Transcriptome analysis in the midgut of the earthworm (Eisenia andrei) using expressed sequence tags

In order to gain insight into the expression profiles of the earthworm midgut, we analyzed 1106 expressed sequence tags (ESTs) derived from the earthworm midgut cDNA library. Among the 1106 ESTs analyzed, 557 (50.4%) ESTs showed significant similarity to known genes and represented 229 unique genes of which 166 ESTs were singletons and 63 ESTs manifest as two or more ESTs. While 552 ESTs (49.9%) were sequenced only once, 230 ESTs (20.8%) appeared two to five times and 324 ESTs (29.3%) were sequenced more than five times. Considering this redundancy of expression, it is likely that the gene expression profile of the earthworm midgut would be polarized. The expression of globin-related proteins, including ferritin and linker chain, and fibrinolytic enzymes appeared to account for 10.1% and 4.7% of the total ESTs analyzed in this study, respectively. This suggests that the prime functions of the midgut in the earthworm would be associated with protein hydrolysis as well as globin formation. Among the recognized protein-coding genes, the gene category involved in protein synthesis appeared to be the largest one accounting for 15.6% of the expression in the midgut, followed by gene categories associated with energy (11.2%), homeostasis (10.8%), metabolism (3.6%), cytoskeleton (2.5%), and protein fate (1.4%). With regard to functional aspects, the most abundantly expressed genes were associated with respiratory pigment (10.1%), cellular respiration (8.6%), and fibrin hydrolysis (4.7%). In addition, we were able to identify novel ESTs in the earthworm, which were related to the innate immune system, including destabilase, a possible antagonist of transglutaminase.     

Transcriptome generation and analysis from spleen of Indian catfish, Clarias batrachus (Linnaeus, 1758) through normalized cDNA library

Catfishes are commercially important fish for both the fisheries and aquaculture industry. Clarias batrachus, an Indian catfish species is economically important owing to its high demand. A normalized cDNA library was constructed from spleen of the Indian catfish to identify genes associated with immune function. One thousand nine hundred thirty seven ESTs were submitted to the GenBank with an average read length of approximately 700 bp. Clustering analysis of ESTs yielded 1,698 unique sequences, including 184 contigs and 1,514 singletons. Significant homology to known genes was found by homology searches against data in GenBank in 576 (34 %) ESTs, including similarity to functionally annotated unigenes for 158 ESTs. Additionally, 433 ESTs revealed similarity to unigenes and ESTs in the dbEST but the remaining 658 EST sequences (39 %) did not match any sequence in GenBank. Of a total of 1,698 ESTs generated, 65 ESTs were found to be associated with immune functions. Gene Ontology and KEGG pathway analyses of C. batrachus ESTs collectively revealed a preponderance of immune relevant pathways apart from the presence of pathways involved in protein processing, localization, folding and protein degradation. This study constitutes first EST analysis of lymphoid organ in aquaculturally important Indian catfish species and could pave the way for further research of immune-related genes and functional genomics in this catfish.     

Development of EST derived SSRs and SNPs as a genomic resource in Indian catfish, <Emphasis Type="Italic">Clarias batrachus</Emphasis>

Clarias batrachus, an Indian catfish species, is endemic to the Indian subcontinent and potential cultivable species. The genomic resources in C. batrachus in the form of ESTs containing microsatellite repeats (EST-SSR) and single nucleotide polymorphisms (SNPs) that are associated with the expressed genes from spleen were mined. From a total of 1,937 ESTs generated, 1,698 unique sequences were obtained, out of which 221 EST-SSRs were identified and 54% could be functionally annotated by similarity searches. A total of 23 contigs containing 3 or more ESTs were found to contain 31 SNP loci, out of which 8 ESTs showed similarity to genes of known function and 1 for hypothetical protein. Nine ESTs with SSRs and/or SNPs identified in this study were reported to be associated with diseases in human and animals. These identified loci can be developed into markers in C. batrachus, which can be useful in linkage mapping, comparative genomics studies and for its genetic improvement programmes.     

Use of a large-scale Triticeae expressed sequence tag resource to reveal gene expression profiles in hexaploid wheat (Triticum aestivum L.).

The US Wheat Genome Project, funded by the National Science Foundation, developed the first large public Triticeae expressed sequence tag (EST) resource. Altogether, 116,272 ESTs were produced, comprising 100,674 5' ESTs and 15 598 3' ESTs. These ESTs were derived from 42 cDNA libraries, which were created from hexaploid bread wheat (Triticum aestivum L.) and its close relatives, including diploid wheat (T. monococcum L. and Aegilops speltoides L.), tetraploid wheat (T. turgidum L.), and rye (Secale cereale L.), using tissues collected from various stages of plant growth and development and under diverse regimes of abiotic and biotic stress treatments. ESTs were assembled into 18,876 contigs and 23,034 singletons, or 41,910 wheat unigenes. Over 90% of the contigs contained fewer than 10 EST members, implying that the ESTs represented a diverse selection of genes and that genes expressed at low and moderate to high levels were well sampled. Statistical methods were used to study the correlation of gene expression patterns, based on the ESTs clustered in the 1536 contigs that contained at least 10 5' EST members and thus representing the most abundant genes expressed in wheat. Analysis further identified genes in wheat that were significantly upregulated (p < 0.05) in tissues under various abiotic stresses when compared with control tissues. Though the function annotation cannot be assigned for many of these genes, it is likely that they play a role associated with the stress response. This study predicted the possible functionality for 4% of total wheat unigenes, which leaves the remaining 96% with their functional roles and expression patterns largely unknown. Nonetheless, the EST data generated in this project provide a diverse and rich source for gene discovery in wheat.     

Classification of sequences expressed during the primordial and basidiome stages of the cultivated mushroom Agaricus bisporus

Two complementary DNA (cDNA) libraries were constructed from tissues isolated from primordia and basidiomes of Agaricus bisporus to characterize genes involved in mushroom development. Using single-pass sequencing of 869 cDNA clones, we found 477 expressed sequence tags (ESTs), including 466 not previously described in the databases for A. bisporus. A BLASTX search revealed that 374 ESTs had similarities with protein sequences available from databases; 193 of these ESTs were categorized according to their putative function. Most ESTs were assigned to one of four roles: metabolism (23%), cell structure (15%), cell growth and division (12%), and protein destination and storage (10%). The remaining ESTs with putative homologues were classified in 10 additional categories. Many ESTs could not be functionally assigned. Based on redundancy levels, at least 4 ESTs were preferentially expressed in each tissue type. Sequence analysis also suggested the presence of paralog tyrosinase genes in the A. bisporus genome.     

Immune gene discovery by expressed sequence tag (EST) analysis of hemocytes in the ridgetail white prawn Exopalaemon carinicauda

The ridgetail white prawn Exopalaemon carinicauda is one of the most important commercial species in eastern China. However, little information of immune genes in E. carinicauda has been reported. To identify distinctive genes associated with immunity, an expressed sequence tag (EST) library was constructed from hemocytes of E. carinicauda. A total of 3411 clones were sequenced, yielding 2853 ESTs and the average sequence length is 436 bp. The cluster and assembly analysis yielded 1053 unique sequences including 329 contigs and 724 singletons. Blast analysis identified 593 (56.3%) of the unique sequences as orthologs of genes from other organisms (E-value < 1e-5). Based on the COG and Gene Ontology (GO), 593 unique sequences were classified. Through comparison with previous studies, 153 genes assembled from 367 ESTs have been identified as possibly involved in defense or immune functions. These genes are categorized into seven categories according to their putative functions in shrimp immune system: antimicrobial peptides, prophenoloxidase activating system, antioxidant defense systems, chaperone proteins, clottable proteins, pattern recognition receptors and other immune-related genes. According to EST abundance, the major immune-related genes were thioredoxin (141, 4.94% of all ESTs) and calmodulin (14, 0.49% of all ESTs). The EST sequences of E. carinicauda hemocytes provide important information of the immune system and lay the groundwork for development of molecular markers related to disease resistance in prawn species.     

应用大规模测序技术和生物信息学研究造血干/祖细胞的基因表达及新基因的识别和克隆

从新生儿脐血和成人骨髓中分选出造血干／祖细胞（ＨＳＣ／ＨＰＣ）,构建成ｃＤＮＡ文库,对其进行大规模表达序列标签（ＥＳＴ）测序,通过生物信息学等手段分析基因表达谱,并进行新基因的全长ｃＤＮＡ克隆。在所测的１０５１２条可分析ＥＳＴ序列中,有９８６６条来自脐血ＣＤ３４＋细胞,其中４６９７条（４７６％）为已知基因,２６０３条（２６４％）为已知ＥＳＴ,１４１５条（１４３％）代表未知ＥＳＴ。在已知基因中,８２％基因与造血相关,２２７％涉及细胞代谢、结构和迁移,１３０％与细胞分裂和防御相关,２６２％与ＲＮＡ、蛋白质的合成相关,１０６％和细胞信号传递有关。对一些已知和未知的ＥＳＴ,综合测序、生物信息学等方法,进行全长克隆,已获得２３个新基因的全长ｃＤＮＡ。     

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.     

EST analysis of mRNAs expressed during embryogenesis in Gallus gallus

Chicken Expressed Sequence Tags (ESTs) were analyzed to identify genes associated with myogenesis during embryonic development. A total of 6,184 ESTs were generated from three cDNA libraries constructed from whole embryos (Stage 26), somites associated with neural tube (Stage 15), and limb buds (Stages 21, 24 and 26). Clustering and assembly of 4,998 valid ESTs resulted in 2,329 unique sequences with 902 clusters (38.7%) and 1,427 singletons (61.3%). There are more than 400,000 chicken ESTs available at GenBank and we were able to identify 143 novel sequences. From these, 45 sequences found either a human EST homolog or a match with conserved regions among proteins. Most of these sequences were found to be expressed in somites, an important tissue for muscle development and not characterized before. This study revealed the value of micro dissected embryonic libraries for describing gene expression profiles associated with myogenesis and gene discovery.