利用有序差异显示技术克隆受稻瘟病菌诱导表达的水稻cDNA的研究

利用有序差异显示技术(ODD)分析受稻瘟病菌诱导表达的水稻基因,通过反式RNA印迹进行辅助筛选,获得了37个在接种稻瘟病菌后表达量增强的水稻cDNA克隆.采用RNA印迹对其中5个克隆在接种稻瘟病菌后的表达分析表明,这些克隆在抗病以及感病的水稻株系中都具有诱导表达的特点.根据序列同源性分析,与这些克隆序列相应的同源基因可能涉及抑制病原菌生长、清除真菌毒素、传递抗病信号以及调节宿主生理状态等几方面的功能.     

Identification of stress-induced genes from the drought-tolerant plant Prosopis juliflora (Swartz) DC. through analysis of expressed sequence tags.

Abiotic stresses such as cold, salinity, drought, wounding, and heavy metal contamination adversely affect crop productivity throughout the world. Prosopis juliflora is a phreatophyte that can tolerate severe adverse environmental conditions such as drought, salinity, and heavy metal contamination. As a first step towards the characterization of genes that contribute to combating abiotic stress, construction and analysis of a cDNA library of P. juliflora genes is reported here. Random expressed sequence tag (EST) sequencing of 1750 clones produced 1467 high-quality reads. These clones were classified into functional categories, and BLAST comparisons revealed that 114 clones were homologous to genes implicated in stress response(s) and included heat shock proteins, metallothioneins, lipid transfer proteins, and late embryogenesis abundant proteins. Of the ESTs analyzed, 26% showed homology to previously uncharacterized genes in the databases. Fifty-two clones from this category were selected for reverse Northern analysis: 21 were shown to be upregulated and 16 downregulated. The results obtained by reverse Northern analysis were confirmed by Northern analysis. Clustering of the 1467 ESTs produced a total of 295 contigs encompassing 790 ESTs, resulting in a 54.2% redundancy. Two of the abundant genes coding for a nonspecific lipid transfer protein and late embryogenesis abundant protein were sequenced completely. Northern analysis (after polyethylene glycol stress) of the 2 genes was carried out. The implications of the analyzed genes in abiotic stress tolerance are also discussed.     

Xylose metabolism in the anaerobic fungus<Emphasis Type="Italic"> Piromyces</Emphasis> sp. strain E2 follows the bacterial pathway

The anaerobic fungus Piromyces sp. strain E2 metabolizes xylose via xylose isomerase and d-xylulokinase as was shown by enzymatic and molecular analyses. This resembles the situation in bacteria. The clones encoding the two enzymes were obtained from a cDNA library. The xylose isomerase gene sequence is the first gene of this type reported for a fungus. Northern blot analysis revealed a correlation between mRNA and enzyme activity levels on different growth substrates. Furthermore, the molecular mass calculated from the gene sequence was confirmed by gel permeation chromatography of crude extracts followed by activity measurements. Deduced amino acid sequences of both genes were used for phylogenetic analysis. The xylose isomerases can be divided into two distinct clusters. The Piromyces sp. strain E2 enzyme falls into the cluster comprising plant enzymes and enzymes from bacteria with a low G+C content in their DNA. The d-xylulokinase of Piromyces sp. strain E2 clusters with the bacterial d-xylulokinases. The xylose isomerase gene was expressed in the yeast Saccharomyces cerevisiae, resulting in a low activity (25±13 nmol min^–1mg protein^-1). These two fungal genes may be applicable to metabolic engineering of Saccharomyces cerevisiae for the alcoholic fermentation of hemicellulosic materials.     

Identification of fungal (Magnaporthe grisea) stress-induced genes in wild rice (Oryza minuta)

To identify fungal stress-related genes in wild rice, Oryza minuta, we constructed a subtracted library using suppression subtractive hybridization in combination with mirror orientation selection. DNA chips containing 960 randomly selected cDNA clones were applied by reverse Northern analysis to eliminate false positive clones from the library and to prescreen differentially expressed genes. In total, 377 cDNA clones were selected on the basis of their signal intensities and expression ratios. Sequence analyses of these 377 cDNA fragments revealed that 180 of them (47.7%) represented unique genes. Of these180 cDNAs, 89 clones (49.6%) showed significant homologies to previously known genes, while the remaining 91 did not match any known sequences. The putative functions of the 180 unique ESTs were categorized by aligning them with MIPS data. They were classified into seven different groups using microarray data-derived expression patterns and verified by Northern blotting.Abbreviations ER: Endoplasmic reticulum - EST: Expressed sequence tag - MIPS: Munich Information Center for Protein Sequences - MOS: Mirror orientation selection - NCBI: National Center for Biotechnology Information - omfi: Oryza minuta fungal-stress induced - PCD: Programmed cell death - PDI: Proteins disulfide isomerase - SSH: Suppression subtractive hybridization Communicated by I.S. ChungK.S. Shim and S.K. Cho contributed equally to this work.     

Identification of heavy metal-induced genes encoding glutathione S-transferases in the arbuscular mycorrhizal fungus Glomus intraradices

Arbuscular mycorrhizal fungi are able to alleviate the stress for plants caused by heavy metal contamination of soil. To analyze the molecular response of arbuscular mycorrhizal fungi to these pollutants, a subtractive cDNA library was constructed using RNA from Glomus intraradices extraradical hyphae of a root organ culture treated with a mixture of Cd, Zn, and Cu. Screening by reverse Northern blot analysis indicated that, among 308 clones, 17% correspond to genes up-regulated by heavy metals. Sequence analysis of part of the clones resulted, amongst others, in the identification of six genes putatively coding for glutathione S-transferases belonging to two different classes of these enzymes. Expression analyses indicated that the genes are differentially expressed during fungal development and that their RNA accumulation dramatically increases in extraradical hyphae grown in a heavy metal-containing solution.     

小麦幼苗期水分胁迫所诱导基因表达谱的初步分析

利用抑制差减杂交(Suppression Subtractive Hybridization,SSH)和高密度点阵膜技术研究小麦2叶幼苗期水分胁迫诱导表达基因。通过筛选具有1530个克隆的SSH文库,获得181个阳性克隆。序列同源性比较和功能查询结果发现,83．2％的水分胁迫诱导表达基因分别与不同逆境胁迫条件下表达的基因具有较高的同源性,这些基因在生物体内的功能都是直接或间接对细胞遭受逆境胁迫起保护作用。其中17个EST未找到同源性较高的匹配序列,已经在GenBank注册。用反向Northern、RT-PCR和Northern进一步检验所获得的功能已知EST,初步建立了小麦幼苗期水分胁迫诱导的基因表达谱。     

Mercury-induced genes in Arabidopsis thaliana: identification of induced genes upon long-term mercuric ion exposure

Mercuric‐ion‐induced gene expression was studied in Arabidopsis thaliana Columbia wild type. Rosettes of plants grown for 21 d on agar medium supplemented with 20, 30 and 40 µm HgCl₂ were pooled and used to isolate cDNAs of induced genes by suppression subtractive hybridization. Of the 576 clones isolated initially, 31 turned out to be mercury‐induced by Northern hybridization. However, kinetic studies using cDNA arrays clearly showed that seven genes were exclusively mercuric‐ion‐induced, 14 were induced by mercury but also affected by a diurnal rhythm, and 10 clones were only modulated by the day–night cycle. The expression levels of the metal‐induced genes increased from 1·5‐fold to 10‐fold. Functional classification resulted in genes encoding proteins for the photosynthetic apparatus and for the antioxidative system. In addition, unexpected genes, whose connection to mercury ion stress is not evident, were identified.     

Molecular characterization and genomic organization of low molecular weight glutenin subunit genes at the Glu-3 loci in hexaploid wheat (Triticum aestivum L.)

In this study, we report on the molecular characterization and genomic organization of the low molecular weight glutenin subunit (LMW-GS) gene family in hexaploid wheat (Triticum aestivum L.). Eighty-two positive BAC clones were identified to contain LMW-GS genes from the hexaploid wheat ‘Glenlea’ BAC library via filter hybridization and PCR validation. Twelve unique LMW glutenin genes and seven pseudogenes were isolated from these positive BAC clones by primer-template mismatch PCR and subsequent primer walking using hemi-nested touchdown PCR. These genes were sequenced and each consisted of a single-open reading frame (ORF) and untranslated 5′ and 3′ flanking regions. All 12 LMW glutenin subunits contained eight cysteine residues. The LMW-m-type subunits are the most abundant in hexaploid wheat. Of the 12 LMW-GS, 1, 2 and 9 are i-type, s-type and m-type, respectively. The phylogenetic analysis suggested that the LMW-i type gene showed greater differences to LMW-s and LMW-m-type genes, which, in turn, were more closely related to one another. On the basis of their N-terminal sequences, they were classified into nine groups. Fingerprinting of the 82 BAC clones indicated 30 BAC clones assembled into eight contigs, while the remaining clones were singletons. BAC end sequencing of the 82 clones revealed that long terminal repeat (LTR) retrotransposons were abundant in the Glu-3 regions. The average physical distance between two adjacent LMW-GS genes was estimated to be 81 kb. Most of LMW-GS genes are located in the d-genome, suggesting that the Glu-D3 locus is much larger than the Glu-B3 locus and Glu-A3 locus. Alignments of sequences indicated that the same type (starting with the same N-terminal sequence) LMW-GS genes were highly conserved in the homologous genomes between hexaploid wheat and its donors such as durum wheat and T. tauschii. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of rice tryptophan decarboxylases and their direct involvement in serotonin biosynthesis in transgenic rice

l-Tryptophan decarboxylase (TDC) and l-tyrosine decarboxylase (TYDC) belong to a family of aromatic l-amino acid decarboxylases and catalyze the conversion of tryptophan and tyrosine into tryptamine and tyramine, respectively. The rice genome has been shown to contain seven TDC or TYDC-like genes. Three of these genes for which cDNA clones were available were characterized to assign their functions using heterologous expression in Escherichia coli and rice (Oryza sativa cv. Dongjin). The purified products of two of the genes were expressed in E. coli and exhibited TDC activity, whereas the remaining gene could not be expressed in E. coli. The recombinant TDC protein with the greatest TDC activity showed a K _m of 0.69 mM for tryptophan, and its activity was not inhibited by phenylalanine or tyrosine, indicating a high level of substrate specificity toward tryptophan. The ectopic expression of the three cDNA clones in rice led to the abundant production of the products of the encoded enzymes, tyramine and tryptamine. The overproduction of TYDC resulted in stunted growth and a lack of seed production due to tyramine accumulation, which increased as the plant aged. In contrast, transgenic plants that produced TDC showed a normal phenotype and contained 25-fold and 11-fold higher serotonin in the leaves and seeds, respectively, than the wild-type plants. The overproduction of either tyramine or serotonin was not strongly related to the enhanced synthesis of tyramine or serotonin derivatives, such as feruloyltyramine and feruloylserotonin, which are secondary metabolites that act as phytoalexins in plants.     

A comparative approach to study inhibition of grazing and lipid composition of a toxic and non-toxic clone of Chrysochromulina polylepis (Prymnesiophyceae)

Since the massive bloom in 1988 in the North Sea, the prymnesiophyte flagellate Chrysochromulina polylepis Manton et Parke has been known for its ichtyotoxicity. Laboratory experiments using two different clones of C. polylepis were conducted in a comparative approach. Both clones were nearly similar in size and shape, but differed in their toxicity, as demonstrated by the Artemia bioassay. In order to study the effects of toxic C. polylepis on protozooplankton grazers, grazing experiments were performed with the heterotrophic dinoflagellate Oxyrrhis marina Dujardin as grazer. A first experiment was carried out in order to follow batch culture growth and initial grazing of O. marina when fed toxic or non-toxic clones of C. polylepis. Ingestion of the toxic clone was 27% of ingestion when fed with the non-toxic clone. When O. marina was fed with the toxic clone, vacuoles within O. marina contained fewer food particles per cell and the cells attained slower division rate (58% of the division rate estimated for the non-toxic clone). A second experiment was conducted to determine the grazing and growth response of O. marina as a function of algal food concentration. Profound differences in ingestion, clearance, division and gross growth efficiency of O. marina when fed the two clones of C. polylepis again were apparent. However, even at algal concentrations of 400×10³ ml⁻¹, O. marina is not killed by the presence or by the ingestion of toxic C. polylepis, indicating that the toxin deters grazers. In addition to grazing experiments, lipid classes and fatty acids of both algal clones were analysed and compared in order to follow the hypothesis that toxicity of C. polylepis is caused by liposaccharides, lipids, or fatty acids. However, the chemical composition with respect to lipid classes and fatty acids of both clones were quite similar, making an involvement of these substances in the toxicity towards Artemia and O. marina unlikely.     

Eine Mutation bei der siphonalen GrünalgeDerbesia marina

Zusammenfassung 1. In einer aus Zoosporen vonDerbesia marina entstandenen Gametophytengeneration trat eine einzelne Pflanze vom Habitus des Sporophyten auf. Ihre Fäden entsprangen einer zentralen kugeligen Anschwellung.2. Diese Mutante erzeugt aus geschlechtlich neutralen Schwärmern immer wieder gleichartige Generationen.3. Die Mutante ist haploid (vgl.Neumann 1969).4. Die Befunde bestätigenFeldmanns (1952) Hypothese der Entstehung neuer Arten durch Trennung und selbständige Entwicklung von Gliedern eines Lebenszyklus.

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Mutation in the siphonaceous green algaDerbesia marina

Over 15 years the heteromorphous alternation of generations inDerbesia marina proved to be obligatory. Only once has a single sporophyte-like plant been found amidst hundreds of gametophytes. Unlike the normal sporophyte, the filaments of this mutant originated from a central globule. Its neutral zoospores re-created the special morphological character of the mother plant. 12Derbesia generations succeeded one another within two years. Characteristic features of theDerbesia mutant are haploidy (Neumann 1969) and absence of sexuality. The observation from the culture experiment is important for our understanding of taxonomic relationships within algal genera, confirmingFeldmann's (1952) hypothesis regarding the origin of new algal species by independent development of life cycle members.

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Herrn Prof. Dr.A. Bückmann zum 70. Geburtstag gewidmet.