利用烟草表达人源性胰岛素样生长因子1

构建了由花椰菜花叶病毒35S启动子引导人源性胰岛素样生长因子1基因(igf-1)的表达载体pCAM-BIA1301-35S promoter-igf-1-nos,并利用根癌农杆菌LAB4404介导,将其导入烟草。经潮霉素抗性筛选、GUS检测和PCR鉴定,获得17棵转基因植株。RT-PCR分析结果显示,igf-1能够在转基因烟草中正常转录。本试验为利用烟草以及其他双子叶植物高效表达便于分离纯化的IGF-1药用蛋白研究奠定了重要的基础。     

Peroxidase-Induced Wilting in Transgenic Tobacco Plants

Peroxidases are a family of isoenzymes found in all higher plants. However, little is known concerning their role in growth, development, or response to stress. Plant peroxidases are heme-containing monomeric glycoproteins that utilize either H2O2 or O2 to oxidize a wide variety of molecules. To obtain more information on possible in planta functions of peroxidases, we have used a cDNA clone for the primary isoenzyme form of peroxidase to synthesize high levels of this enzyme in transgenic plants. We were able to obtain Nicotiana tabacum and N. sylvestris transformed plants with peroxidase activity that is 10-fold higher than in wild-type plants by introducing a chimeric gene composed of the cauliflower mosaic virus 35S promoter and the tobacco anionic peroxidase cDNA. The elevated peroxidase activity was a result of increased levels of two anionic peroxidases in N. tabacum, which apparently differ in post-translational modification. Transformed plants of both species have the unique phenotype of chronic severe wilting through loss of turgor in leaves, which was initiated at the time of flowering. The peroxidase-induced wilting was shown not to be an effect of diminished water uptake through the roots, decreased conductance of water through the xylem, or increased water loss through the leaf surface or stomata. Possible explanations for the loss of turgor, and the significance of these types of experiments in studying isoenzyme families, are discussed.     

转SCaM—GFP融合基因烟草中钙调素分泌特性的研究

钙调素是一种重要的Ｃａ2 结合蛋白 ,在细胞内发挥着重要的生物学功能。一系列实验证实 ,钙调素也普遍存在于动植物细胞外[1- 3 ] ,并且具有重要的生物学功能[4 - 6] 。我们实验室的研究表明 :胞内外钙调素在Ｃａ2 依赖性以及在靶酶激活特性等方面基本相同 ,但在Ｃａ2 亲合力等方面存在差异[2 ,7] 。近年来 ,在植物细胞中发现存在多种钙调素亚型 ,特别是在大豆中对钙调素亚型研究得比较清楚[8] 。通过对大豆中钙调素亚型亚细胞定位的研究 ,一方面可以阐明钙调素是否具有向胞外主动分泌的特性 ,另一方面可以确定向细胞外分泌的钙调素是…     

Potassium-Ammonium Uptake Interactions in Tobacco Seedlings

Short-term (< 12 h) uptake experiments were conducted with6–7-week-old tobacco (Nicotiana tabacum L. cv. Ky 14)seedlings to determine absorption interactions between K⁺ andNH₄⁺. At equal solution concentrations (0.5 mol m^–3) netK⁺ uptake was inhibited 30–35% by NH₄⁺ and NH₄⁺ uptakewas decreased 9–24%. Removal of NH₄⁺ resulted in completerecovery in K⁺ uptake rate, but NH⁴⁺ uptake rate did not recoverwhen K⁺ was removed. In both cases, inhibition of the uptakerate of one cation saturated as the concentration of the othercation was increased up to 0.5 mol m^–3. The relative effectof K⁺-NH₄⁺ interactions was not altered when Cl- was replacedwith SO₄^2–, but the magnitudes of the uptake rates wereless in the absence of Cl-. The V_max for NH₄⁺ uptake was reducedfrom 128 to 105 µmol g^–1 dry wt. h^–1 in thepresence of 0.5 mol m^–3 K⁺ and the K_m for NH₄⁺ doubledfrom 12 to 27 mmol m^–3 in the presence of K⁺. The resultsof these K⁺-NH₄⁺ experiments are interpreted as mixed-noncompetitiveinteractions. However, an enhanced efflux of K⁺ coupled to NH₄⁺influx via an antiporter cannot be ruled out as contributingto the decrease in net K⁺ uptake. Key words: Nicotiana tabacum, K⁺, NH₄⁺, Uptake interactions     

黄瓜花叶病毒及抗病转基因烟草研究进展

黄瓜花叶病毒(Cucumber mosaic virus,CMV)是世界上分布最广的植物病毒之一,其株系繁多,给烟草生产造成很大的损失。近些年来,各国学者对CMV开展了大量研究。该文主要介绍了黄瓜花叶病毒的基因组结构、亚组以及抗CMV的烟草基因工程的研究进展。     

An Analysis of Interactions between Fluorescently-Tagged Mutant and Wild-Type SOD1 in Intracellular Inclusions

Background

By mechanisms yet to be discerned, the co-expression of high levels of wild-type human superoxide dismutase 1 (hSOD1) with variants of hSOD1 encoding mutations linked familial amyotrophic lateral sclerosis (fALS) hastens the onset of motor neuron degeneration in transgenic mice. Although it is known that spinal cords of paralyzed mice accumulate detergent insoluble forms of WT hSOD1 along with mutant hSOD1, it has been difficult to determine whether there is co-deposition of the proteins in inclusion structures.

Methodology/Principal Findings

In the present study, we use cell culture models of mutant SOD1 aggregation, focusing on the A4V, G37R, and G85R variants, to examine interactions between WT-hSOD1 and misfolded mutant SOD1. In these studies, we fuse WT and mutant proteins to either yellow or red fluorescent protein so that the two proteins can be distinguished within inclusions structures.

Conclusions/Significance

Although the interpretation of the data is not entirely straightforward because we have strong evidence that the nature of the fused fluorophores affects the organization of the inclusions that form, our data are most consistent with the idea that normal dimeric WT-hSOD1 does not readily interact with misfolded forms of mutant hSOD1. We also demonstrate the monomerization of WT-hSOD1 by experimental mutation does induce the protein to aggregate, although such monomerization may enable interactions with misfolded mutant SOD1. Our data suggest that WT-hSOD1 is not prone to become intimately associated with misfolded mutant hSOD1 within intracellular inclusions that can be generated in cultured cells.     

Ectopic Overexpression of Asparagine Synthetase in Transgenic Tobacco

Here, we monitor the effects of ectopic overexpression of genes for pea asparagine synthetase (AS1) in transgenic tobacco (Nicotiana tabacum). The AS genes of pea and tobacco are normally expressed only during the dark phase of the diurnal growth cycle and specifically in phloem cells. A hybrid gene was constructed in which a pea AS1 cDNA was fused to the cauliflower mosaic virus 35S promoter. The 35S-AS1 gene was therefore ectopically expressed in all cell types in transgenic tobacco and constitutively expressed at high levels in both the light and the dark. Northern analysis demonstrated that the 35S-AS1 transgene was constitutively expressed at high levels in leaves of several independent transformants. Furthermore, amino acid analysis revealed a 10- to 100-fold increase in free asparagine in leaves of transgenic 35S-AS1 plants (construct z127) compared with controls. Plant growth analyses showed increases (although statistically insignificant) in growth phenotype during the vegetative stage of growth in 35S-AS1 transgenic lines. The 35S-AS1 construct was further modified by deletion of the glutamine-binding domain of the enzyme (gln[delta]AS1; construct z167). By analogy to animal AS, we reasoned that inhibition of glutamine-dependent AS activity might enhance the ammonia-dependent AS activity. The 3- to 19-fold increase in asparagine levels in the transgenic plants expressing gln[delta]AS1 compared with wild type suggests that the novel AS holoenzyme present in the transgenic plants (gln[delta]AS1 homodimer) has enhanced ammonia-dependent activity. These data indicate that manipulation of AS expression in transgenic plants causes an increase in nitrogen assimilation into asparagine, which in turn produces effects on plant growth and asparagine biosynthesis.     

Rice Phytochrome Is Biologically Active in Transgenic Tobacco

To investigate the mechanisms of phytochrome action in vivo, we have overexpressed rice phytochrome in transgenic tobacco plants. A full-length rice phytochrome cDNA was fused to the cauliflower mosaic virus 35S promoter and transferred to tobacco. The progeny of some of the transgenic plants contain large amounts of rice phytochrome mRNA in green leaves. Extracts prepared from overexpressing plants contain twofold to fivefold more spectrophotometrically detectable phytochrome than extracts from control plants. Species-specific, anti-phytochrome monoclonal antibodies were used in immunoblots to discriminate between rice and tobacco phytochrome apoproteins in fractions eluted from a DEAE-Sepharose column. Red minus far-red difference spectra of the partially purified rice phytochrome from the transgenic plants indicate that the rice phytochrome assembles with chromophore and is photoreversible. Analysis of the circadian pattern of Cab mRNA levels in transgenic plants versus controls demonstrates that the overproduction of rice phytochrome extends the duration of the free-running rhythm of Cab gene expression. The rice phytochrome is, therefore, biologically active in the transgenic tobacco plant, which establishes a system for in vivo functional analysis of phytochrome.     

Transgenic Tobacco Plants Low in Phytochrome A Content

Tobacco (Nicotiana tabacum) plants were transformed with a construct encoding phytochrome A (PHYA) antisense RNA. The construct inserted into the tobacco genome contained squash PHYA cDNA in an antisense orientation under the cauliflower mosaic virus 35S promoter providing for gene expression in higher plant tissues. Using immunoblot analysis and Z3-B1 antibodies against PHYA, the authors demonstrated that the PHYA content of the transgenic plants was lower than that of the wild-type plants. The studies of PHYA-dependent inhibition of hypocotyl elongation by high-intensity far-red light showed a considerable decrease in light sensitivity of the transgenic hypocotyl characteristic for aphyAmutation.     

转基因烟草的甘露醇合成和耐盐性

土壤的盐碱性是世界许多地区限制植物生长和作物产量的主要制约因素。长期的研究发现:在高盐或干旱环境下,大多数植物在细胞质中开始积累一些低分子量的代谢物,如脯氨酸、甜菜碱、糖醇等。这些物质通过维持高的细胞质渗透压,有利于植物在高盐或干旱条件下的水分吸收。通过基因工程手段,影响或改变植物体内的生理代谢途径,使得植物细胞产生和积累不同的低分子量有机化合物,能够     

Rearing in Seawater Mesocosms Improves the Spawning Performance of Growth Hormone Transgenic and Wild-Type Coho Salmon

Growth hormone (GH) transgenes can significantly accelerate growth rates in fish and cause associated alterations to their physiology and behaviour. Concern exists regarding potential environmental risks of GH transgenic fish, should they enter natural ecosystems. In particular, whether they can reproduce and generate viable offspring under natural conditions is poorly understood. In previous studies, GH transgenic salmon grown under contained culture conditions had lower spawning behaviour and reproductive success relative to wild-type fish reared in nature. However, wild-type salmon cultured in equal conditions also had limited reproductive success. As such, whether decreased reproductive success of GH transgenic salmon is due to the action of the transgene or to secondary effects of culture (or a combination) has not been fully ascertained. Hence, salmon were reared in large (350,000 L), semi-natural, seawater tanks (termed mesocosms) designed to minimize effects of standard laboratory culture conditions, and the reproductive success of wild-type and GH transgenic coho salmon from mesocosms were compared with that of wild-type fish from nature. Mesocosm rearing partially restored spawning behaviour and success of wild-type fish relative to culture rearing, but remained lower overall than those reared in nature. GH transgenic salmon reared in the mesocosm had similar spawning behaviour and success as wild-type fish reared in the mesocosm when in full competition and without competition, but had lower success in male-only competition experiments. There was evidence of genotype×environmental interactions on spawning success, so that spawning success of transgenic fish, should they escape to natural systems in early life, cannot be predicted with low uncertainty. Under the present conditions, we found no evidence to support enhanced mating capabilities of GH transgenic coho salmon compared to wild-type salmon. However, it is clear that GH transgenic salmon are capable of successful spawning, and can reproduce with wild-type fish from natural systems.     

Uncoupling Auxin and Ethylene Effects in Transgenic Tobacco and Arabidopsis Plants

Overproduction of auxin in transgenic plants also results in the overproduction of ethylene. Plants overproducing both auxin and ethylene display inhibition of stem elongation and growth, increased apical dominance, and leaf epinasty. To determine the relative roles of auxin and ethylene in these processes, transgenic tobacco and Arabidopsis plants expressing the auxin-overproducing tryptophan monooxygenase transgene were crossed to plants expressing an ethylene synthesis-inhibiting 1-aminocyclopropane-1-carboxylate deaminase transgene. Tobacco and Arabidopsis plants with elevated auxin and normal levels of ethylene were obtained by this strategy. Transgenic auxin-overproducing Arabidopsis plants were also crossed with the ethylene-insensitive ein1 and ein2 mutants. Analysis of these plants indicates that apical dominance and leaf epinasty are primarily controlled by auxin rather than ethylene. However, ethylene is partially responsible for the inhibition of stem elongation observed in auxin-overproducing tobacco. Finally, these data show that auxin overproduction can be effectively uncoupled from ethylene overproduction in transgenic plants to enable direct manipulation of plant morphology for agronomic and horticultural purposes.     

Conjugation of Indole-3-Acetic Acid (IAA) in Wild-Type and IAA-Overprodcing Transgenic Tobacco Plants,and Identification of the Main Conjugates by Frit-Fast Atom Bombardment Liquid Chromatography-Mass Spectrometry

Transgenic plants overproducing indole-3-acetic acid (IAA) from expression of the Agrobacterium tumefaciens T-DNA IAA biosynthesis genes were used to study the conjugation of IAA. At the 11-node stage, free IAA, as well as ester- and amide-conjugated IAA, was analyzed in wild-type tobacco SR1 and in transgenic plants denoted 35S-iaaM/iaaH (line C) and 35S-iaaM x 35S-iaaH (line X). The transgenic plants contained increased levels of both free and conjugated IAA, and the main increase in IAA conjugates occurred in amide conjugates. Two amide conjugates were identified by fritfast atom bombardment liquid chromatography-mass spectrometry as indole-3-acetylaspartic acid (IAAsp) and indole-3-acetylglutamic acid (IAGlu), and one ester conjugate was identified as indole-3-acetylglucose. IAAsp and IAGlu were also identified as endogenous substances in wild-type plants. In wild-type plants, the percent of total IAA in the free form was significantly higher in young leaves (73 [plus or minus] 7%, SD) than in old leaves (36 [plus or minus] 8%), whereas there was no difference between young (73 [plus or minus] 8%) and old internodes (70 [plus or minus] 9%). In IAA-overproducing transformants, both free and conjugated IAA levels were increased, but the percent free IAA was maintained constant (57 [plus or minus] 10%) for both leaves and internodes, independent of the total IAA level or tissue age. These results suggest that synthesis or transport of IAA conjugates is regulated in the vegetative wild-type plant, and that different organs possess a unique balance between free and conjugated IAA. The IAA-overproducing plant, however, acquires a lower proportion of free IAA in the stem and younger leaves, presumably determined by a higher conjugation in those tissues compared with wild type.     

鸡α干扰素基因遗传转化烟草研究

利用植物生物反应器生产外源药用蛋白近年来备受关注,本研究通过农杆菌介导法,将人工合成的鸡α干扰素基因（ChIFN-α）转化烟草（Nicotiana tabctcum）无菌苗叶盘。对抗性植株进行的GUS活性鉴定,PCR和RT-PCR检测表明,ChIFN-α基因已整合到烟草基因组中并具有转录活性,ELISA检测和细胞病变（CPE）抑制试验表明转基因烟草表达的干扰素蛋白具有抗病毒活性。     

Inheritance of Altered Flower Morphology and Kanamycin-Resistance in Transgenic Tobacco Plants

Inheritance of altered flower morphology and resistance to antibiotic kanamycin was studied in the first and second generations (T₁and T₂, respectively) of self-pollinated transgenic tobacco plants. In most transformants, kanamycin resistance was closely linked to mutant phenotype. T-DNA-induced mutations were shown to be dominant.