棉花枯萎病菌突变体库的构建及分析

[背景]棉花枯萎病逐渐成为威胁新疆海岛棉产业发展的主要病害,但关于棉花枯萎病菌的致病力、产孢量、生长速度及颜色变化等相关功能基因目前还不是十分明确.[目的]通过构建绿色荧光蛋白(green fluorescent protein,GFP)标记棉花枯萎病菌突变体库,筛选出由于T-DNA的随机插入而导致性状发生变异的突变体...     

Easy determination of ploidy level in Arabidopsis thaliana plants by means of pollen size measurement

Summary Cytogenetic examination of transgenic Arabidopsis thaliana (L.) Heynh. plants obtained by Agrobacterium-mediated gene transfer to cotyledon- and root-explants or by direct gene transfer into protoplasts revealed a high percentage of tetraploid or aneuploid transformants. Depending on the transformation procedure used, 13% (root explant transformation), 33% (cotyledon explant transformation), or 38% (direct gene transfer) of the transformants showed aberrant ploidy levels. A good correlation between the ploidy level of a plant and the size of its pollen grains was observed. This allows quick and simple testing of the ploidy level of transgenic Arabidopsis plants.Abbreviations AM Arabidopsis medium - ANOVA analysis of variance - DAPI 4,6-Diamidino-2-phenylindole - PEG polyethyleneglycol     

Efficient transformation of Arabidopsis thaliana using direct gene transfer to protoplasts

Summary Direct gene transfer has proved to be an efficient transformation method for arabidopsis thaliana, a member of the Brassicaceae. Transgenic Arabidopsis plants resistant to hygromycin B have been regenerated from mesophyll protoplasts treated with polyethylene glycol and plasmid DNA carrying the hygromycin phosphotransferase (HPT) gene under the control of the 35 S promoter of cauliflower mosaic virus. The transformation procedure reproducibly yields transformants at frequencies of approximately 1×10^-4 (based on the number of protoplasts treated) or 5% (based on the number of regenerating calli). DNA from plants regenerated from hygromycin resistant colonies was analysed by Southern blot hybridization demonstrating that the foreign gene is stably integrated into the plant chromosome. Genetic analysis of several hygromycin resistant plants showed that the HPT gene is transmitted to the progeny. Transformation experiments performed with a selectable and a non-selectable gene on separate plasmids resulted in a co-transformation rate of functionally active copies in about 25% of the transformants analysed. Hence this approach can be used to introduce non-selectable genes into the Arabidopsis genome.     

嫁接西瓜对西瓜枯萎病菌侵染的防御机制研究

为了揭示嫁接提高西瓜抗枯萎病的机制,该研究以嫁接西瓜为材料,采用扫描电镜观察了枯萎病菌侵染下寄主的组织结构变化,荧光定量分析了相关防卫基因的表达,比较了嫁接西瓜对枯萎病菌侵染的抗感反应。结果显示:(1)枯萎病菌侵染后,与自根西瓜相比,嫁接西瓜的根部木质部导管通过快速形成膜状物、侵填体及细胞壁增厚阻塞菌丝入侵;自根西瓜防御反应较嫁接西瓜晚,严重侵染时薄壁细胞降解,导管组织脱落导致维管系统空洞,从而使植株呈现萎蔫症状,该现象在嫁接西瓜中没有发现。(2)枯萎病菌侵染后,嫁接西瓜比自根西瓜具有较高的防卫基因表达水平,其中:嫁接西瓜中,CHI、APX和PPO基因的表达随枯萎病菌侵染时间的延长而升高,而PAL呈现先升高后降低的表达趋势,但仍高于本底表达;自根西瓜中,仅PPO基因在枯萎病菌侵染后表达上调,而其他基因的表达则是先升高后降低,与嫁接西瓜中的PAL基因表达一致。研究表明,嫁接植株一方面通过快速的组织结构响应,另一方面从转录水平提高了相关防卫基因的表达,最终使植株具有抗病性;推测防御基因在嫁接植株与枯萎病菌互作中的强烈诱导响应可能是嫁接植株抗病的分子机制之一。     

Management Fusarium wilt on melon and watermelon by Penicillium oxalicum

The potential of the biological control fungus Penicillium oxalicum to suppress wilt caused by Fusarium oxysporum f. sp. melonis and F. oxysporum f. sp. niveum on melon and watermelon, respectively, was tested under different growth conditions. The area under disease progress curve of F. oxysporum f. sp. melonis infected melon plants was significantly reduced in growth chamber and field experiments. In glasshouse experiments, it was necessary to apply P. oxalicum and dazomet in order to reduce Fusarium wilt severity in melons caused by F. oxysporum f. sp. melonis. For watermelons, we found that P. oxalicum alone reduced the area under the disease progress curve by 58% in the growth chamber experiments and 54% in the glasshouse experiments. From these results, we suggested that P. oxalicum may be effective for the management of Fusarium wilt in melon and watermelon plants.     

Development of a system for integrative and stable transformation of the zygomycete<Emphasis Type="Italic"> Rhizopus oryzae</Emphasis> by<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated DNA transfer

Two transformation systems, based on the use of CaCl₂/PEG and Agrobacterium tumefaciens, respectively, were developed for the zygomycete Rhizopus oryzae. Irrespective of the selection marker used, a pyr4 marker derived from R. niveus or a dominant amdS⁺ marker from Aspergillus nidulans, and irrespective of the configuration of the transforming DNA (linear or circular), the transformants obtained with the CaCl₂/PEG transformation method were found to carry multiple copies of tandemly linked vector molecules, which failed to integrate into the genomic DNA. Furthermore, these transformants displayed low mitotic stability. In contrast, transformants obtained by Agrobacterium-mediated transformation were mitotically stable, even under non-selective conditions. Detailed analysis of these transformants revealed that the transforming DNA had integrated into the genome of R. oryzae at a single locus in independently obtained transformants. In addition, truncation of the transforming DNA was observed, resulting in the integration of the R. niveus pyr4 marker gene, but not the second gene located on the transferred DNA. Modification of the transforming DNA, resulting in partial resistance to restriction enzyme digestion, was observed in transformants obtained with the CaCl₂/PEG transformation method, suggesting that a specific genome defence mechanism may exist in R. oryzae. It is likely that the unique mechanism used by A. tumefaciens to deliver its transferred DNA to its hosts facilitates bypass of the host defence mechanisms, thus allowing the DNA to integrate into the chromosomal genome.An erratum to this article can be found at Communicated by C. P. Hollenberg     

Occurrence and distribution of <Emphasis Type="Italic">Microdochium nivale</Emphasis> and <Emphasis Type="Italic">Fusarium</Emphasis> species isolated from barley,durum and soft wheat grains in France from 2000 to 2002

Fusarium Head Blight of small grain cereal is a disease of growing concern in Europe. Along with Microdochium nivale, several species of Fusarium may be associated with the disease, including species that are potentially toxigenic. This paper describes the results of a large scale survey of the variety and frequency of different Fusarium species and M. nivale in France. A total of 749 soft wheat, durum wheat and barley samples were collected and analyzed from 2000 to 2002. The most frequent species isolated were F.graminearum, F. avenaceum and F. poae. The frequency of F. poae seems to have increased while M.nivale and F. culmorum appear less frequent than previously described in France. Other Fusarium species detected in decreasing prevalence were F. tricinctum, F. equiseti, F. acuminatum, F. sambucinum, F.sporotrichioides, F. moniliforme, F. heterosporum, F. subglutinans and F. oxysporum. All the most frequent pathogenic species and also the less pathogenic ones were frequently associated with individual fields. The implications of these associations for the protection of cereals crops and for contamination by mycotoxins are discussed.     

The distribution ofFusarium species in soils planted to millet and sorghum in Lesotho,Nigeria and Zimbabwe

Samples from soils planted to millet and sorghum from Lesotho, Nigeria, and Zimbabwe were processed and a total of 3,291Fusarium cultures were recovered. Of these 1,296 cultures were isolated from plant debris and 1,995 cultures were recovered from soil dilutions. The most prevalent species recovered wereF. oxysporum (37%),F. equiset (30%),F. solani (14%),F. moniliforme (6%),F. compactum (5%),F. nygamai (4%), andF. chlamydosporum (2%). OtherFusarium species isolated wereF. merismoides, F. polyphialidicum, F. graminearum, F. subglutinans, F. sambucinum, F. longipes, F. semitectum, F. dimerum, F. lateritium, and a group of cultures designated as population A which resembleF. camptoceras. Fusarium equiseti was the predominant species in soil samples from Nigeria and Zimbabwe, whileF. oxysporum was the predominant species recovered from soil from Lesotho.Contribution No. 1881, Fusarium Research Center, Department of Plant Pathology, The Pennsylvania State University.     

Silicon Carbide Whisker-Mediated Embryogenic Callus Transformation of Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) and Regeneration of Salt Tolerant Plants

A silicon carbide whisker-mediated gene transfer system with recovery of fertile and stable transformants was developed for cotton (Gossypium hirsutum L.) cv. Coker-312. Two-month-old hypocotyl-derived embryogenic/non-embryogenic calli at different days after subculture were treated with silicon carbide whiskers for 2 min in order to deliver pGreen0029 encoding GUS gene and pRG229 AVP1 gene, encoding Arabidopsis vacuolar pyrophosphatase, having neomycin phosphotransferaseII (nptII) genes as plant-selectable markers. Three crucial transformation parameters, i.e., callus type, days after subculture and selection marker concentration for transformation of cotton calli were evaluated for optimum efficiency of cotton embryogenic callus transformation giving upto 94% transformation efficiency. Within six weeks, emergence of kanamycin-resistant (km^r) callus colonies was noted on selection medium. GUS and Southern blot analysis showed expression of intact and multiple transgene copies in the transformed tissues. Kanamycin wiping of leaves from T₁, T₂, and T₃ progeny plants revealed that transgenes were inherited in a Mendelian fashion. Salt treatment of T₁ AVP1 transgenic cotton plants showed significant enhancement in salt tolerance as compared to control plants. Thus far, this is first viable physical procedure after particle bombardment available for cotton that successfully can be used to generate fertile cotton transformants.     

Heterologous expression of IAP1, a seed protein from bean modified by indole-3-acetic acid,in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Medicago truncatula</Emphasis>

The seed protein IAP1 from bean (PvIAP1; Phaseolus vulgaris L.) that is modified by the phytohormone indole-3-acetic acid (IAA) was heterologously expressed in the two reference plant species Arabidopsis thaliana and Medicago truncatula. For the transformation of Medicago we devised a novel protocol using seedling infiltration. When PvIAP1 was overexpressed under the control of the constitutive 35SCaMV promoter in Arabidopsis, the plants showed signs of earlier bolting and enhanced branching. Expression of a fusion protein of PvIAP1 with both a green fluorescence protein (GFP) as reporter and 6× histidine (His) tag under the control of the native bean IAP1 promoter resulted in the accumulation of the protein in both plant species exclusively in seeds as shown by immunoblotting and by fluorescence microscopy. During seed development, PvIAP1 was first expressed in the vascular bundle of Arabidopsis, whereas in later stages GFP fluorescence was visible essentially in all tissues of the seed. Fluorescence decreased rapidly after imbibition in the seeds for both Arabidopsis and Medicago, although the fluorescence persisted longer in Arabidopsis. GFP fluorescence was distributed evenly between an organelle fraction, the microsomal membrane fraction, and the cytosol. This was also confirmed by immunoblot analysis. Clusters of higher GFP fluorescence were observed by confocal microscopy. Although PvIAP1 protein accumulated in seeds of both Arabidopsis and Medicago, neither species post-translationally modified the protein with an indoleacyl moiety as shown by quantitative GC–MS analysis after alkaline hydrolysis. These results indicate an apparent specificity for IAA attachment in different plant species. Alexander Walz and Claudia Seidel contributed equally to the paper.     

Optimization of biolistic bombardment parameters for <Emphasis Type="Italic">Dendrobium</Emphasis> Sonia 17 calluses using GFP and GUS as the reporter system

Genetic transformation system of Dendrobium Sonia 17 was optimized using green fluorescent protein (GFP) and -glucuronidase (GUS) gene as the reporter systems. The 35S-sgfp-TYG-nos (p35S) and pSMDFR, carrying sgfp and gusA gene, respectively, were co-bombarded into the calluses. Parameters optimized were acceleration pressure, target distance, gold particle size, pre-bombardment cultured time, plasmid DNA precipitation, total plasmid DNA and the ratio of the plasmids co-bombarded. Both reporter systems responded similarly to the bombardment parameters investigated. Based on the GUS/GFP spot counts, the GFP expression rate was higher than that for GUS under the control of the same promoter, CaMV 35S. GFP could be used as the reporter system for the co-bombardment as it was rapid and non-destructive system to monitor the transformed tissues. A combination of GFP and antibiotic resistance gene was used to select stable putative transformants.     

Molecular characterization of pathogenic Fusarium species in cucurbit plants from Kermanshah province, Iran

Fusarium is one of the important phytopathogenic genera of microfungi causing serious losses on cucurbit plants in Kermanshah province, the largest area of cucurbits plantation in Iran. Therefore, the objectives in this study were to isolate and identify disease-causing Fusarium spp. from infected cucurbit plants, to ascertain their pathogenicity, and to determine their phylogenetic relationships. A total of 100 Fusarium isolates were obtained from diseased cucurbit plants collected from fields in different geographic regions in Kermanshah province, Iran. According to morphological characters, all isolates were identified as Fusarium oxysporum, Fusarium proliferatum, Fusarium equiseti, Fusarium semitectum and Fusarium solani. All isolates of the five Fusarium spp. were evaluated for their pathogenicity on healthy cucumber (Cucumis sativus) and honeydew melon (Cucumis melo) seedlings in the glasshouse. F. oxysporum caused damping-off in 20–35 days on both cucurbit seedlings tested. Typical stem rot symptoms were observed within 15 days after inoculation with F. solani on both seedlings. Based on the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) restriction fragment length polymorphism (RFLP) analysis, the five Fusarium species were divided into two major groups. In particular, isolates belonging to the F. solani species complex (FSSC) were separated into two RFLP types. Grouping among Fusarium strains derived from restriction analysis was in agreement with criteria used in morphological classification. Therefore, the PCR-ITS-RFLP method provides a simple and rapid procedure for the differentiation of Fusarium strains at species level. This is the first report on identification and pathogenicity of major plant pathogenic Fusarium spp. causing root and stem rot on cucurbits in Iran.     

Gene targeting in Arabidopsis thaliana.

Summary Gene targeting of a chromosomally integrated transgene in Arabidopsis thaliana is reported. A chimeric gene consisting of the promoter of the 35S RNA of CaMV, the polyadenylation signal of the octopine synthase gene and the coding region of the bacterial hygromycin phosphotransferase gene (hpt), which was rendered non-functional by deletion of 19 bp, was introduced into the genome of A. thaliana using Agrobacterium-mediated gene transfer. A total of 3.46 x 10⁸ protoplasts isolated from 17 independent transgenic Arabidopsis lines harbouring the defective chimeric hpt gene were transformed via direct gene transfer using various DNA forms containing only the intact coding region of the hpt gene. Out of 150 hygromycin-resistant colonies appearing in the course of these experiments, four were the result of targeted recombination of the incoming DNA with the defective chromosomal locus as revealed by PCR and Southern blot analysis. Comparison with the number of transformants obtained when an hpt gene controlled by a promoter and terminator from the nopaline synthase gene was employed results in a maximal ratio of homologous to non-homologous transformation in A. thaliana of 1 x 10^–4.     

A large-scale<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated transformation procedure with a strong positive-negative selection for gene targeting in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A large-scale transformation procedure handling an adequate number of stable transformants with highly efficient positive-negative selection is a necessary prerequisite to successful gene targeting by homologous recombination, as the integration of a transgene by somatic homologous recombination in higher plants has been reported to be 10^-3 to 10^-5 compared with random integration by non-homologous end joining. We established an efficient and large-scale Agrobacterium-mediated rice transformation protocol that generated around 10³ stable transformants routinely from 150 seeds and a strong positive-negative selection procedure that resulted in survivors at 10^-2 using the gene for diphtheria toxin A fragment as a negative marker. The established transformation procedure provides a basis for efficient gene targeting in rice.Abbreviations AS: Acetosyringone - 5-FU: 5-Fluorouracil - FW: Fresh weight - GT: Gene targeting - HR: Homologous recombination - NHEJ: Non-homologous end joining Communicated by H. Ebinuma     

Stable transformation and regulated expression of an inducible reporter construct inCandida albicans using restriction enzyme-mediated integration

To allow the regulated expression of cloned genes inCandida albicans, a plasmid was constructed using the inducible promoter of theC. albicans MAL2 gene. To demonstrate that theMAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of theC. albicans URA3 gene. This plasmid was introduced into a Ura^– strain ofC. albicans using the process of restriction enzyme-mediated integration (REMI). This procedure involves the transformation of theBamHI-linearized plasmid in the presence ofBamHI enzyme. The majority of transformants generated contained insertions of the plasmid at chromosomalBamHI sites. All transformants examined were inducible forURA3 expression, which was determined by growth analysis and by measuring the level ofURA3 gene product activity. The Ura⁺ phenotype of the transformants was stable during growth under nonselective conditions. This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes inC. albicans.Deceased, December 15, 1995     

Overexpression of barley <Emphasis Type="Italic">hva1</Emphasis> gene in creeping bentgrass for improving drought tolerance

The objectives of this study were to test the feasibility of introducing barley hva1 gene, a LEA3 member, into perennial grass species using the Agrobacterium-mediated transformation technique and to determine whether heterologous expression of hva1 would alleviate water-deficit injury in grass species. Creeping bentgrass (Agrostis stolonifera var. palustris), a drought-intolerant grass species, was transformed transiently or stably using three different promoters in conjunction with the downstream report/target genes. Two abscisic acid (ABA)-inducible promoters, ABA1 and ABA2 derived from ABA-response complex (ABRC3) were used to examine stress-responsive expression of the green fluorescent protein (GFP). Transient expression of GFP demonstrated the inducibility of ABA1 and ABA2 promoters in response to exogenous ABA application. The ABA2 promoter was further studied for stress-responsive expression of hva1 and a maize Ubi-1 promoter was tested for constitutive expression of the gene. In the T₀ generation, the Ubi-1::hva1 transformants displayed variable expression levels of HVA1 protein under normal growth conditions. The hva1 gene in the ABA2::hva1 transformants maintained low expression under well-watered conditions, but was upregulated under water-deficit conditions. The tolerance to water deficit of T₀ transgenic lines was assessed by measuring leaf relative water content and visually rating the severity of leaf wilting during to water stress. Under water-stressed conditions, some transgenic lines maintained high water content in leaves and showed significantly less extent of leaf wilting compared with non-transgenic control plants. These results indicated that the introduction of barley hva1 gene using constitutive or stress-inducible promoters lessened water-deficit injury in creeping bentgrass, suggesting that heterologous expression of LEA3 protein genes may enhance the survival ability of creeping bentgrass in water limiting environments.     

Heterologous expression of Fusarium oxysporum tomatinase in Saccharomyces cerevisiae increases its resistance to saponins and improves ethanol production during the fermentation of Agave tequilana Weber var. azul and Agave salmiana must

This paper describes the effect of the heterologous expression of tomatinase from Fusarium oxysporum f. sp lycopersici in Saccharomyces cerevisiae. The gene FoTom1 under the control of the S. cerevisiae phosphoglycerate kinase (PGK1) promoter was cloned into pYES2. S. cerevisiae strain Y45 was transformed with this vector and URA3 transformant strains were selected for resistance to α-tomatine. Two transformants were randomly selected for further study (designated Y45-1 and Y45-2). Control strain Y45 was inhibited at 50 μM α-tomatine, in contrast, transformants Y45-1 and Y45-2 did not show inhibition at 200 μM. Tomatinase activity was detected by HPLC monitoring tomatine disappearance and tomatidine appearance in the supernatants of culture medium. Maximum tomatinase activity was observed in the transformants after 6 h, remaining constant during the following 24 h. No tomatinase activity was detected in the parental strain. Moreover, the transformants were able to grow and produce ethanol in a mix of Agave tequilana Weber var. azul and Agave salmiana must, contrary to the Y45 strain which was unable to grow and ferment under these conditions.