell line PTK₁. When the laser power delivered to sample surface was 90–190 mW, irradiation of one of the two nucleoli in the prophase cell did not inhibit the mitotic progress, but resulted in the loss of the irradiated nucleolus in daughter cells. When the laser power was increased to 360–420 mW, either irradiation of the nucleolus or chromosome in midprophase caused a blockage of mitosis at terminal midprophase. The irradiated cells returned morphologically to early prophase. No mitotic reversion occurred in the case of irradiation of chromosomes at late prophase, prometaphase, metaphase, and anaphase. Irradiation of the cytoplasm in prophase cells caused a 50–70 min mitotic delay at prophase. However, the irradiated cells underwent successive mitotic divisions. The mechanism of laser-induced mitotic prophase reversion is discussed.     

高等植物根细胞高亲和性吸收钾的机制

K^ 是高等植物所必需的大量元素,它在植物的膨压调节、电荷平衡、叶片运动和蛋白质合成中都具有重要的作用。高等植物根细胞吸收K^ 通过高亲和性K^ 吸收系统和低亲和性K^ 吸收系统和低亲和性K^ 吸收系统两条途径。高新和性K^ 吸收系统,是在微摩尔浓度的外界K^ 水平时起作用,K^ 的吸收必须消耗能量。近年来,随着分子生物学技术和电生理技术的飞速发展,植物根细胞吸收K^ 的机制取得了较大进展。本文对高等植物根细胞高亲和性吸收K^ 的机制的研究进展进行了综述。     

Effects of seeding rate and plant density on sclerotinia stem rot incidence in canola

Abstract

Sclerotinia stem rot (Sclerotinia sclerotiorum Lib. deBary) affects canola wherever it is grown. Seeding rates, which are believed to affect the microclimate beneath the canopy, were evaluated for their impact on sclerotinia stem rot incidence. A study with five seeding rates (2.2 kg/ha, 3.3 kg/ha, 6.7 kg/ha, 13.3 kg/ha and 20.0 kg/ha) and four canola cultivars chosen for their variation in canopy structure and lodging resistance was conducted in Carman, Manitoba, Canada, in 2001 to 2003. A significant relationship between sclerotinia stem rot disease incidence (DI) and seeding rate was found. With an increase in seeding rate, the DI was significantly increased in the mean of the canola cultivars, and individually, only in the lodging-prone cultivar AC Excel. Lodging significantly increased for all cultivars when seeding rates exceeded the standard 6.7 kg/ha. Multiple regression analysis revealed that both plant density and lodging explain the majority of the variation in DI. Both plant density and lodging resistance varied in having a larger influence on DI depending on the year and cultivar analysed. Our results indicate that increasing seeding rate does modify the microenvironment and increases the potential for lodging, which may be responsible for plant-to-plant spread of this disease.     

Physiological Effects of Rapeseed Transformation with the <Emphasis Type="Italic">acb</Emphasis> Gene as Affected by the Genetic Vector Structure

Rapeseed (Brassica napus L.) plants transformed with the acb gene encoding acyl-CoA-binding protein from Arabidopsis thaliana were used to demonstrate the physiological effects of transformation as related to the genetic vector structure. The opportunities and limitations of the genetic engineering technologies in the studies of particular proteins are under discussion.     

植物原生质体在分子细胞生物学研究中的应用

植物原生质体是去除了细胞壁的裸露细胞,其具有细胞全能性,现广泛应用于植物分子细胞生物学的研究中,可以大大缩减实验周期,并有助于得到体内实验的实时检测数据。该文除了介绍植物原生质体的提取和纯化方法外,还对国内外利用各种植物的原生质体进行细胞瞬时转化、亚细胞定位、细胞融合和大分子复合物相互作用等试验进行了总结和讨论。植物原生质体还可用于基因表达模式的实时检测,并作为生物反应器的受体细胞进行代谢物的体外生产。此外,还对当前该技术所面临的瓶颈进行了分析,为植物原生质体在分子细胞生物学领域的应用提供帮助,为技术的优化和推广提供参考。     

Ultraviolet microbeam irradiations of spindle fibres in crane-fly spermatocytes and newt epithelial cells: Resolution of previously conflicting observations

Summary In order to resolve apparent differences in reported experiments, we directly compared the effects of ultraviolet (UV) microbeam irradiations on the behaviour of spindle fibres in newt epithelial cells and crane-fly spermatocytes, using the same apparatus for both cell types. This work represents the first time that irradiated crane-fly spermatocytes have been followed using a high-NA objective and video-enhancement of images. In both cell types, irradiation of a kinetochore fibre in metaphase produced an area of reduced birefringence (ARB), known to be devoid of spindle microtubules (MTs). Subsequently the kinetochore-ward edge of the ARB moved poleward with average velocities of 0.5 m/min (n=20) in spermatocytes and 1.1 m/min (n=6) in epithelial cells. The poleward edge of the ARB rapidly disappeared when viewed using a ×100, high-NA objective but generally remained visible when viewed with a ×32, low-NA objective; this difference suggests that MTs poleward from the ARB disperse vertically out of the narrow depth of field of the ×100 objective but that many remain encompassed by that of the ×32 objective. The primary difference in response between the two cell types was in the behaviour of the spindle poles after an ARB formed. In spermatocytes the spindle maintained its original length whereas in epithelial cells the pole on the irradiated side very soon moved towards the chromosomes, after which the other pole did the same and a much shortened functional metaphase spindle was formed.     

水稻胚乳特异性启动子Gt1的克隆及其在玉米中的功能验证

以水稻基因组DNA为模板,用PCR方法克隆了水稻谷蛋白基因G t1的启动子序列,并将其构建到带有绿色荧光蛋白基因(gfp)的植物表达载体上,用微束激光穿刺法转化玉米的受体组织,通过外源基因瞬时表达的方法验证了该启动子的功能,结果表明在胚乳中有较强的表达,而在其它组织中表达很弱;证明了水稻谷蛋白基因G t1的启动子在不同物种玉米中同样具有胚乳特异表达的功能。为这一胚乳特异启动子的广泛利用提供了理论依据。     

High molecular weight protein detected in higher plant cells by antibodies against dynein is associated with vesicular organelles including Golgi apparatus

The cytoplasmic dynein is a multisubunit complex driving organelles along microtubules to their minus-end. We used antibodies against two functional domains (motor and microtubule-binding) of one of principal components of the complex—dynein heavy chain of slime mould Dictyostelium discoideum—to test root meristem cells of wheat Triticum aestivum. The antibodies reacted with a high molecular weight protein (>500 kDa) in the total cell extract and the band recognized by the antibodies in plant extracts had a lower electrophoretic mobility than the high molecular weight band of mammalian dynein. Antibodies coupled to protein A-Sepharose precipitated the high molecular weight protein from the purified cell extracts. Immunocytochemical analysis demonstrated that the antigen recognized by antibodies against dynein heavy chains is associated with the vesicles whose localization depends on the cell cycle stage. The antigen-positive vesicles were localized to the perinuclear region in interphase and early prophase, to the spindle periphery and to spindle pole region during mitosis, and to the interzonal region in the period of fragmoplast and cell plate formation. Some antigen-positive vesicles also reacted with antibodies against Golgi protein markers. The obtained data indicate that higher plant cells contain a high molecular weight protein interacting with antibodies against the motor and microtubules-binding domains of Dictyostelium dynein heavy chain. The revealed antigen was associated with the vesicular structures in the cytoplasm including the Golgi apparatus.     

Preparatory studies for the use of plant protoplasts in space research

An experiment using plant protoplasts has been accepted for the IML-1 mission to be flown on a space shuttle in 1991. Preparatory experiments include studies of cell wall formation, cell division, the effect of simulated weightlessness using fast and slow rotating clinostats, and the development and testing of hardware for the IML-1 mission. After 24 h at 25°C, protoplasts isolated from hypocotyls or leaves of rapeseed seedlings, or from carrot suspension cells, show 60, 20 and 15% cell wall formation, respectively. The time course of formation of the cell wall and cell division could be delayed by treatment at low temperatures or immobilization in alginate or agarose. This aspect is of importance in connection with problems of late access to the space shuttle before launch. At 4°C only 18% of the rapeseed hypocotyl protoplasts had formed cell walls after 24 h. Protoplasts immobilised in agarose or alginate gradually regain their cell division capacity and after 72 h the frequencies are 51 and 26%, respectively, compared to non-immobilised control protoplasts. A significant decrease in cell division activity is observed after rotation for 6 h on the slow clinostat. A similar effect is not observed on the fast clinostat. Protoplasts, cultured in the specially designed plant chamber for up to 14 days established cell aggregates which have further developed into plants.     

Studies on plant regeneration from cotyledonary protoplasts in Brassica campestris

Protoplasts isolated from both 7-day-old light-grown and 4-day-old dark/dim light-grown cotyledons of four Brassica campestris varieties (Arlo, Sonja, Bunyip and Wonk Bok) were cultured in three liquid media: modified K8P, modified MS and modified Pelletier's B to compare the capacities for cell division and plant regeneration. Following cell wall regeneration the cultured protoplasts from dark/dim light-grown cotyledons of four varieties showed rapid division and high frequency of cell division compared with those isolated from light-grown cotyledons. The frequencies of cell division were significantly influenced by varieties and culture media but only in cultured protoplasts isolated from dark/dim light-grown cotyledons. The interaction between varieties and media was also significant. Cell colonies formed within 7–14 days in protoplast cultures from dark/dim light-grown cotyledons, and calli subsequently grown on a solid medium developed shoots when transferred onto a regeneration medium. Three of four tested varieties (Arlo, Sonja and Bunyip) showed shoot regeneration within 2–3 months after protoplast isolation, with a high degree of reproducibility in Arlo and Bunyip. Regenerated shoots, which were induced to root on half-strength MS medium with 0.1 mg.l^–1 IBA, survived in soil and grew to produce siliques and set viable seeds in the greenhouse. The present report is the first to document the production of regenerated plants that set seeds in Brassica campestris from cotyledonary protoplasts.Abbreviations BAP benzylaminopurine - CPW Composition of Protoplast Washing-solution - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediamine-tetraacetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - KT kinetin - FDA fluorescein diacetate - SDS sodium dodecyl sulfate     

Construction of a specialized cDNA library from plant cells isolated by laser capture microdissection: toward comprehensive analysis of the genes expressed in the rice phloem

Laser capture microdissection (LCM) is a powerful system which allows the isolation of selectively targeted cells from a tissue section for the analysis of gene-expression profiles of individual cells. The technique has been successfully used for the isolation of specific mammalian cells, mainly cancer cells. However, LCM has never been reported to be applied to the gene expression analysis of plant cells. We used a modified LCM system and successfully applied it to target and isolate phloem cells of rice leaf tissue whose morphology is apparently different from the surrounding cells. Total RNA was extracted from microdissected (approximately 150) phloem cells and the isolated RNA was used for the construction of a cDNA library following the T7 RNA polymerase amplification. Sequence analysis of 413 randomly chosen clones from the library revealed that there was a high level of redundancy in the population and the clones could be subclassified into 124 different groups that contained related sequences. Approximately 37% of both the redundant population and the non-redundant subgroups had novel components while approximately 63% were either homologues to the known genes reported to be localized in phloem of different plant species, or were homologues to other known genes. In situ hybridization revealed that putative amino acid permease, one of the non-redundant clones, was specifically expressed in the phloem. The results proved the effectiveness of construction of a specialized cDNA library from the specific plant cells.     

Membrane deletion during plasmolysis in hardened and non-hardened plant cells

Abstract Video recordings of interference phase contrast microscopy were used to study plasmalemma deletion during plasmolysis in hardened and non-hardened suspension cultured cells of Brassica napus, alfalfa, and cells isolated from rye seedlings. Although different hardening regimes and different cells were used, the responses to plasmolysis were consistent. Hardened cells uncoupled the volume to surface area ratio during plasmolysis both by forming a large number of strands between the cell wall and protoplast and by leaving rivulet-like networks of membranes on the cell wall surface. Tonoplast membrane was deleted as sac-like intrusions into the vacuole. Non-hardened cells produced few strands during plasmolysis. They also deleted plasmalemma and tonoplast into the vacuole as endocytotic vesicles. During deplasmolysis of hardened cells both the individual membrane strands and the rivulets of membrane material vesiculated into strings of vesicles. The vesicles were osmotically active and were re-incorporated into the expanding protoplast. Conversely, deplasmolysis in non-hardened cells resulted in few osmotically active vesicles and many broken strands. The vacuolar sac-like intrusions in hardened cells were re-incorporated into the vacuole whereas the endocytotic vesicles in non-hardened cells were not re-incorporated. Therefore, the non-hardened cells underwent expansion-induced lysis.     

Actin polymerization processes in plant cells

Growing evidence shows that the actin cytoskeleton is a key effector of signal transduction, which controls and maintains the shape of plant cells, as well as playing roles in plant morphogenesis. Recently, several signaling pathways, including those triggered by hormones, Ca(2+), and cAMP, have been reported to be connected to the reorganization of the actin cytoskeleton. The molecular mechanisms involved in such signaling cascades are, however, largely unknown. The Arabidopsis genome sequence is a valuable tool for identifying some of the highly conserved molecules that are involved in such signaling cascades. Recent work has begun to unravel these complex pathways using a panoply of techniques, including genetic analysis, live-cell imaging of intracellular actin dynamics, in vivo localization of factors that are involved in the control of actin dynamics, and the biochemical characterization of how these factors function.