中国地方品种香猪的肌肉特异组织表达序列标签(ESTs)的

通过构建香猪肌肉组织cDNA文库,并在文库中随机挑选克隆进行测序的方法,获得了131个香猪肌肉EST序列.在这131个EST序列所代表的109个单一克隆中,有99个为人类及其他物种的同源序列,3个为已知的猪的ESTs,7个为未知ESTs.对这10个已知、未知ESTs进行开放阅读框预测并进行B1ast分析,没有找到高度同源的氨基酸序列.对上述EST所对应的基因功能分析结果表明,除去27.27%的EST未能分类外,克隆到的EST大多来自与基因/蛋白的表达调控相关的基因(占45.46%).来自具有其他功能的基因的EST依次是细胞代谢占10.10%、细胞结构/迁移占10.10%、细胞/机体防御占5.05%和细胞信号/传导占2.02%.没有发现和细胞分裂相关的已知功能基因.本研究结果为中国地方品种香猪提供了第一个骨骼肌的基因表达谱,为今后寻找猪肌肉生长和肉用品质的候选基因奠定了基础.     

Expressed Sequences from Conidial, Mycelial, and Sexual Stages ofNeurospora crassa

In theNeurosporaGenome Project at the University of New Mexico, expressed sequence tags (ESTs) corresponding to three stages of the life cycle of the filamentous fungusNeurospora crassaare being analyzed. The results of a pilot project to identify expressed genes and determine their patterns of expression are presented. 1,865 partial complementary DNA (cDNA) sequences for 1,409 clones were determined using single-pass sequencing. Contig analysis allowed the identification of 838 unique ESTs and 156 ESTs present in multiple cDNA clones. For about 34% of the sequences, highly or moderately significant matches to sequences (of known and unknown function) in the NCBI database were detected. Approximately 56% of the ESTs showed no similarity to previously identified genes. Among genes with assigned function, about 43.3% were involved in metabolism, 32.9% in protein synthesis and 8.4% in RNA synthesis. Fewer were involved in defense (6%), cell signalling (3.4%), cell structure (3.4%) and cell division (2.6%).     

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。     

Development of Expressed Sequence Tags from the Bay Scallop, Argopecten irradians irradians

The bay scallop, Argopecten irradians irradians, introduced from North America, has become one of the most important aquaculture species in China. Inan effort to identify scallop genes involved in host defense, a high-quality cDNA library was constructed from whole body tissues of the bay scallop. A total of 5828 successful sequencing reactions yielded 4995 expressed sequence tags (ESTs) longer than 100 bp. Cluster and assembly analyses of the ESTs identified 637 contigs (consisting of 2853 sequences) and 2142 singletons, totaling 2779 unique sequences. Basic Local Alignment Search Tool (BLAST) analysis showed that the majority (73%) of the unique sequences had no significant homology (E-value ≤ 0.005) to sequences in GenBank. Among the 748 sequences with significant GenBank matches, 160 (21.4%) were for genes related to metabolism, 131 (17.5%) for cell/organism defense, 124 (16.6%) for gene/protein expression, 83 (11.1%) for cell structure/motility, 70 (9.4%) for cell signaling/communication, 17 (2.3%) for cell division, and 163 (21.8%) matched to genes of unknown functions. The list of host-defense genes included many genes with known and important roles in innate defense such as lectins, defensins, proteases, protease inhibitors, heat shock proteins, antioxidants, and Toll-like receptors. The study provides a significant number of ESTs for gene discovery and candidate genes for studying host defense in scallops and other molluscs.     

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。     

Analysis of expressed sequence tags from two starvation, time-of-day-specific libraries of Neurospora crassa reveals novel clock-controlled genes

In an effort to determine genes that are expressed in mycelial cultures of Neurospora crassa over the course of the circadian day, we have sequenced 13,000 cDNA clones from two time-of-day-specific libraries (morning and evening library) generating approximately 20,000 sequences. Contig analysis allowed the identification of 445 unique expressed sequence tags (ESTs) and 986 ESTs present in multiple cDNA clones. For approximately 50% of the sequences (710 of 1431), significant matches to sequences in the National Center for Biotechnology Information database (of known or unknown function) were detected. About 50% of the ESTs (721 of 1431) showed no similarity to previously identified genes. We hybridized Northern blots with probes derived from 26 clones chosen from contigs identified by multiple cDNA clones and EST sequences. Using these sequences, the representation of genes among the morning and evening sequences, respectively, in most cases does not reflect their expression patterns over the course of the day. Nevertheless, we were able to identify four new clock-controlled genes. On the basis of these data we predict that a significant proportion of the expressed Neurospora genes may be regulated by the circadian clock. The mRNA levels of all four genes peak in the subjective morning as is the case with previously identified ccgs.     

Analysis of genes expressed during rice-Magnaporthe grisea interactions.

Expressed sequence tag (EST) analysis was applied to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe grisea and fungal genes involved in growth within the host during a compatible interaction. A total of 511 clones was sequenced from a cDNA library constructed from rice leaves (Oryza sativa cv. Nipponbare) infected with M. grisea strain 70-15 to generate 296 nonredundant ESTs. The sequences of 293 clones (57.3%) significantly matched National Center for Biotechnology Information database entries; 221 showed homologies with previously identified plant genes and 72 with fungal genes. Among the genes with assigned functions, 32.8% were associated with metabolism, 29.4% with cell/organism defense or pathogenicity, and 18.4% with gene/protein expression. cDNAs encoding a type I metallothionein (MTs-1) of rice and a homolog of glucose-repressible gene 1 (GRG1) of Neurospora crassa were the most abundant representatives of plant and fungal genes, comprising 2.9 and 1.6% of the total clones, respectively. The expression patterns of 10 ESTs, five each from rice and M. grisea, were analyzed. Five defense-related genes in rice, including four pathogenesis-related genes and MTs-1, were highly expressed during M. grisea infection. Expression of five stress-inducible or pathogenicity-related genes of the fungus, including two hydrophobin genes, was also induced during growth within the host. Further characterization of the genes represented in this study would be an aid in unraveling the mechanisms of pathogenicity of M. grisea and the defense responses of rice.     

Expressed sequence tag analysis of the soybean rust pathogen Phakopsora pachyrhizi

Soybean rust is caused by the obligate fungal pathogen Phakopsora pachyrhizi Sydow. A unidirectional cDNA library was constructed using mRNA isolated from germinating P. pachyrhizi urediniospores to identify genes expressed at this physiological stage. Single pass sequence analysis of 908 clones revealed 488 unique expressed sequence tags (ESTs, unigenes) of which 107 appeared as multiple copies. BLASTX analysis identified 189 unigenes with significant similarities (Evalue<10(-5)) to sequences deposited in the NCBI non-redundant protein database. A search against the NCBI dbEST using the BLASTN algorithm revealed 32 ESTs with high or moderate similarities to plant and fungal sequences. Using the Expressed Gene Anatomy Classification, 31.7% of these ESTs were involved in primary metabolism, 14.3% in gene/protein expression, 7.4% in cell structure and growth, 6.9% in cell division, 4.8% in cell signaling/cell communication, and 4.8% in cell/organism defense. Approximately 29.6% of the identities were to hypothetical proteins and proteins with unknown function.     

Generation of a total of 6483 expressed sequence tags from 60 day-old bovine whole fetus and fetal placenta

Expressed sequence tags (ESTs) generated based on characterization of clones isolated randomly from cDNA libraries are used to study gene expression profiles in specific tissues and to provide useful information for characterizing tissue physiology. In this study, two directionally cloned cDNA libraries were constructed from 60 day-old bovine whole fetus and fetal placenta. We have characterized 5357 and 1126 clones, and then identified 3464 and 795 unique sequences for the fetus and placenta cDNA libraries: 1851 and 504 showed homology to already identified genes, and 1613 and 291 showed no significant matches to any of the sequences in DNA databases, respectively. Further, we found 94 unique sequences overlapping in both the fetus and the placenta, leading to a catalog of 4165 genes expressed in 60 day-old fetus and placenta. The catalog is used to examine expression profile of genes in 60 day-old bovine fetus and placenta.     

Identification by subtractive suppression hybridization of bacteria-induced genes expressed in Manduca sexta fat body

Insect immune processes are mediated by programs of differential gene expression. To understand the molecular regulation of the immune response in the tobacco hornworm, Manduca sexta, the relevant subset of differentially expressed genes of interest must be identified, cloned and studied in detail. In this study, suppression subtractive hybridization, a PCR-based method for cDNA subtraction was performed to identify mRNAs from fat body of immunized larvae that are not present (or present at a low level) in control larvae. A subtracted cDNA library enriched in immune-inducible genes was constructed. Northern blot analysis of a sample of clones from our subtracted library indicated that >90% of the clones randomly selected from the subtracted library are immune inducible. Sequence analysis of 238 expressed sequence tags (ESTs) revealed that 120 ESTs, representing 54 distinct genes or gene families, had sequences identical or similar to previously characterized genes, some of which have been confirmed to be involved in innate immunity. These ESTs were categorized into seven groups, including pattern recognition proteins, serine proteinases and their inhibitors, and antimicrobial proteins. 112 ESTs, about 47.5% of the library, showed no significant similarity to any known genes. The sequences identified in this M. sexta library reflect our knowledge of insect immune strategies and may facilitate better understanding of insect immune responses.     

Expressed sequence tags from the zhikong scallop (Chlamys farreri): Discovery and annotation of host-defense genes

A high-quality cDNA library was constructed from whole body tissues of the zhikong scallop, Chlamys farreri, challenged by Listonella anguillarum. A total of 5720 clones were sequenced, yielding 5123 expressed sequence tags (ESTs). Among the 3326 unique genes identified, 2289 (69%) genes had no significant (E-value < 1e?5) matches to known sequences in public databases and 194 (6%) matched proteins of unknown functions. The remaining 843 (25%) genes that exhibited homology with genes of known functions, showed broad involvement in metabolic processes (31%), cell structure and motility (20%), gene and protein expression (12%), cell signaling and cell communication (8%), cell division (4%), and notably, 25% of those genes were related to immune function. They included stress response genes, complement-like genes, proteinase and proteinase inhibitors, immune recognition receptors and immune effectors. The EST collection obtained in this study provides a useful resource for gene discovery and especially for the identification of host-defense genes and systems in scallops and other molluscs.     

Analysis of expressed sequence tags of flower buds in Lotus japonicus.

In order to study gene expression in a reproductive organ, we constructed a cDNA library of mature flower buds in Lotus japonicus, and characterized expressed sequence tags (ESTs) of 842 clones randomly selected. The EST sequences were clustered into 718 non-redundant groups. From BLAST and FASTA search analyses of both protein and DNA databases, 58.5% of the EST groups showed significant sequence similarities to known genes. Several genes encoding these EST clones were identified as pollen-specific genes, such as pectin methylesterase, ascorbate oxidase, and polygalacturonase, and as homologous genes involved in pollen-pistil interaction. Comparison of these EST sequences with those derived from the whole plant of L. japonicus, revealed that 64.8% of EST sequences from the flower buds were not found in EST sequences of the whole plant. Taken together, the EST data from flower buds generated in this study is useful in dissecting gene expression in floral organ of L. japonicus.     

Analysis of expressed sequence tags of the water flea Daphnia magna.

To study gene expression in the water flea Daphnia magna we constructed a cDNA library and characterized the expressed sequence tags (ESTs) of 7210 clones. The EST sequences clustered into 2958 nonredundant groups. BLAST analyses of both protein and DNA databases showed that 1218 (41%) of the unique sequences shared significant similarities to known nucleotide or amino acid sequences, whereas the remaining 1740 (59%) showed no significant similarities to other genes. Clustering analysis revealed particularly high expression of genes related to ATP synthesis, structural proteins, and proteases. The cDNA clones and EST sequence information should be useful for future functional analysis of daphnid biology and investigation of the links between ecology and genomics.     

Cloning and characterization of chitin synthase gene fragments from Penicillium chrysogenum

Abstract We constructed a 3'-directed cDNA library of cleistothecia and Hülle cells of Aspergillus nidulans to examine gene expression patterns of the sexual structures and to have probes necessary to isolate sexual structure-specific genes. Sequencing of 360 randomly selected cDNA clones yielded 272 expressed sequence tags (ESTs), most of which probably represent frequently or less expressed genes in sexual structures of A. nidulans . Among the 272 ESTs, 33 ESTs (87 cDNA clones) appeared more than once and 2 ESTs appeared 6 times; 9 ESTs matched GenBank entries. When compared with sequences obtained from a mycelial 3'-directed cDNA library of A. nidulans , 28 out of 33 ESTs seem to be sexual structure-specific. Northern blot analyses of 20 ESTs showed that 17 are sexual structure-specific. The remaining three ESTs also hybridized with RNA isolated from vegetative mycelia. These results suggest that analyses of ESTs from different cell types or tissues can readily demonstrate gene expression patterns of specific cell types and identify cell type-specific cDNA probes.     

Generation of 919 expressed sequence tags from immature flower buds and gene expression analysis using expressed sequence tags in the model plant Lotus japonicus

Lotus japonicus has received increased attention as a potential model legume plant. In order to study gene expression in reproductive organs and to identify genes that play a crucial function in sexual reproduction, we constructed a cDNA library from immature flower buds containing anthers at the stage of developing tapetum cells in L. japonicus, and characterized 919 expressed sequence tags (ESTs) randomly selected from a cDNA library of the immature flower buds. The 919 ESTs analyzed were clustered into 821 non-redundant EST groups. As a result of a database search, 436 groups (53%) out of the 821 groups showed sequence similarity to genes registered in the public database. Out of these 436 groups, 109 groups showed similarity to genes encoding hypothetical proteins whose function had not yet been estimated. Three hundred eighty five groups (47%) showed no significant homology to known sequences and were classified as novel sequences. A comparison of 821 non-redundant EST sequences and EST sequences derived from the whole plant L. japonicus revealed that 474 EST sequences derived from immature flower buds were not found in the EST sequences of the whole plant. In order to confirm the expression pattern of potential reproductive-organ specific EST clones, nine clones, which were not matched to ESTs derived from the whole plant, were selected, and RT-PCR analysis was performed on these clones. As a result of RT-PCR, we found two novel anther specific clones. One clone was homologous to a gene encoding human cleft lip and palate associated transmembrane protein (CLPTM1) like protein, and the other clone did not show a significant similarity to any genes deposited in the public database. These results indicate that ESTs analyzed here represent a valuable resource for finding reproductive-organ specific genes in Lotus japonicus.