Stable expression of a single-copy rolA gene in transgenic Arabidopsis thaliana plants allows an exhaustive mutagenic analysis of the transgene-associated phenotype

Several publications have documented the instability of transgene expression in plants. Previous genetic approaches to the study of transgene-associated phenotypes in plants were limited by this phenomenon. Here we show that a transgene can be expressed in plants with sufficient stability to allow an exhaustive mutagenic analysis of the resulting phenotype. We have expressed the morphogenic rolA gene from the TL-DNA of Agrobacterium rhizogenes Ri plasmid in transgenic Arabidopsis thaliana plants. The resulting pleiotropic RolA phenotype allows a visual screen for reversion to detect germinal as well as somatic instability of transgene expression. However no spontaneous reversions of the RolA phenotype were observed in 65 000 progeny of two independent transgenic A. thaliana lines, each carrying a single homozygous rolA locus. In contrast, 12 revertants of the RolA phenotype were isolated from 360000 ethyl methane sulphonate (EMS)-mutagenized M2 progeny. All revertants were shown genetically to carry stable recessive mutations in the rolA locus, thus establishing a series of loss-of-function alleles. Molecular characterization revealed that the loss-of-function alleles were structurally intact and expressed in all rolA mutants. A wild-type rolA locus and two loss-of-function alleles were reisolated and sequenced; base pair substitutions were found in each loss-of-function allele leading to single amino acid substitutions in the rolA open reading frame. Therefore no instability of expression of the rolA locus was detected in any of the 425 000 individuals studied in this analysis. Furthermore even under conditions of saturation mutagenesis, no extragenic suppressor locus was detected.     

Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants

Summary We characterized the expression of genes that correspond to a cDNA clone, RD29, which is induced by desiccation, cold and high-salt conditions in Arabidopsis thaliana. Northern analysis of desiccation-induced expression revealed a two-step induction process. Early induction occurs within 20 min and secondary induction occurs 3 h after the start of desiccation. Exogenous abscisic acid (ABA) induces RD29 mRNA within 3 h. Two genes corresponding to RD29, rd29A and rd29B, are located in tandem in an 8 kb region of the Arabidopsis genome and encode hydrophilic proteins. Desiccation induces rd29A mRNA with two-step kinetics, while rd29B is induced only 3 h after the start of desiccation. The expression of both genes is stimulated about 3 h after application of ABA. It appears that rd29A has at least two cis-acting elements, one involved in the ABA-associated response to desiccation and the other induced by changes in osmotic potential. The -glucuronidase (GUS) reporter gene driven by the rd29A promoter was induced at significant levels by desiccation, cold, high-salt conditions and ABA in both transgenic Arabidopsis and tobacco. Histochemical analysis of GUS activity revealed that the rd29A promoter functions in almost all the organs and tissues of vegetative plants during water deficiency.     

拟南芥GH3.9基因的过表达及其表型分析

为研究GH3.9基因在植物生长发育过程中的作用,利用RT-PCR成功克隆到GH3.9基因,该基因全长为1 750bp。通过构建pEGAD-GH3.9过表达载体转化拟南芥,获得过表达GH3.9基因纯系转基因株系GH3.9ox-3和GH3.9ox-7。对拟南芥野生型(WT)和转基因株系(GH3.9ox-3和GH3.9ox-7)幼苗用不同光强和光质进行处理,结果显示:在蓝光、红光、远红光等不同光照强度下培养,过表达株系幼苗下胚轴的生长均明显受到抑制,且较野生型明显;采用不同光周期处理拟南芥幼苗,过表达幼苗下胚轴的伸长明显低于野生型;对成年植株表型进行观察,发现过表达株系植株矮小、雄蕊变短、果荚短小。研究表明:GH3.9基因参与了拟南芥生长发育调控,过表达GH3.9基因对拟南芥生长有抑制作用。     

Insertional mutagenesis in Arabidopsis thaliana

Recently, a number of mutant gene loci in the Arabidopsis thaliana plant genome have been identified through insertional mutagenesis. In this review, we evaluate different methods used for Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis with regard to their mutation frequencies and conclude that a major breakthrough in the isolation of genes involved in plant development has been acheived. To provide a specific example, we summarize recent progress made in the understanding of flower morphogenesis at the molecular level through the study of homeotic genes obtained via gene tagging. T-DNA gene fusion vectors are being discussed that will allow the isolation of plant regulatory sequences with particular cell or tissue specificity, or that are controlled by specific external stimuli. Finally, we report on the approaches followed to convert the maize transposons Ac/Ds into valuable gene tags for use in a heterologous host such as Arabidopsis.     

In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.National Research Council of Canada Publication No. 38003     

Differential expression of a polygalacturonase gene family in Arabidopsis thaliana

By systematic sequencing of a flower bud cDNA library from Arabidopsis thaliana, we have identified four cDNAs encoding polygalacturonase. The corresponding genes, together with seven other A. thaliana genes present in the databases, form a small gene family. Sequence comparisons of the deduced polypeptides within the gene family or with other plant polygalacturonases allow classification of the genes into different clades. Five polygalacturonases, including all those isolated from the flower buds, are closely related to the enzyme in pollen. Of the six remaining polygalacturonases, three are more closely related to the abscission-specific type of enzyme and two others to the fruit polygalacturonase. The last one is more distantly related to the others and might correspond to a new type of polygalacturonase. Expression of the different genes was analysed on Northern blots and by a PCR-based strategy. Results indicate that if, as expected, the cDNAs isolated from the flower bud library are strongly expressed in pollen, other genes are expressed at a low level in young developing tissues, such as in seedlings and roots, suggesting that they could be implicated in the cell wall modifications observed during cell elongation and/or expansion which occur in these tissues. Received: 7 December 1998 / Accepted: 1 April 1999     

Genotoxicity of 2,4-D and dicamba revealed by transgenic Arabidopsis thaliana plants harboring recombination and point mutation markers

The phenoxy herbicides 2,4-D and dicamba are released daily into the environment in large amount. The mechanisms of genotoxicity and mutagenicity of these herbicides are poorly understood, and the available genotoxicity data is controversial. There is a cogent need for a novel genotoxicity monitoring system that could provide both reliable information at the molecular level, and complement existing systems.We employed the transgenic Arabidopsis thaliana ‘point mutation’ and ‘recombination’ plants to monitor the genetic effects of the herbicides 2,4-D and dicamba. We found that both herbicides had a significant effect on the frequency of homologous recombination A→G mutation. Neither herbicides affected the T→G mutation frequency. Interestingly, these transgenic biomonitoring plants were able to detect the presence of phenoxy herbicides at concentrations that were lower than the guideline levels for Drinking Water Quality. The results of our studies suggest that our transgenic system may be ideal for the evaluation of the genotoxicity of herbicide-contaminated water. Moreover, the unique ability of the plants to detect both double-strand breaks (homologous recombination) and point mutations provides tremendous potential in the study of molecular mechanisms of genotoxicity and mutagenicity of phenoxy herbicides.     

High intensity and blue light regulated expression of chimeric chalcone synthase genes in transgenic Arabidopsis thaliana plants

Summary To establish a genetic system for dissection of light-mediated signal transduction in plants, we analyzed the light wavelengths and promoter sequences responsible for the light-induced expression of the Arabidopsis thaliana chalcone synthase (CHS) promoter fused to the -glucuronidase (GUS) marker gene. Transgenic A. thaliana lines carrying 1975, 523, 186, and 17 by of the CHS promoter fused to the GUS gene were generated, and the expression of these chimeric genes was monitored in response to high intensity light in mature plants and to different wavelengths of light in seedlings. Fusion constructs containing 1975 and 523 by of CHS promoter sequence behaved identically to the endogenous CHS gene under all conditions. Expression of these constructs was induced specifically in response to high intensity white light and blue light. The response to blue light was seen in the presence of the P_fr form of phytochrome. Fusion constructs containing 186 by of promoter sequence showed reduced basal levels of expression and only weak stimulation by blue light but were induced significantly by high intensity white light. These analyses showed that the expression of the A. thaliana CHS gene is responsive to a specific blue light receptor and that sequences between — 523 and — 186 by are required for optimal basal and blue light-induced expression of this gene. The experiments lay the foundation for a simple genetic screen for light response mutants.     

拟南芥AtCDPK1基因克隆与转化马铃薯的研究

以野生拟南芥生态型(Col-0)为材料,利用PCR和DNA重组技术,克隆了拟南芥钙依赖蛋白激酶基因(AtCDPK1)。AtCDPK1基因全长为2 269bp,碱基序列与已知基因At1g18890完全一致。构建AtCDPK1基因的植物表达载体pCHF3-CDPK1,并利用农杆菌介导法将pCHF3-CDPK1转入马铃薯品种‘费乌瑞它’的脱毒试管微型薯中,经筛选与植株再生,共获得50个抗性再生植株。PCR检测显示,有36个植株基因组中含有AtCDPK1基因。RT-PCR分析证实,AtCDPK1基因在这些马铃薯植株的基因组中均能正常转录表达。这一结果为进一步开展AtCDPK1基因功能分析及转基因抗旱马铃薯新品种选育研究奠定了基础。     

Regeneration of fertile plants from protoplasts of different Arabidopsis thaliana genotypes

Summary A protocol for obtaining regenerated fertile plants from mesophyll protoplasts of Arabidopsis thaliana is reported. Protoplasts were isolated from leaves of 21-to 28-day-old Arabidopsis plants grown in a controlled environment. Sustained divisions were achieved when protoplasts were embedded in beads formed by 1.4% sodium alginate in the presence of 50mM CaCl₂ in 0.4 mannitol, which was then exchanged againts modified B5 medium. About 0.4%–0.6% of the protoplasts developed into colonies of which 80%–90% formed shoots and subsequently regenerated to fertile plants. Seeds harvested from more than 200 independently regenerated plants were sown and germination frequencies of more than 95% were obtained. Furthermore, the F₁ plants did not show any evidence of somaclonal variation on visual inspection. This protocol was originally developed for Arabidopsis thaliana Columbia; however it was shown to be applicable also for the genotypes Wassilewskija, Landsberg erecta and Estland though with differing efficiencies.Abbreviations FDA fluorescein diacetate - CM culture medium - SRM shoot regeneration medium - SEM shoot elongation medium - RM rooting medium - PE plating effciency - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine - Kin kinetin - 2-iP 2-isopentenyladenine - GA₃ gibberetic acid     

Development of an efficient two-element transposon tagging system in Arabidopsis thaliana

Summary Modified Ac and Ds elements, in combination with dominant markers (to facilitate monitoring of excision, reinsertion and segregation of the elements) were introduced into Arabidopsis thaliana ecotype Landsberg erecta. The frequencies of somatic and germinal transactivation of the Ds elements were monitored using a streptomycin resistance assay. Transactivation was significantly higher from a stable Ac (sAc) carrying a 537 by deletion of the CpG-rich 5 untranslated leader of the transposase mRNA than from a wild-type sAc. However, substitution of the central 1.77 kb of the transposase open reading frame (ORF) with a hygromycin resistance marker did not alter the excision frequency of a Ds element. -Glucuronidase (GUS) or iaaH markers were linked to the transposase source to allow the identification of plants in which the transposase source had segregated away from the transposed Ds element, eliminating the possibility of somatic or germinal re-activation. Segregation of the excision marker, Ds and sAc was monitored in the progeny of plants showing germinal excision of Ds. 29% of the plants inheriting the excision marker carried a transposed Ds element.     

UV-B预处理诱导拟南芥耐旱性的提高

为了解UV-B提高拟南芥(Arabidopsis thaliana)耐旱性的生理机制,将2周龄的野生型拟南芥(WT)和sto突变体幼苗用不同剂量UV-B预处理1周,再用30%PEG模拟干旱处理24 h,对植株的表型进行统计,并测定类黄酮、脯氨酸和MDA含量。结果表明,低剂量UV-B预处理能够提高拟南芥的耐旱性,植株的类黄酮与脯氨酸含量分别提高了20%～40%和50%～65%,细胞膜受损程度降低,从而提高了保水性。低剂量UV-B提高拟南芥耐旱性的效应在sto突变体中消失,证明这种效应在分子机制上可能与STO蛋白相关。     

Structure and expression of AtS1, an Arabidopsis thaliana gene homologous to the S-locus related genes of Brassica

Summary Genetic and molecular analysis of the self-incompatibility locus (S-locus) of the crucifer Brassica has led to the characterization of a multigene family involved in pollen-stigma interactions. While the crucifer Arabidopsis thaliana does not have a self-incompatibility system, S-related sequences were detected in this species by cross-hybridization with Brassica DNA probes. In this paper, we show that an A. thaliana S-related sequence, designated AtS1, is expressed specifically in flower buds. Sequence analysis suggests that AtS1 encodes a secreted glycoprotein that is most similar to the Brassica S-locus related protein SLR1. As has been proposed for SLR1, this gene may be involved in determining some fundamental aspect of pollen-stigma interactions during pollination. The molecular and genetic advantages of the Arabidopsis system will provide many avenues for testing this hypothesis.     

拟南芥基因密码子偏爱性分析

密码子偏爱性对外源基因的表达强度有一定影响,特别是编码蛋白质N端7～8个氨基酸残基的密码子.通过对拟南芥染色体中26 827个蛋白质对应的基因密码子进行分析,得到了编码氨基酸的61种密码子在拟南芥中的使用频率,并与大肠杆菌和哺乳动物进行了比较,结果表明三者间的密码子偏爱性有较大差异.这一分析结果对于动物基因在植物中的表达,及植物基因在微生物中的表达具有一定指导意义.同时提供了一种直接以XML文档为数据源解析巨型XML格式染色体数据的方法.     

Cloning and expression analyses of AtMRP4 , a novel MRP -like gene from Arabidopsis thaliana

In all organisms glutathione-conjugate transporters (GS-X pumps) mediate the detoxification of a number of xenobiotics by removing them from the cytosol. In addition, GS-X pumps appear to play a role in the processing of endogenous compounds. We have isolated a novel genomic clone from Arabidopsis thaliana that encodes a putative GS-X pump, AtMRP4, which is part of a recently defined gene family. The derived amino acid sequence shares high levels of similarity (55–63%) with human, yeast, and other Arabidopsis homologues. The expression of the different members of the AtMRP gene family in Arabidopsis cell suspensions after treatment with chemicals that modify glutathione metabolism (compounds that induce different types of stress and that act as herbicide antidotes – safeners – in monocotyledonous species) revealed that the members of this gene family are differentially regulated. Received: 20 February 1998 / Accepted: 9 March 1998     

GFP基因在新疆小拟南芥和拟南芥中的表达分析

以质粒pMCB30为模板,扩增GFP基因,连接到载体pCMBIA2300-35S-OCS上,构建过量表达载体p35S:GFP,将其转入农杆菌GV3101.通过农杆菌介导法将p35S:GFP载体分别转入新疆特色植物小拟南芥和拟南芥中.T0代经含有卡那霉素的1/2MS培养基筛选,获得了T1代转基因小拟南芥2株,T1代转基因拟南芥9株.通过激光共聚焦显微镜观察,在转基因小拟南芥和拟南芥的根尖细胞中均可检测到GFP绿色荧光蛋白;对转基因植株进行PCR扩增,均可检测到GFP基因,表明GFP基因已成功转入小拟南芥和拟南芥中.该研究建立了小拟南芥的遗传转化体系,为进一步利用GFP基因和进一步研究小拟南芥的功能基因奠定基础.     

拟南芥叶形态建成基因ABL1的图位克隆及鉴定

前期研究表明ABL1可能在植物叶发育过程中扮演重要的角色,其突变表现为叶片生长迟缓、成熟叶片叶缘缺刻明显等生长缺陷特征。该研究利用图位克隆及其精细定位技术,将ABL1基因锁定在2个SSLP标记T23K8和T8F5之间,该区间包含44个基因。通过生物信息学成功找到ABL1突变基因为拟南芥FAS1,该基因编码染色质组装因子CAF1的一个亚基,在植物顶端分生组织生长调控中扮演重要角色。RT-PCR结果显示,该基因表达受阻,功能互补实验证实abl1突变体的确是FAS1基因的一个新等位突变。研究结果暗示,ABL1/FAS1在植物叶形态建成中也起着重要作用。     

A two-element Enhancer-Inhibitor transposon system in Arabidopsis thaliana

The Enhancer-Inhibitor (En-I), also known as Suppressor-mutator (Spm-dSpm), transposable element system of maize was modified and introduced into Arabidopsis by Agrobacterium tumefaciens transformation. A stable En/Spm transposase source under control of the CaMV 35S promoter mediated frequent transposition of I/dSpm elements. Transposition occurred continuously throughout plant development over at least seven consecutive plant generations after transformation. New insertions were found at both linked and unlinked positions relative to a transposon donor site. The independent transposition frequency was defined as a transposition parameter, which quantified the rate of unique insertion events and ranged from 7.8% to 29.2% in different populations. An increase as well as a decrease in I/dSpm element copy number was seen at the individual plant level, but not at the population level after several plant generations. The continuous, frequent transposition observed for this transposon system makes it an attractive tool for use in gene tagging in Arabidopsis.