Identification of expressed genes during infection of Chinese cabbage (Brassica rapa subsp. pekinensis) by Plasmodiophora brassicae

Plasmodiophora brassicae is an obligate, biotrophic pathogen causing the club-root disease of crucifers. Despite its importance as a plant pathogen, little is known about P. brassicae at the molecular level as most of its life cycle takes place inside the plant host, and axenic culturing is impossible. Discovery of genes expressed during infection and gene organization are the first steps toward a better understanding of the pathogen-host interaction. Here, suppression subtractive hybridization was used to search for the P. brassicae genes expressed during plant infection. One-hundred and forty ESTs were found of which 49% proved to be P. brassicae genes. Ten novel P. brassicae genes were identified, and the genomic sequences surrounding four of the ESTs were acquired using genome walking. Alignment of the ESTs and the genomic DNA sequences confirmed that P. brassicae genes are intron rich and that the introns are small. These results show that it is possible to discover new P. brassicae genes from a mixed pool of both plant and pathogen cDNA. The results also revealed that some of the P. brassicae genes expressed in Chinese cabbage (Brassica rapa subsp. pekinensis) were identical to the genes expressed in the infection of Arabidopsis plants, indicating that these genes play an important role in P. brassicae infection.     

A new set of Arabidopsis expressed sequence tags from developing seeds. The metabolic pathway from carbohydrates to seed oil

Large-scale single-pass sequencing of cDNAs from different plants has provided an extensive reservoir for the cloning of genes, the evaluation of tissue-specific gene expression, markers for map-based cloning, and the annotation of genomic sequences. Although as of January 2000 GenBank contained over 220,000 entries of expressed sequence tags (ESTs) from plants, most publicly available plant ESTs are derived from vegetative tissues and relatively few ESTs are specifically derived from developing seeds. However, important morphogenetic processes are exclusively associated with seed and embryo development and the metabolism of seeds is tailored toward the accumulation of economically valuable storage compounds such as oil. Here we describe a new set of ESTs from Arabidopsis, which has been derived from 5- to 13-d-old immature seeds. Close to 28,000 cDNAs have been screened by DNA/DNA hybridization and approximately 10,500 new Arabidopsis ESTs have been generated and analyzed using different bioinformatics tools. Approximately 40% of the ESTs currently have no match in dbEST, suggesting many represent mRNAs derived from genes that are specifically expressed in seeds. Although these data can be mined with many different biological questions in mind, this study emphasizes the import of photosynthate into developing embryos, its conversion into seed oil, and the regulation of this pathway.     

高山植物黄管秦艽cDNA文库构建与表达序列标签（EST）分析

黄管秦艽（Gentiana officinalis）是一种重要的藏药高山植物,本研究构建了该物种开花期的eDNA文库。经检测达到中等cDNA文库水平,文库滴度为1．2×10^7pfu／ml,重组率95．9％,插入片段平均长度大于500bp。对343个随机挑选的重组克隆进行部分测序,获得的ESTs经编辑后共有181条有效序列。经生物信息学方法分析181条表达序列标签（EST）代表144个单克隆序列,其中55个与已鉴定的基因同源,35个序列与未鉴定的EST匹配,54个未找到同源序列;后两者共有89个EST序列未发现功能相似的蛋白。对已鉴定的EST进行功能分析发现,相关基因主要编码以下蛋白：与蛋白表达相关的占35％;光合作用相关的占笠％;新陈代谢相关的占18％;抗性相关的占11％;质膜运输和细胞分裂相关的分别占5％;染色体变化和细胞信号转导的分别占2％。根据有效EST序列设计引物,通过RT-PCR进一验证了所得EST的准确性。这些研究结果为将来研究黄管秦艽的功能基因以及该物种与相关物种的群体遗传学、进化生物学等方面提供了基础。     

Gene expression profiling of the plant pathogenic basidiomycetous fungus Rhizoctonia solani AG 4 reveals putative virulence factors

Rhizoctonia solani is a ubiquitous basidiomycetous soilborne fungal pathogen causing damping-off of seedlings, aerial blights and postharvest diseases. To gain insight into the molecular mechanisms of pathogenesis a global approach based on analysis of expressed sequence tags (ESTs) was undertaken. To get broad gene-expression coverage, two normalized EST libraries were developed from mycelia grown under high nitrogen-induced virulent and low nitrogen/methylglucose-induced hypovirulent conditions. A pilot-scale assessment of gene diversity was made from the sequence analyses of the two libraries. A total of 2280 cDNA clones was sequenced that corresponded to 220 unique sequence sets or clusters (contigs) and 805 singlets, making up a total of 1025 unique genes identified from the two virulence-differentiated cDNA libraries. From the total sequences, 295 genes (38.7%) exhibited strong similarities with genes in public databases and were categorized into 11 functional groups. Approximately 61.3% of the R. solani ESTs have no apparent homologs in publicly available fungal genome databases and are considered unique genes. We have identified several cDNAs with potential roles in fungal pathogenicity, virulence, signal transduction, vegetative incompatibility and mating, drug resistance, lignin degradation, bioremediation and morphological differentiation. A codon-usage table has been formulated based on 14694 R. solani EST codons. Further analysis of ESTs might provide insights into virulence mechanisms of R. solani AG 4 as well as roles of these genes in development, saprophytic colonization and ecological adaptation of this important fungal plant pathogen.     

Identification and characterization of differentially expressed ESTs of Gossypium barbadense infected by Verticillium dahliae with suppression subtractive hybridization

Cotton wilt defense reaction is a complicated continuous process and involves a battery of genes. In this study, we adopted suppression subtractive hybridization (SSH) technique to isolate differentially expressed ESTs from Gossypium barbadense variety 7124 during Verticillium wilt defense process. An array of 1165 clones from the subtractive library has been screened with reverse northern blotting, of which 131 ESTs were considered as over-expressed and 16 ESTs were down-regulated. Sequence analysis and blast search showed that 83 ESTs were homologous to 45 unique sequences in the databases. Among all these differentially expressed ESTs, at least three kinds of genes were characterized. The majority of ESTs with deduced identity to aerobic metabolism enzymes strongly expressed in the infection process. Likewise, ESTs similar to those reported for pathogen-related protein genes were also picked out in this study. These ESTs in combination with other kinase-like genes and a defensin-like EST constituted an assembly of genes responded during pathogens' infection. These results imply that sea-island cotton undergoes strong oxidative stress and results in a series of defense responses when attacked by V. dahliae. To our knowledge, this is the first report on the isolation of global ESTs during sea-island cotton defense reaction.     

高山植物黄管秦艽cDNA 文库构建与表达序列标签(EST) 分析

黄管秦艽( Gentiana officinalis) 是一种重要的藏药高山植物, 本研究构建了该物种开花期的cDNA 文库。经检测达到中等cDNA 文库水平, 文库滴度为1 . 2×107 pfu􊄯ml , 重组率95.9% , 插入片段平均长度大于500 bp。对343 个随机挑选的重组克隆进行部分测序, 获得的ESTs 经编辑后共有181 条有效序列。经生物信息学方法分析181 条表达序列标签(EST) 代表144 个单克隆序列, 其中55 个与已鉴定的基因同源, 35 个序列与未鉴定的EST 匹配, 54 个未找到同源序列; 后两者共有89 个EST 序列未发现功能相似的蛋白。对已鉴定的EST进行功能分析发现, 相关基因主要编码以下蛋白: 与蛋白表达相关的占35%; 光合作用相关的占22%; 新陈代谢相关的占18%; 抗性相关的占11%; 质膜运输和细胞分裂相关的分别占5% ; 染色体变化和细胞信号转导的分别占2%。根据有效EST 序列设计引物, 通过RT-PCR 进一验证了所得EST 的准确性。这些研究结果为将来研究黄管秦艽的功能基因以及该物种与相关物种的群体遗传学、进化生物学等方面提供了基础。     

Sequence analysis of expressed sequence tags from an ABA-treated cDNA library identifies stress response genes in the moss Physcomitrella patens.

Partial cDNA sequencing was used to obtain 169 expressed sequence tags (ESTs) in the moss, Physcomitrella patens. The source of ESTs was a random cDNA library constructed from 7 day-old protonemata following treatment with 10(-4) M abscisic acid (ABA). Analysis of the ESTs identified 69% with homology to known sequences, 61% of which had significant homology to sequences of plant origin. More importantly, at least 11 ESTs had significant similarities to genes which are implicated in plant stress-responses, including responses which may involve ABA. These included a cDNA associated with desiccation tolerance, two heat shock protein genes, one cold acclimation protein cDNA and five others that may be involved in either oxidative or chemical stress or both, i.e., Zn/Cu-superoxide dismutase, NADPH protochlorophyllide oxidoreductase (PorB), selenium binding protein, glutathione peroxidase and glutathione S transferase. Analysis of codon usage between P. patens and seed plants indicated that although mosses and higher plants are to a large extent similar, minor variations also exists that may represent the distinctiveness of each group.     

Analysis of expressed genes in ethylene-treated potato leaves using expressed sequence tags

To isolate useful and interesting plant genes in large quantities, random sequencing of cDNA clones from potato leaf library treated with ethylene was performed. Partial sequences of randomly selected 210 clones with the insert of longer than 500 base pair (bp) as well as poly (A) tail have been compared with sequences in GeneBank, EMBL and DDBJ nucleic acid databases and fostered 193 expressed sequence tags (ESTs). The 210 cDNA clones identified are related to various aspect of metabolic pathways such as glycolysis, amino acid synthesis, translation mechanism, ribosome synthesis, hormone response, stress response, regulation of gene expression, and signal transduction. Among the 193 ESTs, 12 ESTs (29 cDNA clones) appeared more than once and 181 ESTs appeared once regarded as a solitary group. Out of 210 clones, 29 clones (13.8%) have no similarity to the known nucleotide sequences and could serve as a potentially useful resource for plant molecular biology referring to particular genes. Nucleotide sequencing to generate more ESTs from ethylene-induced as well as non-induced potato leaf is in progress as well.     

Generation of 7137 non-redundant expressed sequence tags from a legume, Lotus japonicus.

For comprehensive analysis of genes expressed in a model legume, Lotus japonicus, a total of 22,983 5' end expressed sequence tags (ESTs) were accumulated from normalized and size-selected cDNA libraries constructed from young (2 weeks old) plants. The EST sequences were clustered into 7137 non-redundant groups. Similarity search against public non-redundant protein database indicated that 3302 groups showed similarity to genes of known function, 1143 groups to hypothetical genes, and 2692 were novel sequences. Homologues of 5 nodule-specific genes which have been reported in other legume species were contained in the collected ESTs, suggesting that the EST source generated in this study will become a useful tool for identification of genes related to legume-specific biological processes. The sequence data of individual ESTs are available at the web site: http://www.kazusa.or.jp/en/plant/lotus/EST/.