Global Identification of Significantly Expressed Genes in Developing Endosperm of Rice by Expression Sequence Tags and cDNA Array Approaches

Rice endosperm plays a very important role in seedling germination and determines the qualities of rice grain. Although studies on specific gene categories in endosperm have been carried out, global view of gene expression at a transcription level In rice endosperm Is still limited. To gain a better understanding of the global and tissue-specific gene expression profiles In rice endosperm, a cDNA library from rice endosperm of immature seeds was sequenced. A cDNA array was constructed based on the tentative unique transcripts derived from expression sequence tag （EST） assembling results and then hybridized with cDNAs from five different tissues or organs including endosperm, embryo, leaf, stem and root of rice. Significant redundancy was found for genes encoding prolamin, glutelin, allergen, and starch synthesis proteins, accounting for ~34% of the total ESTs obtained. The cDNA array revealed 87 significantly expressed genes In endosperm compared with the other four organs or tissues. These genes included 13 prolamin family proteins, 17 glutelin family proteins, 12 binding proteins, nine catalytic proteins and four ribosomal proteins, indicating a complicated biological processing in rice endosperm. In addition, Northern verification of 1,4-alpha-glucan branching enzyme detected two isoforms in rice endosperm, the larger one of which only existed in endosperm.

THE ABNORMAL EXPRESSION OF CLONED REPEATED SEQUENCE DNA, L5B-4, IN RAT HEPATOMA BERH-2

A repeated sequence DNA fragment, L5B-4, was cloned from the 5 kb BamHI DNA fragments of rat genomic DNA. The expressions of the L5B-4 DNA fragment are different in liver and hepatoma cells. The amounts of transcripts in hepatoma cells are lower in nucleus and higher in cytoplasm, especially in polysomal RNA, as compared with that in liver cells. The alteration shown in polysomal RNA of hepatoma cells seems to be specific. These results are discussed with respect to the possible function of this repeated DNA and its variation in hepatoma cells.

Full-length transcriptome sequence and SSR marker development for genetic diversity research in yellowfin seabream Acanthopagrus latus

Yellowfin seabream Acanthopagrus latus is an important economic fish in Chinese coastal areas.Given its narrow distribution and overfishing,the genetic diversity of yellowfin seabream has been restricted for artificial breeding and reproduction.We performed full-length transcriptome sequencing and assembly of the genome of yellowfin seabream.A total of 68086 unigenes were obtained,with an N50 of 3391 bp on average length of 2933 bp.A total number of 50593 expressed sequence tags linked to simple sequence repeats(EST-SSR)were identified,among them dinucleotide repeats(40.6%)and AC/GT motifs(38.5%)were the most frequent.Of the 190 EST-SSRs for which PCR primer pairs were designed,150 primer pairs successfully amplified target loci and 15 SSRs showed high polymorphism.The alleles per locus ranged 6-50 on average of 25.3.The expected and observed heterozygosity varied from 0.632 to 0.969 and from 0.519 to 0.953,respectively.The polymorphic index content(PIC)values of each locus ranged 0.587-0.966 on average of 0.851.Among six yellowfin seabream population samples preliminarily tested for genetic diversity and differentiation,the Fangchenggang(FCG)population in Guangxi Province had the highest mean observed heterozygosity(H_(o))value(0.786),whereas the Zhangzhou(ZZ)population in Fujian Province had the lowest(0.678).The pairwise fixation index(Fst)values indicated significant population differentiation among six yellowfin seabream populations.This study provided evidence for the usefulness of the transcriptomic resource information and EST-SSR markers for natural resource conservation,population genetics,and breeding studies of yellowfin seabream in South China.

Analysis of Resistance to Powdery Mildew in Wheat Based on Expressed Sequence Tags (EST) Technique

Powdery mildew is a serious disease of wheat in China. As part of ITEC (International Triteace EST cooperation), EST (expressed sequence tags) technique was used to explore the gene expression in leaf induced by Erysiphe graminis DC. A conventional cDNA library was constructed, and a total of 1 500 clones picked randomly from the library were sequenced, three hundred and eighty_seven ESTs of them were unique, which got the Accession Number in GenBank. About 49.4% ESTs showed significant similarity to functions of known sequences in GenBank. There are 196 ESTs' with functions not able to be determined, and eighty_four ESTs were demonstrated to be novel sequences. High_density dot membranes from unique clones were produced, and several disease resistance related genes were screened by differential hybridization.

Generation and analysis of expressed sequence tags from the salt-tolerant eelgrass species, Zostera marina

Zostera marina, a monocotyledonous angiosperm, is one of the most important seagrass species. To inves- tigate the salt-tolerance mechanism and discover salt-tolerant genes in Z. marina, a cDNA library was con- structed. Single-pass sequencing of the 5＇ ends of 4 081 clones yielded 4 002 high quality expressed sequence tags （ESTs）, which were assembled into 241 contigs and 1 673 singletons, representing 1 914 unigenes. The average length of the ESTs was 582 bp, with sizes ranging from 100-1 500 bp. Basic Local Alignment Search Tool （BLASTX） analysis revealed that 1 664 unigenes had significant homology to known genes in the Na- tional Center for Biotechnology Information （NCBI） non-redundant （nr） database （E-value≤5-10）. Among them, the two most abundant genes encoded metallothionein （157 ESTs） and chlorophyll a/b-binding pro- tein （38 ESTs）, accounting for 7.1% and 1.7% of the total ESTs, respectively. Using Kyoto Encyclopedia of Genes and Genomes （KEGG）, 1 462 unigenes were assigned to 1 161 pathways （E-value≤5-10）. A total of 938 unigenes were assigned Gene Ontology （GO） terms based on the GO hierarchy analysis, and InterProScan searches recognized 1 003 InterPro families. Three genes for metallothionein in Z. marina that belonged to Class II was identified. Results of this study will improve understanding of the molecular mechanisms of saline tolerance in Z. marina.

Analysis of expressed sequence tags from the Ulva prolifera (Chlorophyta)

In 2008, a green tide broke out before the sailing competition of the 29th Olympic Games in Qingdao. The causative species was determined to be Enteromorpha prolifera （Ulva prolifera O. F. Miiller）, a familiar green macroalga along the coastline of China. Rapid accumulation of a large biomass of floating U. prolifera prompted research on different aspects of this species. In this study, we constructed a nonnormalized cDNA library from the thalli of U. prolifera and acquired 10072 high-quality expressed sequence tags （ESTs）. These ESTs were assembled into 3 519 nonredundant gene groups, including 1 446 clusters and 2 073 singletons. After annotation with the nr database, a large number of genes were found to be related with chloroplast and ribosomal protein, GO functional classification showed 1 418 ESTs participated in photosynthesis and 1 359 ESTs were responsible for the generation of precursor metabolites and energy. In addition, rather comprehensive carbon fixation pathways were found in U. prolifera using KEGG. Some stress-related and signal transduction-related genes were also found in this study. All the evidences displayed that U. prolifera had substance and energy foundation for the intense photosynthesis and the rapid proliferation. Phylogenetic analysis of cytochrome c oxidase subunit I revealed that this green-tide causative species is most closely affiliated to Pseudendoclonium akinetum （Ulvophyceae）.

Generation and Analysis of Expressed Sequence Tags(ESTs) from Muscle Full-Length cDNA Library of Wujin Pig

Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. Construction of a full-length cDNA library is an effective way to understand the expression of functional genes in muscle tissues. In addition, novel genes for further research could be identified in the library. In this study, we constructed a full-length cDNA library from porcine muscle tissue. The estimated average size of the cDNA inserts was 1 076 bp, and the cDNA fullness ratio was 86.2%. A total of 1 058 unique sequences with 342 contigs （32.3%） and 716 singleton （67.7%） expressed sequence tags （EST） were obtained by clustering and assembling. Meanwhile, 826 （78.1%） ESTs were categorized as known genes, and 232 （21.9%） ESTs were categorized as unknown genes. 65 novel porcine genes that exhibit no identity in the TIGR gene index of Sus scrofa and 124 full-length sequences with unknown functions were deposited in the dbEST division of GenBank （accession numbers： EU650784-EU650788, GE843306, GH228978-GH229100）. The abundantly expressed genes in porcine muscle tissue were related to muscle fiber development, energy metabolism and protein synthesis. Gene ontology analysis showed that sequences expressed in porcine muscle tissue contained a high percentage of binding activity, catalytic activity, structural molecule activity and motor activity, which involved mainly in metabolic, cellular and developmental process, distributed mainly in intracellular region. The sequence data generated in this study would provide valuable information for identifying porcine genes expressed in muscle tissue and help to advance the study on the structure and function of genes in pigs.

Development and Validation of 89 Novel Expressed Sequence Tag-Derived Microsatellite Markers in Blood Clam,Tegillarca granosa

Blood clam, Tegillarca granosa, is an important shellfish in Chinese mariculture industry. Investigative research in this species, such as genetic linkage mapping, requires a large panel of molecular markers. In present study, a total of 89 polymorphic microsatellite markers were developed in T. granosa using the sequence database of Life Sciences Technology 454 next generation sequencing technology. All 89 loci were characterized in 20 individual clams from a natural population inhabiting Yueqing Gulf, Zhejiang Province, China. The number of alleles per polymorphic locus varied between 2 and 15, while the observed heterozygosity, expected heterozygosity and polymorphic information content varied between 0.000 and 1.000, 0.102 and 0.921, and 0.048 and 0.886, respectively. Of the 89 loci identified, 32 loci deviated significantly from Hardy-Weinberg equilibrium following Bonferroni correction. Thirty nine markers, which were shown to be polymorphic in a full-sibling family, were tested in Mendelian segregations. As expected, 32 loci were co-dominantly segregated in a Mendelian fashion. These novel developed microsatellite markers represent useful research tools for investigation of population genetic structure and genetic diversity in this species.

Screening microsatellite markers from EST sequences of bay scallop Argopecten irradians

Seventy-five simple sequence repeats （SSRs） were identified by the bioinformatic analysis from 5008 expressed sequence tags （ESTs） of Argopecten irradians. Among the SSRs, the number of repeat nucleotide varied from 2 to 6. Dinucleotide and trinueleotide repeat motifs were dominant in EST-SSRs of bay scallop, with a proportion of 80% over the total screened SSRs. Twenty-nine pairs of primer were designed based on the flank sequences of the selected ESTs using the software of Primet 5, and verified under the given PCR reaction condition. Eighteen of the 29 primer pairs resulted in the expected products, while the remaining either failed to produce any fragments or yielded products over expected size. Thirteen of the 18 SSRs, accounting for 72%, were detected to show polymorphism in the examined scallop samples. A preliminary test in this study indicated that the majority of the identified SSRs were informative in the cultured bay scallops, making them suitable for the population and other genetic analysis. EST-SSR markers have more advantages than the traditional genomic-derived SSRs and there is a wide range of application in comparative mapping, functional gene cloning and marker assisted selection. This research provides a reference to the identification of EST-SSRs with relative bioinformatic analysis from aquaculture species, as well as to those with a large number of ESTs.

Characterization of genic microsatellite markers derived from expressed sequence tags in Pacific abalone (Haliotis discus hannai)

Simple sequence repeat (SSR) markers were developed from the expressed sequence tags (ESTs) of Pacific abalone (Haliotis discus hannai).Repeat motifs were found in 4.95% of the ESTs at a frequency of one repeat every 10.04 kb of EST sequences,after redundancy elimination.Seventeen polymorphic EST-SSRs were developed.The number of alleles per locus varied from 2-17,with an average of 6.8 alleles per locus.The expected and observed heterozygosities ranged from 0.159 to 0.928 and from 0.132 to 0.922,respectively.Twelve of the 17 loci (70.6%) were successfully amplified in H.diversicolor.Seventeen loci segregated in three families,with three showing the presence of null alleles (17.6%).The adequate level of variability and low frequency of null alleles observed in H.discus hannai,together with the high rate of transportability across Haliotis species,make this set of EST-SSR markers an important tool for comparative mapping,marker-assisted selection,and evolutionary studies,not only in the Pacific abalone,but also in related species.

Generation and characterization of expressed sequence tags(ESTs) from coralloid root cDNA library of Cycas debaoensis

A normalized full-length cDNA library was constructed from the coralloid roots of Cycas debaoensis by the DSN （duplex-specific nuclease） normalization method combined with the SMART （Switching Mechanism At 5＇ end of the RNA Transcript） technique. The titer of the original cDNA library was about 1.5 × 10^6 cfu·mL^-1 and the average insertion size was about 1 kb with a high recombination rate （97%）. The 5011 high-quality expressed sequence tags （ESTs） were obtained from 5393 randomly picked cDNA clones. Clustering and assembly of ESTs resulted in 2984 unique sequences, consisting of 618 contigs and 2366 singlets. EST sequence annotation revealed that 2333 and 1901 unigenes were functionally anno- tated in the NCBI non-redundant database and Swiss-Prot protein database, respectively. Functional analysis demonstrated that 1495 （50.1%） unigenes were associated with 4082 Gene Ontology （GO） terms. A total of 847 unigenes were grouped into 22 Cluster of Orthologous Groups （COG） functional categories. Based on the EST dataset, 22 ESTs that encoded putative receptor-like protein kinase （RLK） genes were screened. Furthermore, a total of 94 simple sequence repeats （SSRs） were discovered, of which 20 loci were successfully amplified in C debaoensis. This study is the first EST analysis for the coralloid roots of C debaoensis and provides a valuable genomic resource for novel gene discovery, gene expression and comparative genomics, conservation and management studies as well as applications in C debaoensis and related cycad species.

Expressed sequence tags (ESTs) analysis of the ripening Vitis amurensis cv. Shuang Hong berry skins

Vitis amurensis is a valuable resource for wine production. Ripening of the grape berry is the key phase which determines the com- position of wine. To better understand the gene expression that manifest in V. amurensis berry skins during the ripening, cDNA library of V. amurensis berry skins was constructed. A total of 935 high quality ex- pressed sequence tags （ESTs） were obtained from the library. These ESTs represent 636 unigenes, including 108 contigs and 528 singletons. The EST analysis was performed and genes were assigned to functional categories according to their primary BLAST match. Of these 25.35% were involved with metabolism, 6.27% with cell rescue and defense, 6.84% energy, 11.68% protein synthesis, 18.8% protein activity regula- tion, 11.11% cell structure, 7.98% transport, 6.27% transcription and the remaining 5.7% were signal transduction. The generated ESTs were characterized by the gene ontology analysis and were categorized ac- cording to its cellular component, molecular function and biological process. In the cDNA library, some genes are relevant to the biosynthesis of anthocyanins, while some genes are related to grape berry maturation.

Analysis of Expressed Sequence Tags from Liver Tissue in Swine

In order to study the expression of function gene and its effect on metabolic control and other physiological function in liver, 438 expressed sequence tags (ESTs) were determined, which were from a cD-NA library of porcine liver tissue. The results showed that the nucleotide sequences of 186 ESTs have already presented in GenBank database, and 37 ESTs could be found the homology with human and other species, while the others were not identified. 45 full length insertion of the clones randomly isolated from cDNA library were also completely sequenced with different size, and the results showed that 19 of them were function-known genes, 11 had no open reading frame ( ORF )at all and 15 had ORF but the function were not elucidated yet.

Microsatellite Analysis of Expressed Sequence Tags and Development of EST-SSR Markers for Melampsora spp.

In this study,64 498 ESTs of Melampsoraspp.was assembled into 1 998 contigs,and 604SSR loci were detected on these contigs,with 736.6 bp containing one SSR on average.Among these SSRs,trinucleotide repeats were the most abundant repeats（44.70%）.As for the composition of microsatellites, AC and AT repeats were the richest motif in dinucleotide repeats,and AGT and AAG repeats were the most frequent motifs in trinucleotide repeats,whereas（AAAN） n and（AAAAN） n repeats were dominant in tetranucleotide and pentanucleotide repeats,respectively.All the dominant repeat motifs of different types of SSRs were rich in A and T.In EST sequences of Melampsoraspp.genome,microsatellites longer than 20 bp accounted for about 15.07%.It was noticeable that microsatellites were highly rich in the expressed sequences of Melampsoraspp.genome,which implied that SSRs played a significant role in triggering the gene mutation in Melampsoraspp.genome.A total of 455 SSR primers were designed according to the detected microsatellites using Primer 5.0 and Oligo 6. 0,and 30 primer pairs were randomly selected for amplification test.Among these primer pairs,27 primer pairs succeed in amplification,with a successful rate of 90%.Eight primer pairs generated polymorphic fingerprints in Melampsoraspp.collected from different poplar genotypes,accounting for 26.7% of the total primer pairs.The EST-SSR markers developed fromMelampsoraspp.EST sequences provided important marker resources for studying Melampsoraspp.from the aspects of pathogen identification and survey of genetic variation.

Molecular cloning, characterization and expression analysis of a catalase gene in Paphia textile

Catalase is an important antioxidant protein that can protect organisms against various forms of oxidative damage by eliminating hydrogen peroxide. In this study, the catalase c DNA of Paphia textile（Pt CAT） was cloned using RTPCR and rapid amplification of c DNA ends（RACE）. Pt CAT is 1 921 bp long and consists of a 5′-UTR of 50 bp, a 3′-UTR of 349 bp, and an ORF of 1 542 bp that encodes 513 amino acids with a molecular weight of 58.4 k D and an estimated isoelectric point of 8.2. Sequence alignment indicated that Pt CAT contained a highly conserved catalytic signature motif（^（61）FNRERIPERVVHAKGAG^（77））, a proximal heme-ligand signature sequence（^（352）RLFSYSDP^（359））, and three catalytic amino acid residues（H^（72）, N^（145）, and Y^（356））. Pt CAT also contains two putative N-glycosylation sites（^（34）NKT^（36） and ^（437）NFT^（439）） and a peroxisome-targeting signal（^（511）AQL^（513））. Furthermore, Pt CAT shares 53%–88% identity and 29%–89% similarity with other catalase amino acid sequences. Pt CAT m RNA was present in all tested organs, including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, but its expression was highest in the digestive gland. High-temperature-induced stress produced two expression patterns of Pt CAT m RNA： first, an initial up-regulation followed by a down-regulation in the heart, digestive gland, and gonad and, second, consistent down-regulation in all other organs. These results demonstrate that Pt CAT is a typical member of the catalase family and might be involved in the responses to harmful environmental factors.

Microarray-Based Differential Expression Monitoring of 79 Novel Genes in Human Fetal Tissues

ESTs fragments which represents corresponding novel genes were obtained by sequencing and bioinformatics analysis of human fet al kidney cDNA library. Microarray was prepared by using these novel EST fragmen ts by automatic spotting. Expression patters of 79 ESTs of novel genes from huma n fetal kidney were analyzed in fetal brain and fetal heart tissues of 20\|week\ | and 26\|week\|age fetus by performing of cDNA chip hybridization. This provide s clues for studying exact functions of the novel genes. 8 genes were obtained w hich were expressed differentially in the fetal brain and heart of 20\|week\| an d 26\|week\|age respectively. Then differentially expressed genes were identifie d by Northern analysis. The more exact function of the novel genes is under stud y.

Isolation, Identification of Differentially Expressed Sequence Tags in the Backfat Tissue from Meishan, Large White and MeishanLarge White Cross Pigs

In order to detect the molecular mechanism of heterosis in pigs, the mRNA differential display technique was performedto investigate the differences of gene expression in the backfat tissue from Meishan, Large White and MeishanLargeWhite cross pigs. Nine 3'-end anchored primers in combination with ten 5'-end arbitrary primers were used to perform thedifferential display PCR and nearly 3 000 reproducible bands were examined. Fifteen expressed sequence tags that weredifferentially expressed were isolated and then identified through semi-quantitative RT-PCR. BLAST analysis revealedthat the fifteen expressed sequence tags (ESTs) were not homologous to any of the known porcine genes or ESTs. Thesenovel ESTs were then submitted to GenBank.

Molecular Cloning and Characterization of Porcine Indoleamine 2,3-Dioxygenase and Its Expression in Various Tissues

In order to confirm the existence of indoleamine 2,3-dioxygenase(IDO) gene in swine,and to clone the novel gene followed by the molecule structure properties and expression pattern analysis,the porcine mRNA sequences homologous to human IDO were obtained from GenBank database by bioinformatics method.By using RT-PCR,the IDO gene was cloned from porcine endothelial cell line and the accuracy of the nucleic acid sequence was confirmed,and the expression pattern of the gene was detected.The three-dimensional structure model of porcine IDO was built referring to the tertiary structure of human IDO using biological sequence analysis software and database.The results showed that the porcine IDO was identified by sequencing.The nucleotide sequences were confirmed as a novel gene after submitted to Genbank.Porcine IDO was expressed in the lung,thymus,epididymis and anterior chamber with a basic level,however in peripheral blood mononuclear cells(PBMCs) the IDO gene was highly expressed.The three-dimensional structure model of porcine IDO was similar to that of human IDO.It was suggested that identification of the structure information of porcine IDO is essential to further investigate the immunologic function of the gene.Study of IDO on NK cells-mediated xenograft rejection will be a novel therapeutic target for the development of xenotransplantation.

IN SILICO EXPRESSION ANALYSIS OF HUMAN NOVEL GENE UBAP1 IN MULTIPLE CANCERS

Objective: To identify the differential expression profile of human novel gene UBAP1, a putative nasopharyngeal neoplasms (NPC) relate gene, in multiple cancers. Methods: We first present an EST approach for electronic Northern in silico to analyse expression patterns of UBAP1 in tumor and normal tissues. Full length cDNA of UBAP1 gene was taken as a “probe” sequence, and a blastn search was performed against human EST Database. The Blastn report can be used to determine the fold differences between the pedigree ESTs in different libraries. Especially, the ESTs corresponding to UBAP1 present in fifteen tumor-derived libraries were compared against their normal counterpart to produce an electronic differential expression profile. Second, the distinct down-regulation of UBAP1 in meningioma, glioma, and colorectal tumor was confirmed by differentially RT-PCR analysis. Results: Database surveys indicated that UBAP1 gene was not only ubiquitously expressed in many normal tissues with various levels but also differentially expressed in different tumor tissues, especially down-regulated in multiple neoplastic tissues such as brain, breast, skin, colon, testis and uterus tumor tissues. Furthermore, differential RT-PCR analysis demonstrated that expression of UBAP1 was down-regulated or absent in 7 of 12 (58%) meningioma samples, 6 of 9 (66%) glioma and 7 of 11 (63%) colorectal tumor tissues respectively. Conclusion: we described a data mining procedure in silico that proved to be useful for the identification of differential expression patterns of UBAP1. These findings could be valuable for the investigation of the mechanism the differential expression of UBAP1 gene and its significance in the progression of multiple cancers.

Genome Sequence and Genetic Diversity Studies of Melon

Cucumis melo is one of the most important fruit vegetables cultivated in tropical and temperate regions, as it is highly valued for the quality of its fruits. Traditionally, C. melo is considered to be divided into two subspecies according to ovary hairiness, i.e., subspecies melo （long hairs, distributed from India to Europe and in America） and subspecies agrestis （short hairs, in Eastern Asia from India to Japan and in Africa）. Today, the species comprises wild, feral and cultivated varieties, including sweet melons used for dessert and non-sweet ones consumed raw, pickled or cooked. The goal of this paper was to summarize melon genome sequencing and the genetic diversity studies from different references.