致病疫霉效应蛋白Pi16275的功能研究

[目的]分析致病疫霉效应蛋白Pi16275的超量表达对病原菌致病性的影响,明确Pi16275的亚细胞定位,筛选Pi16275在植物中的互作靶标蛋白及靶标蛋白在抵御病原菌侵染过程中的作用,初步揭示Pi16275在病原菌侵染植物过程中的作用机制.[方法]利用农杆菌介导的烟草瞬时表达系统在烟草叶片表皮细胞中瞬时表达Pi162...     

Failure of Ethylene to Change the Distribution of Indoleacetic Acid in the Petiole of Coleus blumei X frederici during Epinasty

The effect of ethylene on the distribution of applied indoleacetic acid in the petiole of Coleus blumei Benth. X C. frederici G. Taylor has been investigated during the development of epinastic curvature. Using intact plants, ¹⁴C-IAA was applied to the distal region of the leaf lamina and the accumulation of label in the abaxial and adaxial halves of 5 mm petiole sections was determined after 1.5, 3, and 6 hours. Over this period the label was transported out of the lamina into the petiole at a rate of at least 66 mm hr⁻¹. Of the total amount of label in the petiole sections, 24 to 30% was located in the adaxial half and this distribution was not altered significantly by exposing plants to an atmosphere containing 50 μl/l ethylene. Thus when epinastic curvature is induced by ethylene there is no associated increase in the IAA content of the expanding adaxial half. The role of endogenous IAA in petiole epinasty was studied by restricting its movement with DPX 1840 (3,3a-dihydro-2-[p-methoxyphenyl]-8H-pyrozolo{5,1-a}isoindol-8-one). The leaf petioles still showed an initial epinastic response to ethylene. It is concluded that ethylene-induced epinasty is not dependent upon either any change in the transport of IAA or its redistribution within the petiole.     

转彩色马铃薯StAN1基因的烟草幼苗耐旱性分析

该研究以转彩色马铃薯StAN1基因烟草为材料、野生型烟草(WT)为对照,测定分析转StAN1基因烟草在种子萌发期、幼苗期和苗期对干旱(甘露醇)处理的耐受情况,并对苗期旱热共同胁迫的耐受情况进行测定分析,以探讨彩色马铃薯StAN1基因的功能,为耐旱彩色马铃薯育种提供新路径。结果显示:(1)转StAN1基因烟草鉴定显示,阳性率为82.6%,且转基因烟草的叶片明显变紫,花青素含量极显著高于野生型烟草。(2)在培养基甘露醇浓度为150 mmol/L时,点播在培养基上的转基因烟草种子第5天时的萌发率达到了7%,是野生型烟草萌发率的2.3倍。(3)在甘露醇浓度为0和100 mmol/L的培养基上竖直培养时,转基因烟草的根长分别是野生型烟草的1.46和1.30倍,根长比野生型烟草显著增长。(4)在干旱胁迫下,转基因烟草幼苗叶片中的脯氨酸含量以及超氧化物歧化酶活性均显著高于野生型烟草,丙二醛含量均显著低于野生型烟草。(5)转基因烟草LEA基因和ERF基因在干旱和旱热处理中的相对表达量均高于野生型烟草。研究表明,StAN1基因在提高植物花青素含量的同时也提高了植物的耐旱性。     

Expression of E. coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partitioning

Transgenic plants were constructed expressing a novel cytosolic inorganic pyrophosphatase in order to reduce the cytosolic pyrophosphate content. To this end the Escherichia coli gene ppa encoding inorganic pyrophosphatase was cloned between the 35S CaMV promoter and the poly(A) site of the octopine synthase gene and transferred into tobacco and potato plants by Agrobacterium-mediated gene transfer. Regenerated plants were tested for the expression of the ppa gene by Northern blots and activity gels. Plants expressing active inorganic pyrophosphatase showed a dramatic change in photoassimilate partitioning. In both transgenic tobacco and potato plants the ratio between soluble sugars and starch was increased by about 3-4-fold in source leaves as compared with the wild-type. However, whereas source leaves of transgenic tobacco plants accumulated much higher levels of glucose (up to 68-fold), fructose (up to 24-fold), sucrose (up to 12-fold) and starch (up to 8-fold) this was not observed in potato plants where the change in assimilate partitioning in source leaves was due to an increase of about 2-fold in sucrose and a reduction in starch content. Expression of the cytosolic inorganic pyrophosphatase in tobacco results in stunted growth of vegetatively growing plants due to a reduced internode distance. Upon flowering the transgenic plants increase their growth rate, reaching almost the same height as control plants at the end of the growth period. Old source leaves accumulate up to 100-fold more soluble sugars than control leaves. This increase in soluble sugars is accompanied by a reduction in chlorophyll content (up to 85%). Transgenic potato plants showed a less dramatic change in their growth behaviour. Plants were slightly reduced in size, with stems more highly branched. Tuber number increased 2-3-fold, but tuber weight was lower resulting in no net increase in fresh weight.     

Transgenic potato plants overexpressing the pathogenesis-related STH-2 gene show unaltered susceptibility to Phytophthora infestans and potato virus X

The STH-2 gene is rapidly activated in potato leaves and tubers following elicitation or infection by Phytophthora infestans. However, its biochemical function remains unknown. In order to ascertain if STH-2 protein is directly involved in the defense of potato against pathogens, the STH-2 coding sequence under the control of the CaMV 35S promoter was introduced into potato plants. Transgenic plants expressing the STH-2 gene were analyzed for an altered pattern of susceptibility to a compatible race of P. infestans and to potato virus X. Results indicate that constitutive expression of the STH-2 gene did not reduce susceptibility of potato to these pathogens.     

铁皮石斛DoSMT2基因的功能分析

为了揭示铁皮石斛(Dendrobium officinale)甾醇C-24甲基转移酶2基因(DoSMT2)在甾醇代谢过程的功能,该研究通过根癌农杆菌介导法将来源于铁皮石斛的DoSMT2基因转化烟草(Nicotiana tabacum),并采用qRT-PCR技术检测DoSMT2基因在转基因烟草叶片中的表达,采用气相色谱质谱法分析菜油甾醇和谷甾醇的含量。结果显示:(1)成功获得DoSMT2基因的开放阅读框(1 119 bp),并成功构建正义植物表达载体质粒pCXSN-DoSMT2,经农杆菌介导的烟草叶盘转化法转化烟草并鉴定,获得4株阳性转基因烟草植株。(2)Southern blot结果表明,4株转基因烟草植株都有1条杂交信号带,而非转基因烟草植株没有,说明外源DoSMT2基因都以单拷贝整合到4株转基因烟草基因组中。(3)qRT-PCR检测显示,非转基因烟草未检测到外源DoSMT2基因的表达,4株转基因烟草都能检测到DoSMT2基因的表达,且表达水平差异极显著,各株系表达量高低依次为P3P1P2(P4)。(4)气相色谱质谱分析显示,转DoSMT2基因烟草叶片的菜油甾醇含量均极显著低于非转基因烟草叶片,而谷甾醇含量均极显著高于非转基因烟草叶片。研究表明,DoSMT2具有催化24-亚甲基胆甾烯醇转化形成24-亚乙基胆甾烯醇活性。     

Phenotypic instability of transgenic tobacco plants and their progenies expressing Arabidopsis thafiana small GTP-binding protein genes

Chimeric genes consisting of the cauliflower mosaic virus 35S promoter, a CDNA encoding a small GTP-binding protein from Arabidopsis thaliana (ara-2 or ara-4) and the terminator of the nopaline synthase gene were cloned into a binary vector. Tobacco leaf tissues were transformed with this plasmid via Agrobacterium-mediated transformation. Transgenic plants possessing either ara-2 or ara-4 occasionally showed morphological abnormalities in leaves and other organs. However, such alterations were not always associated with co-transferred characters, such as kanamycin tolerance, and they arose in no more than 10% of the transgenic plants. Such phenomena were also observed in the progenies of the primary transgenic plants. Despite such unusual inheritance of the phenotypic abnormalities, GTP-binding activity of the inserted ara gene products was detected in all plants tested.     

Epinasty of Poinsettias-the Role of Auxin and Ethylene

Upward physical restraint of the normally horizontal bracts of poinsettia (Euphorbia pulcherrima Willd.) resulted in increased ethylene production and epinastic curvature of the petioles after 5 days. Downward restraint caused little change in ethylene production or epinasty, indicating that the enhanced ethylene production observed in petioles bent upwards is not due to the bending stress alone. Epinasty, measured upon removal of upward physical restraint, was not affected by spraying plants with aminoxyacetic acid to reduce ethylene production or with silver thiosulfate to prevent ethylene action. Removal of the bract blades prevented the epinastic response of the petiole, and the response was restored by applying indoleacetic acid to the cut petiole end. Redistribution of auxin appears to be responsible for both the epinasty and the increased ethylene production of reoriented poinsettia bracts.     

过表达IbOr基因甘薯增强抗旱性的生理机制

以超表达甘薯橙色基因(IbOr)的转基因甘薯(TS)以及非转基因甘薯(NT)为实验材料,通过15%聚乙二醇6000(PEG-6000)模拟干旱条件,研究转基因与非转基因甘薯幼苗在水分胁迫不同时间的光合系统,膜脂过氧化及抗氧化防御系统中主要指标的变化情况,探讨转基因甘薯耐旱性的生理机制。结果显示:(1)随PEG-6000胁迫时间延长,甘薯叶片的叶绿素、类胡萝卜素含量及其叶片净光合速率、气孔导度、胞间CO2浓度、蒸腾速率都显著降低,但转基因株系降低幅度小于非转基因植株。(2)在正常供水和水分胁迫下,超表达IbOr基因甘薯叶片中O-·2、MDA含量均低于非转基因甘薯,即转基因甘薯具有较低的活性氧水平且脂膜受损伤较小。(3)PEG-6000胁迫24h后,甘薯叶片中SOD、POD酶活性均增加,48h达到最大值,且转基因甘薯中2种酶活性显著高于非转基因甘薯。研究表明,过表达IbOr基因可以有效减轻甘薯在水分胁迫条件下受损害的程度,且可能主要通过提高甘薯的抗氧化胁迫能力来完成。     

Analysis of an abscisic acid (ABA)-responsive gene promoter belonging to the Asr gene family from tomato in homologous and heterologous systems

Asr is a family of genes that maps to chromosome 4 of tomato. Asr2, a recently reported member of this family, is believed to be regulated by abscisic acid (ABA), stress and ripening. A genomic Asr2 clone has been fully sequenced, and candidate upstream regulatory elements have been identified. To prove that the promoter region is functional in vivo, we fused it upstream of the β-glucuronidase (GUS) reporter gene. The resulting chimeric gene fusion was used for transient expression assays in papaya embryogenic calli and leaves. In addition, the same construct was used to produce transgenic tomato, papaya, tobacco, and potato plants. Asr2 upstream sequences showed promoter function in all of these systems. Under the experimental conditions tested, ABA stimulated GUS expression in papaya and tobacco, but not in tomato and potato systems. Received: 24 March 1997 / Accepted: 26 November 1997     

Expression of a luteoviral movement protein in transgenic plants leads to carbohydrate accumulation and reduced photosynthetic capacity in source leaves

Elucidating the role of viral genes in transgenic plants revealed that the movement protein (MP) from tobacco mosaic virus is responsible for altered carbohydrate allocation in tobacco and potato plants. To study whether this is a general feature of viral MPs, the movement protein MP17 of potato leafroll virus (PLRV), a phloem-restricted luteovirus, was constitutively expressed in tobacco plants. Transgenic lines were strongly reduced in height and developed bleached and sometimes even necrotic areas on their source leaves. Levels of soluble sugars and starch were significantly increased in source leaves. Yet, in leaf laminae the hexose—phosphate content was unaltered and ATP reduced to only a small extent, indicating that these leaves were able to maintain homeostatic conditions by compartmentalization of soluble sugars, probably in the vacuole. On the contrary, midribs contained lower levels of soluble sugars, ATP, hexose—phosphates and UDP-glucose supporting the concept of limited uptake and catabolism of sucrose in the phloem. The accumulation of carbohydrates led to a decreased photosynthetic capacity and carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) probably owing to decreased expression of photosynthetic proteins. In parallel, levels of pathogenesis-related proteins were elevated which may be the reason for the obtained limited resistance against the unrelated potato virus Y (PVY)^N in the transgenic tobacco plants. Ultrathin sections of affected leaves harvested from 2-week-old plants revealed plasmodesmal alterations in the phloem tissue while plasmodesmata between mesophyll cells were indistinguishable from wild-type. These data favour the phloem tissue to be the primary site of PLRV MP17 action in altering carbohydrate metabolism.     

Wound-response regulation of the sweet potato sporamin gene promoter region

Sporamin, a tuberous storage protein of sweet potato, was systemically expressed in leaves and stems by wound stimulation. In an effort to demonstrate the regulatory mechanism of wound response on the sporamin gene, a 1.25 kb sporamin promoter was isolated for studying the wound-induced signal transduction. Two wound response-like elements, a G box-like element and a GCC core-like sequence were found in this promoter. A construct containing the sporamin promoter fused to a -glucuronidase (GUS) gene was transferred into tobacco plants by Agrobacterium-mediated transformation. The wound-induced high level of GUS activity was observed in stems and leaves of transgenic tobacco, but not in roots. This expression pattern was similar to that of the sporamin gene in sweet potatoes. Exogenous application of methyl jasmonate (MeJA) activated the sporamin promoter in leaves and stems of sweet potato and transgenic tobacco plants. A competitive inhibitor of ethylene (2,5-norbornadiene; NBD) down-regulated the effect of MeJA on sporamin gene expression. In contrast, salicylic acid (SA), an inhibitor of the octadecanoid pathway, strongly suppressed the sporamin promoter function that was stimulated by wound and MeJA treatments. In conclusion, wound-response expression of the sporamin gene in aerial parts of plants is regulated by the octadecanoid signal pathway.     

Hormonal content and sensitivity of transgenic tobacco and potato plants expressing single rol genes of Agrobacterium rhizogenes T-DNA

The expression of single rol genes of the T_L-DNA of Agrobacterium rhizogenes strain A4 in transgenic tobacco (Nicotiana tabacum L.) and potato (Solanum tuberosum L.) plants alters the internal concentrations of, and the sensitivity to, several plant hormones. The levels of immunoreactive cytokinins, abscisic acid, gibberellins and indole-3-acetic acid were analysed in tissues of the apical shoots, stems, leaves, roots and undifferentiated callus tissue. The addition of the dominant and morphogenetically active rolA, rolB, or rolC genes resulted in alterations in the content of several hormones. rolC overexpression in particular led to an up to fourfold increase in the content of isopentenyladenosine, dihydrozeatin riboside and trans-zeatin riboside-type cytokinins in potato plants. This increase correlated well with different levels of expression of the rolC gene in different transgenic plants. Furthermore it was shown that the dwarfism of P_35s-rolC transgenic tobacco and potato plants is correlated with a 28–60% reduction of gibberellic acid A₁ concentration in apical shoots. Exogenous addition of gibberellic acid completely restored stem elongation in P_35s-_rolC transgenic plants. Apical shoots of dwarf rolA transgenic tobacco plants also contained 22% less gibberellic acid A₁ than control plants, but growth cannot be restored completely by exogenously added gibberellic acid. Similarly, the sensitivity of transgenic tobacco seedlings or callus tissues towards different phytohormone concentrations can be altered by the expression of single rol genes. The overexpression of the rolC gene in seedlings led to an altered response to auxins, cytokinins, abscisic acid, gibberellic acid and the ethylene precursor 1-aminocyclopropane-carboxylic acid. The overexpression of the rolB gene in tobacco calli led to necrosis at lower auxin concentrations than in the wild-type, while other parameters of auxin action, like the induction of cell growth, remained unchanged.     

Expression of the B subunit of <Emphasis Type="Italic">E. coli</Emphasis> heat-labile enterotoxin in tobacco using a herbicide resistance gene as a selection marker

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) has been transformed to plants for use as an edible vaccine. We have developed a simple and reliable Agrobacterium-mediated transformation method to express synthetic LTB gene in N. tabacum using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. The synthetic LTB gene adapted to the coding sequence of tobacco plants was cloned to a plant expression vector under the control of the ubiquitin promoter and transformed to tobacco by Agrobacterium-mediated transformation. Transgenic plants were selected in the medium supplemented with 5 mg l^-1 phosphinothricin (PPT). The amount of LTB protein detected in the transgenic tobacco was approximately 3.3% of the total soluble protein, approximately 300-fold higher than in the plants generated using the native LTB gene under the control of the CaMV 35S promoter. The transgenic plants that were transferred to a greenhouse had harvested seeds that proved to be resistant to herbicide. Thus, the described protocol could provide a useful tool for the transformation of tobacco plants.     

AtNHX1基因对荞麦的遗传转化及抗盐再生植株的获得

通过农杆菌介导法将拟南芥液泡膜Na⁺/H⁺反向转运蛋白基因AtNHX1转入荞麦中,在2.0mg/L 6-BA、0.1mg/L IAA、1mg/L KT、50mg/L卡那霉素和500mg/L头孢霉素的MS培养基上进行选择培养,从来源于864块外植体的36块抗性愈伤组织中共获得426棵再生植株（转化频率为4.17%）。经PCR、Southern印迹分析、RT-PCR和Northern检测,初步证实AtNHX1基因已整合至荞麦基因组中。用200mmol/L的盐水对转基因植株和对照植株进行胁迫处理6周,转基因植株能够生存,而对照植株死亡。用不同浓度的NaCl溶液处理转基因植株和对照植株,发现Na⁺及脯氨酸含量在转基因植株中的积累水平显著高于对照植株,而K⁺的含量在转基因植株中的积累水平低于对照植株。次生代谢产物黄酮类化合物芦丁在转基因植株根、茎和叶片中的含量也比对照植株明显要高。这些结果表明利用基因工程手段提高作物的耐盐性是可行的。     

Expression of Cry1Ac in Transgenic Tobacco Plants Under the Control of a Wound-Inducible Promoter (AoPR1) Isolated from Asparagus officinalis to Control Heliothis virescens and Manduca sexta

Expression of cry1Ac gene from Bacillus thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1 promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of Cry1Ac protein increased during 6–72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay.