Comparative analysis of sequences expressed during the liquid-cultured mycelia and fruit body stages of Pleurotus ostreatus

To characterize genes involved in fruit body development, two complementary DNA (cDNA) libraries were constructed from RNA isolated from liquid-cultured mycelia and fruit bodies of Pleurotus ostreatus. Using single-pass sequencing of cDNA clones, 952 and 1069 expressed sequence tags (ESTs) were generated from liquid-cultured mycelia and fruit body cDNA library, respectively. A BLASTX search revealed that 390 of the liquid-cultured mycelia ESTs (41%) and 531 of the fruit body ESTs (50%) showed significant similarity to protein sequences described in the nonredudant database (E values < or =1 x 10(-5)). When liquid-cultured mycelia and fruit body ESTs were compared by the SeqMan II program, among the total of 2021 ESTs, 1256 ESTs were unigenes, and 66 unigenes (5.3%) were commonly expressed during both stages. The functional catalogs of the ESTs were made by comparison with functionally identified Saccharomyces cerevisiae genes. Liquid-cultured mycelium ESTs were compared with fruit body ESTs and changes of the expressed genes during fruit body development were analyzed.     

日本通草蛉cDNA文库构建及部分ESTs分析

本研究以日本通草蛉Chrysoperla nipponensis (Okamoto)为材料,采用Oligo(dT)引物定向克隆构建cDNA文库并进行EST序列测定,旨在以基因库的形式进行种质资源的保存,为其遗传改良奠定基础,并为探讨其分类地位提供分子依据。对该文库质量分析表明：库容量为1.0×10⁶,重组率为80.0％,平均插入片段为512 bp。测序后最终成功得到323条表达序列标签（expressed sequence tags,ESTs）序列,经Phrap程序聚类拼接后得到236条单基因簇（unigene）,包括86个重叠群（congtigs）和150个单拷贝（singlets）。使用NCBI中的BlastN和BlastX程序对236条ESTs进行本地化搜索,BlastN的结果表明：180条ESTs（76.3%）没有注解,56条ESTs（23.7%）与GenBank上公布的序列有较高的同源性,其中一条序列被确定为该种的16S rRNA基因,利用MEGA软件构建了基于该16S rRNA序列草蛉科的系统发育树,结果显示通草蛉属Chrysoperla与叉草蛉属Dichochrysa、玛草蛉属Mallada、草蛉属Chrysopa的亲缘关系比较近,这与传统分类相吻合。BlastX的比对结果为197条ESTs（83.5%）有功能注解,39条ESTs（16.5%）无注解或score值小于100。使用GO(gene ontology)数据库对236条ESTs序列进行功能注释,结果表明：142条ESTs（59.7％）有注解,并表达出40多种基因产物。     

Large-scale identification of expressed sequence tags involved in rice and rice blast fungus interaction

To better understand the molecular basis of the defense response against the rice blast fungus (Magnaporthe grisea), a large-scale expressed sequence tag (EST) sequencing approach was used to identify genes involved in the early infection stages in rice (Oryza sativa). Six cDNA libraries were constructed using infected leaf tissues harvested from 6 conditions: resistant, partially resistant, and susceptible reactions at both 6 and 24 h after inoculation. Two additional libraries were constructed using uninoculated leaves and leaves from the lesion mimic mutant spl11. A total of 68,920 ESTs were generated from 8 libraries. Clustering and assembly analyses resulted in 13,570 unique sequences from 10,934 contigs and 2,636 singletons. Gene function classification showed that 42% of the ESTs were predicted to have putative gene function. Comparison of the pathogen-challenged libraries with the uninoculated control library revealed an increase in the percentage of genes in the functional categories of defense and signal transduction mechanisms and cell cycle control, cell division, and chromosome partitioning. In addition, hierarchical clustering analysis grouped the eight libraries based on their disease reactions. A total of 7,748 new and unique ESTs were identified from our collection compared with the KOME full-length cDNA collection. Interestingly, we found that rice ESTs are more closely related to sorghum (Sorghum bicolor) ESTs than to barley (Hordeum vulgare), wheat (Triticum aestivum), and maize (Zea mays) ESTs. The large cataloged collection of rice ESTs in this study provides a solid foundation for further characterization of the rice defense response and is a useful public genomic resource for rice functional genomics studies.     

柔嫩艾美耳球虫孢子发育阶段虫体抑制性消减文库的构建

为了分离鉴定柔嫩艾美耳球虫(Eimeria tenella)孢子发育阶段虫体的差异表达基因,分别以柔嫩艾美耳球虫未孢子化卵囊和孢子化卵囊为驱动组、子孢子为实验组,或未孢子化卵囊为驱动组、孢子化卵囊为实验组,利用抑制性消减杂交(SSH)技术,构建了2个子孢子cDNA消减文库和1个孢子化卵囊cDNA消减文库。随机从3个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个子孢子cDNA消减文库的重组率都为96%,孢子化卵囊cDNA消减文库的重组率为98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示:从孢子化卵囊cDNA消减文库中获得了13个单一有效序列,其中8个EST与已知蛋白同源性很高;从2个子孢子cDNA消减文库中共获得了40个单一有效序列,其中9个EST与已知蛋白同源,其余可能为柔嫩艾美耳球虫的新基因。这些结果为分离柔嫩艾美耳球虫新功能基因和进一步探索防治球虫病的方法提供了理论基础。     

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&#1089839;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。     

日本血吸虫期别差异表达基因文库的构建及分析

为从期别差异表达基因分析入手研究血吸虫的生长发育机制,应用抑制性消减杂交 (suppressed subtractive hybridization , SSH) 技术首次构建了日本血吸虫尾蚴、虫卵和成虫的期别差异表达基因文库 . 经消减效率分析和三种文库克隆的 EST 的期别差异性鉴定,表明所建文库质量较高,为在整个基因组水平分离血吸虫的差异表达基因提供了重要材料 . 由三个文库选择 257 个插入片段大于 500 bp 的克隆测定了 EST 序列 . 同源性分析结果表明 257 个 EST 代表 182 种血吸虫基因,其中有 22 种为血吸虫已知基因,有 128 种为血吸虫已知 EST ,有 32 种为新发现的血吸虫基因 . 对 EST 编码蛋白的功能预测结果显示：尾蚴消减文库的基因多与运动、能量代谢、转录调节及致病性相关;虫卵消减文库的基因可能参与信号转导、细胞粘附、蛋白质和碳水化合物的代谢以及抗氧化反应;成虫消减文库的基因多参与蛋白质的合成、转运及分解代谢,参与虫体的运动等 . 大规模分离、分析血吸虫期别差异表达基因将对从分子水平去解读血吸虫的生长发育机制,筛选高效疫苗候选抗原、药物靶标及诊断制剂有重要意义 .     

Comparative analysis of expressed sequence tags (ESTs) from Triticum monococcum shoot apical meristem at vegetative and reproductive stages

Triticum monococcum has recently drawn the attention of biologists to discover and utilize novel genes and alleles. To explore the molecular features of the genetic network governing floral transition in shoot apical meristem (SAM) of spring growth habit T. monococcum, two expressed sequence tag (EST) libraries containing 3,031 ESTs from vegetative SAM (VS) and 2,647 ESTs from early reproductive SAM (RS) were analyzed. Assembly of ESTs resulted in 2,303 unigenes for VS library (368 contigs and 1,935 singletons) and 1,890 unigenes (337 contigs and 1,553 singletons) for RS library. The 67.05 % of VS unigenes and 66.30 % of RS unigenes showed significant similarity with genes of known, putative and or unknown function, whereas the remaining 32.95 % of the VS unigenes and 33.7 % of RS unigenes displayed no significant match with the public protein database. The 1,064 and 866 unigenes of VS and RS libraries were assigned to functional categories using Pageman ontology tool. Further analysis revealed that the switch from VS to RS caused significant changes in the abundance of unigenes assigned to some functional categories. A total of 37 genes were identified which were significantly differentially expressed between vegetative and reproductive stages of T. monococcum SAM. Investigation of the differentially expressed genes revealed the importance of the genes involved in energy metabolism, ubiquitin/26S proteasome system, polyamines biosynthesis and signaling of reactive oxygen species in SAM differentiation towards floral transition in T. monococcum.     

Expressed Sequence Tags from Amoebophrya sp. Infecting Karlodinium veneficum: Comparing Host and Parasite Sequences

ABSTRACT. Parasitic dinoflagellates of the genus Amoebophrya play important roles in the ecology of estuaries and open ocean environments. Little is known of the cell and molecular biology of Amoebophrya , but the genus is intermediate on phylogenetic trees between apicomplexans and typical dinophycean dinoflagellates. Here, we constructed four cDNA libraries, from different stages after infecting the host, Karlodinium veneficum , with Amoebophrya sp. These libraries were used to generate 898 expressed sequence tags (ESTs), with sequences attributed to either the host or parasite, based on AT bias, codon usage, and occurrence during infection. Overall, 209 sequences were attributable to the parasite and 685 to the host. The 50 putative parasite sequences with good protein matches in GenBank were used to find the same protein from host ESTs. For 26 genes, both host and parasite sequences were identified, of which 20 encoded ribosomal proteins. PCR for seven predicted parasite and two host genes were used to confirm attributions. The most common host and parasite ESTs were compared to see if multiple gene copies were present. The host plastocyanin gene had multiple sequence variants, but parasite rps 27 a contained only one polymorphism, likely due to an amplification error. Amplification, cloning, and sequencing of five parasite protein-coding genes suggested that the parasite has a single sequence for each gene, but three host genes were found to have multiple variants. The genome of Amoebophrya sp. infecting K. veneficum appears to have an organization more similar to other eukaryotes than to the tandem gene arrangements found in dinoflagellates.     

The Dictyostelium developmental cDNA project: generation and analysis of expressed sequence tags from the first-finger stage of development.

In an effort to identify and characterize genes expressed during multicellular development ill Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNA from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, library S, consisting of 9984 clones, carries relatively short inserts, and the other, library L, which consists of 8448 clones, has longer inserts. We sequenced all the selected clones in library S from their 3'-ends, and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs hit known Dictyostelium genes and 910 showed significant similarity to genes of Dictyostelium and other organisms. For library L, 1132 clones were randomly sequenced and 471 non-redundant ESTs were obtained. In combination, the ESTs from the two libraries represent approximately 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will provide a useful resource for investigating the genetic networks that regulate multicellular development of this organism.     

Potato Expressed Sequence Tag Generation and Analysis using Standard and Unique cDNA Libraries

To help develop an understanding of the genes that govern the developmental characteristics of the potato (Solanum tuberosum), as well as the genes associated with responses to specified pathogens and storage conditions, The Canadian Potato Genome Project (CPGP) carried out 5′ end sequencing of regular, normalized and full-length cDNA libraries of the Shepody potato cultivar, generating over 66,600 expressed sequence tags (ESTs). Libraries sequenced represented tuber developmental stages, pathogen-challenged tubers, as well as leaf, floral developmental stages, suspension cultured cells and roots. All libraries analysed to date have contributed unique sequences, with the normalized libraries high on the list. In addition, a low molecular weight library has enhanced the 3′ ends of our sequence assemblies. Using the combined assembly dataset, unique tuber developmental, cold storage and pathogen-challenged sequences have been identified. A comparison of the ESTs specific to the pathogen-challenged tuber and foliar libraries revealed minimal overlap between these libraries. Mixed assemblies using over 189,000 potato EST sequences from CPGP and The Institute for Genomics Research (TIGR) has revealed common sequences, as well as CPGP- and TIGR-unique sequences. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Analysis of Schistosoma mansoni genes using the expressed sequence tag approach

Expressed sequence tags (ESTs) are partial cDNA sequences read from both ends of random expressed gene fragments used for discovering new genes. DNA libraries from four different developmental stages of Schistosoma mansoni used in this study generated 141 ESTs representing about 2.5% of S. mansoni sequences in dbEST. Sequencing was done by the dideoxy chain termination method. The sequences were submitted to GenBank for homology searching in nonredundant databases using Basic Local Alignment Search Tool for DNA (BLASTN) alignment and for protein (BLASTX) alignment at the National Center for Biotechnology Information (NCBI). Among submitted ESTs, 29 were derived from lambdagt11 sporocyst library, 70 from lambdaZap adult worm library, 31 from lambdaZap cercarial library, and 11 from lambdaZap female B worm library. Homology search revealed that eight (5.6%) ESTs shared homology to previously identified S.mansoni genes in dbEST, 15 (10.6%) are homologous to known genes in other organisms, 116 (81.7%) showed no significant sequence homology in the databases, and the remaining sequences (2.1%) showed low homologies to rRNA or mitochondrial DNA sequences. Thus, among the 141 ESTs studied, 116 sequences are derived from noval, uncharactarized S. mansoni genes. Those 116 ESTs are important for identification of coding regions in the sequences, helping in mapping of schistosome genome, and identifying genes of immunological and pharmacological significance.     

Comparative analysis of genes expressed in regenerating intestine and non-eviscerated intestine of Apostichopus japonicus Selenka (Aspidochirotida: Stichopodidae) and cloning of ependymin gene

The regeneration of the intestine of sea cucumber (Apostichopus japonicus) was studied by describing historically the changes that occurred during intestine regeneration on the fifth day after chemically-induced evisceration. An expressed sequence tag (EST) analysis was undertaken to identify major genes, which might be involved in intestine regeneration of A. japonicus. Two cDNA libraries were constructed with directional cloning method, one for regenerating intestine collected on the third, fourth and fifth day after evisceration (post-evisceration, PE), and the other for the non-eviscerated (NE). A total of 730 ESTs were generated by sequencing cDNA clones from the two libraries (372 from PE and 358 from NE). The results showed that the number of genes that were involved in primary metabolism of PE library was less than that of NE library, while the number of genes involved in cell defense/immunity, cell division, cell signal transduction/communication of PE library was more than that of NE library. The results also revealed that the expression of the genes which might be involved in regeneration was enhanced to some extent after evisceration. Only about 11.54% of the sequenced clones were shared by two libraries, which provided some clues for the existence of differential gene expression between PE and NE intestines. A gene named epenAj was also characterized in this study.