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 ...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.展开更多
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. amurens...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.展开更多
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 mo...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.展开更多
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...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 about1.5 x 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 and2366 singlets.EST sequence annotation revealed that 2333 and 1901 unigenes were functionally annotated 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.展开更多
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- struct...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.展开更多
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.Müller),a familiar green ma...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.Müller),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 10 072 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).展开更多
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 p...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.展开更多
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 ...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.展开更多
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 prese...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.展开更多
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 a...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.展开更多
Papaya (Carica papaya L.) is one of the most economically, medicinally and nutritionally important tropical fruit crops. Expressed sequence tags (ESTs) derived simple sequence repeat (SSR) markers are more valuable as...Papaya (Carica papaya L.) is one of the most economically, medicinally and nutritionally important tropical fruit crops. Expressed sequence tags (ESTs) derived simple sequence repeat (SSR) markers are more valuable as they are derived from conserved genic portion. Development of EST-SSRs markers through in silico approach is cheaper, less time consuming and labour-intensive. In this study, we aimed to mine SSRs and developed EST-SSR primers from papaya floral ESTs. A total of 75,846 papaya floral ESTs were downloaded from public database National Centre for Biotechnology Information (NCBI). A total of 26,039 floral unigenes (7961 contigs and 18,078 singletons) were generated after assembly of these ESTs. From these floral unigenes, 433,782 perfect SSRs, 204,968 compound SSRs and 6061 imperfect SSRs were mined, respectively. In perfect SSRs, mononucleotide repeats were most abundant (94.7%) followed by tri- (3.1%) and di-nucleotide repeats (1.7%). The frequencies of tetra-, hexa- and penta-nucleotide repeats accounted for only (0.17%), (0.04%) and (0.03%), respectively. In mononucleotide repeats, the most abundant motif was A/T (69.3%) and in di- and tri-nucleotide repeats were AG/CT (61%) and AAG/CTT (31%), respectively. In imperfect SSRs, mononucleotide repeats (56.5%) were most abundant. 176 different types of motifs were identified. A total of 3807 primer pairs for floral papaya ESTs were successfully designed. These developed EST-SSR primers are being used for the genetic improvement of papaya such as study of cross-transferability across genera/species, evaluation of genetic diversity, and identification of sex-specific markers. These EST derived SSRs can also be used in filling gaps in existing linkage maps in papaya.展开更多
Gymnadenia conopsea,an alpine Orchidaceae plant,was one of the widely used Tibetan traditional medicines.In this study,we sequenced total 105 expressed sequence tags (ESTs) from a full-length cDNA expression library c...Gymnadenia conopsea,an alpine Orchidaceae plant,was one of the widely used Tibetan traditional medicines.In this study,we sequenced total 105 expressed sequence tags (ESTs) from a full-length cDNA expression library constructed by the Oligo-capping technique.The further bioinformatic analyses suggested that the 65 represented unique sequences showed high homology to previously identified genes in other plants:30 sequences matched to other uncharacterized expressed sequence tags (ESTs),and 10 sequences showed no good matches to available sequences in DNA databases.Gene ontology annotation by InterProScan indicated that many of these cDNAs (7 percent) have no known molecular functions and may be unique to G.conopsea.Fifty-five ESTs with matched proteins were involved in a series of diverse functions,in which molecular function such as 'binding' (42.9 percent) and 'catalytic activity' (25.0 percent) were the most frequent functions of the cDNAs.This cDNA library provided a critical basis for further investigation of functional genes expression under cold stress in this alpine species.In addition,13 ESTs-based polymerase chain reaction (PCR) primers were designed and can also be used for genotypic identification and for the genetic diversity analysis of G.conopsea and its closely related species.展开更多
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 artificia...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.展开更多
Objective: To screen the stage-specific expression genes from rat spermatogonia, pachytene spermatocytes and round spermatids. Methods: Highly purified spermatogonia were isolated from 9-day-old rats, pachytene sperma...Objective: To screen the stage-specific expression genes from rat spermatogonia, pachytene spermatocytes and round spermatids. Methods: Highly purified spermatogonia were isolated from 9-day-old rats, pachytene spermatocytes and round spermatids from adult rats by sedimentation velocity at unit gravity, using 2%-4% BSA gradient in DMEM/F12 medium. A mRNA differential display method was used for screening the stage-specific expression gene. Results: Nineteen differentially expressed cDNA fragments were obtained. After excluding the false positive cDNA fragments by dot blot, 13 cDNAs were selected to clone and sequence. To obtain longer cDNAs, six ESTs were used to screen the rat testis λ-zap II cDNA library. Two longer cDNA fragments, designated as LY21 and LM66, were obtained. The analysis with DNAMAN software indicated that LY21 had a long open reading frame coding 372 amino acids while LM66 had no long open reading frame. LY21 were highly homologous with hnRNP H1. To observe the expression patterns of LY21 gene in the testicular cells, we performed in situ hybridization on testis sections from adult rats. The LY21 gene expression was found in the spermatogonia and primary spermatocytes.Conclusion: This study indicated that LY21 gene was associated with spermatogenesis. Further studies will be needed to explore the function of LY21.展开更多
The phytopathogenic fungus Venturia inaequalis causes scab of apple. Once this fungus penetrates the plant surface, it forms a specialized body called a stroma between the inner cuticle surface and the epidermal cell ...The phytopathogenic fungus Venturia inaequalis causes scab of apple. Once this fungus penetrates the plant surface, it forms a specialized body called a stroma between the inner cuticle surface and the epidermal cell wall. A novel Venturia inaequalis 5704 (Cin3) and three expressed sequence tags (ESTs);38, 6987, and 4010 are strongly up-regulated in the early stages of infection. The CIN3 and three ESTs using two vectors pMAL-c2 and pET 21 were expressed in Escherichia coli. Recombinant proteins expression, solubility and yields were analyzed. 38, 5704 (Cin3) and 6987 re- combinant proteins were expressed in soluble form and while 4010 was expressed in inclusion bodies. Re- solution on native-PAGE, the recombinant proteins;38, 5704 (Cin3), 6987 were shown to be present in dimmer, tetramer and polymer. A method was de- veloped, consisting of induction of expression at va- rious temperatures, and using enriched broth with 4% glycerol together with slow shaking, led to a decrease in concentration of nascent polypeptide and production of soluble recombinant proteins of;38, 5704 (Cin3), 6987 and 4010. Resolution on native- PAGE, the recombinant proteins were shown to be present as monomer.展开更多
Prospects for deploying perennial grasses that are currently considered leading candidates for dedicated energy crops over large acreages are debatable because of several limitations, including vegetative propagation ...Prospects for deploying perennial grasses that are currently considered leading candidates for dedicated energy crops over large acreages are debatable because of several limitations, including vegetative propagation or small seed size, low biomass production during the first growing season, and incomplete assessments of crop invasiveness risk. Pearl Millet-Napiergrass hybrids (“PMN”;Pennisetum glaucum [L.] R. Br. × P. purpureum Schumach.), in contrast, are large-seeded, sterile feedstocks capable of high biomass production during establishment year. Novel methods are warranted for confirmation of PMN hybrids, as traditional morphological observations can be inconclusive and chromosome number determination using cytological methods is laborious and time consuming. Six putative PMN lines were produced in this study, and 10 progeny from each line were evaluated using morphological traits, seed fertility, flow cytometry, and expressed sequence tag-simple sequence repeat (EST-SSR) markers. All putative hybrid lines were sterile and failed to produce seed. The PMN hybrids could not be distinguished from either parent using flow cytometry due to highly similar nuclear genome DNA contents. A number of paternal napiergrass-specific EST-SSRs were identified for each PMN line, and four paternal-specific EST-SSRs conserved across all napiergrass accessions were selected to screen the putative PMN hybrids. These EST-SSRs confirmed that all F1 individuals analyzed were PMN hybrids. The use of paternal-specific markers therefore provides a valuable tool in the development of both “Seeded-yet-Sterile” biofuel PMN feedstocks and additional PMN cultivar-and parental species-specific markers.展开更多
Objective: To screen and analyze key express sequence tags (ESTs) which were differentially displayed in every period of SD rats' primary hepatic carcinoma and reveal the molecular mechanism of carcinogenesis. Met...Objective: To screen and analyze key express sequence tags (ESTs) which were differentially displayed in every period of SD rats' primary hepatic carcinoma and reveal the molecular mechanism of carcinogenesis. Methods: Using diethylnitrosamine (DENA) as a cancerigenic agent, animal models with different phases of primary hepatic cancer were constructed in SD rats. Rats were respectively sacrificed at d 14, d 28, d 56, d 77, d 105 and d 112 after the rats received DENA by gavage, then the livers were harvested. One part of the livers was classified according to their pathological changes, while the other was reserved for molecular mechanism studies on hepatocarcinogenesis. The differentially expressed genes were isolated from both normal and morbid tissues by mRNA differential display technique (DDRT-PCR). After the fragments were sequenced, bioinformatics were .used to analyze the results. Results: Twelve differentially expressed cDNA fragments were obtained. Nine fragments had the homology with known cDNA clones, especially EST-7 was similar to BN/SsNHsdMCW mitochondrion gene and the identity was 100% which suggested EST-7 may be the part of BN/SsNHsdMCW mitochondrion gene. In contrast, other three fragments (EST-1, EST-3 and EST-5) had extremely low identity to any genes registered in GENBANK databases. Conclusions: BN/SsNHsdMCW mitochondrion gene was expressed in different periods of hepatocarcinogenesis. Moreover, EST-I, EST-3 and EST-5 were suggested to contribute to the development of rat hepatocarcinogenesis, and thus may be candidates of new targets of oncogenes or cancer suppressor genes.展开更多
Molecular genetic maps were commonly constructed by analyzing the segregation of restriction fragment length polymorphisms (RFLPs). Here we described methodology-marker sequences in a new mapping based on recent docum...Molecular genetic maps were commonly constructed by analyzing the segregation of restriction fragment length polymorphisms (RFLPs). Here we described methodology-marker sequences in a new mapping based on recent documents. With the methods they were unique sequences detected by the polymerase chain reaction (PCR). Each of the methods had its Iimitations and the current trend was to integrate the maps produced by the different methods. Marker sequences contained mainly expressed sequence tags (ESTs),polymorphie sequence-tagged sites (STSs), randomly amplified polymorphic DNA (RAPDs), cIeaved amplified polymorphic sequences (CAPS), amplified fragment Iength pofymorphism (AFLPs), genorne sequence sampling (GSS) and sequence-tagged connectors (STCs) in this paper.展开更多
基金supported by the National Basic Research Program of China(2007CB116201)
文摘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.
基金supported by the China Agriculture Research System (CARS-30)
文摘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.
基金Supported by the Key Forestry Public Welfare Project(201304102)the Key Project for Universities of Jiangsu Province(10KJA180018)+1 种基金enabled by the Program for Innovative Research Team in Universities of Jiangsu Province and the Educational Department of Chinathe Priority Academic Program Development of Jiangsu Higher Education Institutions
文摘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.
基金supported by the Grant(201522)from Shenzhen Urban Management
文摘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 about1.5 x 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 and2366 singlets.EST sequence annotation revealed that 2333 and 1901 unigenes were functionally annotated 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.
基金The Key Science and Technology Program of Shandong Province under contract No. 2012GHY11527Natural Science Foundation of Shandong Province under contract No. Q2007E02+1 种基金Specialized Research Fund for the Doctoral Program of Higher Education (New Teachers) under contract No. 20070423027the Public Science and Technology Research Funds Projects of Ocean, State Oceanic Administration of the People’s Republic of China under contract No. 201105021-8
文摘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.
基金Supported by the Scientific and Technical Supporting Programs of China (2008BAC49B01)the National Natural Science Foundation of China (No. 30830015)
文摘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.Müller),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 10 072 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).
基金supported by the Chinese National Key Project of Basic Research(G20000161)
文摘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.
基金Supported by the National High Technology Research and Development Program of China (863 Program) (No. 2007AA09Z433)the Cultivation Fund of the Key Scientific and Technical Innovation Project Ministry of Education of China (No. 707041)
文摘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.
基金supported by the National High Technology Research and Development Program of China (863 Program) (No. 2012AA10A410)the National Project for Agricultural Technology System (No. CARS-48)+1 种基金National Infrastructure of Fishery Germplasm Resources of China (No. 2015DKA30470)Zhejiang Major Program of Science and Technology (No. 2012C12907-4)
文摘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.
文摘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.
文摘Papaya (Carica papaya L.) is one of the most economically, medicinally and nutritionally important tropical fruit crops. Expressed sequence tags (ESTs) derived simple sequence repeat (SSR) markers are more valuable as they are derived from conserved genic portion. Development of EST-SSRs markers through in silico approach is cheaper, less time consuming and labour-intensive. In this study, we aimed to mine SSRs and developed EST-SSR primers from papaya floral ESTs. A total of 75,846 papaya floral ESTs were downloaded from public database National Centre for Biotechnology Information (NCBI). A total of 26,039 floral unigenes (7961 contigs and 18,078 singletons) were generated after assembly of these ESTs. From these floral unigenes, 433,782 perfect SSRs, 204,968 compound SSRs and 6061 imperfect SSRs were mined, respectively. In perfect SSRs, mononucleotide repeats were most abundant (94.7%) followed by tri- (3.1%) and di-nucleotide repeats (1.7%). The frequencies of tetra-, hexa- and penta-nucleotide repeats accounted for only (0.17%), (0.04%) and (0.03%), respectively. In mononucleotide repeats, the most abundant motif was A/T (69.3%) and in di- and tri-nucleotide repeats were AG/CT (61%) and AAG/CTT (31%), respectively. In imperfect SSRs, mononucleotide repeats (56.5%) were most abundant. 176 different types of motifs were identified. A total of 3807 primer pairs for floral papaya ESTs were successfully designed. These developed EST-SSR primers are being used for the genetic improvement of papaya such as study of cross-transferability across genera/species, evaluation of genetic diversity, and identification of sex-specific markers. These EST derived SSRs can also be used in filling gaps in existing linkage maps in papaya.
基金the Program for New Century Excellent Talents in University (NCET-06-0810)Young Scholars Foundation from the Science and Technology Committee of Sichuan,China (2008-31-371) for their financial support
文摘Gymnadenia conopsea,an alpine Orchidaceae plant,was one of the widely used Tibetan traditional medicines.In this study,we sequenced total 105 expressed sequence tags (ESTs) from a full-length cDNA expression library constructed by the Oligo-capping technique.The further bioinformatic analyses suggested that the 65 represented unique sequences showed high homology to previously identified genes in other plants:30 sequences matched to other uncharacterized expressed sequence tags (ESTs),and 10 sequences showed no good matches to available sequences in DNA databases.Gene ontology annotation by InterProScan indicated that many of these cDNAs (7 percent) have no known molecular functions and may be unique to G.conopsea.Fifty-five ESTs with matched proteins were involved in a series of diverse functions,in which molecular function such as 'binding' (42.9 percent) and 'catalytic activity' (25.0 percent) were the most frequent functions of the cDNAs.This cDNA library provided a critical basis for further investigation of functional genes expression under cold stress in this alpine species.In addition,13 ESTs-based polymerase chain reaction (PCR) primers were designed and can also be used for genotypic identification and for the genetic diversity analysis of G.conopsea and its closely related species.
基金Supported by the National Key R&D Program of China (No. 2019YFD0901202)the Key-Area Research and Development Program of Guangdong Province (No. 2021B0202020002)+1 种基金the China Postdoctoral Science Foundation (No. 2021M693677)the Yellow Fin Bream Seed System Building Project (2021)
文摘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.
基金Supported by the High Technology Research and Development Programme of China (No.2002AA626020) and the National Natural Science Foundation of China (40276045).
基金the Special Funds for Major State Basic Research project (G199055901).
文摘Objective: To screen the stage-specific expression genes from rat spermatogonia, pachytene spermatocytes and round spermatids. Methods: Highly purified spermatogonia were isolated from 9-day-old rats, pachytene spermatocytes and round spermatids from adult rats by sedimentation velocity at unit gravity, using 2%-4% BSA gradient in DMEM/F12 medium. A mRNA differential display method was used for screening the stage-specific expression gene. Results: Nineteen differentially expressed cDNA fragments were obtained. After excluding the false positive cDNA fragments by dot blot, 13 cDNAs were selected to clone and sequence. To obtain longer cDNAs, six ESTs were used to screen the rat testis λ-zap II cDNA library. Two longer cDNA fragments, designated as LY21 and LM66, were obtained. The analysis with DNAMAN software indicated that LY21 had a long open reading frame coding 372 amino acids while LM66 had no long open reading frame. LY21 were highly homologous with hnRNP H1. To observe the expression patterns of LY21 gene in the testicular cells, we performed in situ hybridization on testis sections from adult rats. The LY21 gene expression was found in the spermatogonia and primary spermatocytes.Conclusion: This study indicated that LY21 gene was associated with spermatogenesis. Further studies will be needed to explore the function of LY21.
文摘The phytopathogenic fungus Venturia inaequalis causes scab of apple. Once this fungus penetrates the plant surface, it forms a specialized body called a stroma between the inner cuticle surface and the epidermal cell wall. A novel Venturia inaequalis 5704 (Cin3) and three expressed sequence tags (ESTs);38, 6987, and 4010 are strongly up-regulated in the early stages of infection. The CIN3 and three ESTs using two vectors pMAL-c2 and pET 21 were expressed in Escherichia coli. Recombinant proteins expression, solubility and yields were analyzed. 38, 5704 (Cin3) and 6987 re- combinant proteins were expressed in soluble form and while 4010 was expressed in inclusion bodies. Re- solution on native-PAGE, the recombinant proteins;38, 5704 (Cin3), 6987 were shown to be present in dimmer, tetramer and polymer. A method was de- veloped, consisting of induction of expression at va- rious temperatures, and using enriched broth with 4% glycerol together with slow shaking, led to a decrease in concentration of nascent polypeptide and production of soluble recombinant proteins of;38, 5704 (Cin3), 6987 and 4010. Resolution on native- PAGE, the recombinant proteins were shown to be present as monomer.
文摘Prospects for deploying perennial grasses that are currently considered leading candidates for dedicated energy crops over large acreages are debatable because of several limitations, including vegetative propagation or small seed size, low biomass production during the first growing season, and incomplete assessments of crop invasiveness risk. Pearl Millet-Napiergrass hybrids (“PMN”;Pennisetum glaucum [L.] R. Br. × P. purpureum Schumach.), in contrast, are large-seeded, sterile feedstocks capable of high biomass production during establishment year. Novel methods are warranted for confirmation of PMN hybrids, as traditional morphological observations can be inconclusive and chromosome number determination using cytological methods is laborious and time consuming. Six putative PMN lines were produced in this study, and 10 progeny from each line were evaluated using morphological traits, seed fertility, flow cytometry, and expressed sequence tag-simple sequence repeat (EST-SSR) markers. All putative hybrid lines were sterile and failed to produce seed. The PMN hybrids could not be distinguished from either parent using flow cytometry due to highly similar nuclear genome DNA contents. A number of paternal napiergrass-specific EST-SSRs were identified for each PMN line, and four paternal-specific EST-SSRs conserved across all napiergrass accessions were selected to screen the putative PMN hybrids. These EST-SSRs confirmed that all F1 individuals analyzed were PMN hybrids. The use of paternal-specific markers therefore provides a valuable tool in the development of both “Seeded-yet-Sterile” biofuel PMN feedstocks and additional PMN cultivar-and parental species-specific markers.
基金supported by the Key Program for Science and Technology Development of Henan Province [122102310174]the Zoology Key Subject of Henan Province
文摘Objective: To screen and analyze key express sequence tags (ESTs) which were differentially displayed in every period of SD rats' primary hepatic carcinoma and reveal the molecular mechanism of carcinogenesis. Methods: Using diethylnitrosamine (DENA) as a cancerigenic agent, animal models with different phases of primary hepatic cancer were constructed in SD rats. Rats were respectively sacrificed at d 14, d 28, d 56, d 77, d 105 and d 112 after the rats received DENA by gavage, then the livers were harvested. One part of the livers was classified according to their pathological changes, while the other was reserved for molecular mechanism studies on hepatocarcinogenesis. The differentially expressed genes were isolated from both normal and morbid tissues by mRNA differential display technique (DDRT-PCR). After the fragments were sequenced, bioinformatics were .used to analyze the results. Results: Twelve differentially expressed cDNA fragments were obtained. Nine fragments had the homology with known cDNA clones, especially EST-7 was similar to BN/SsNHsdMCW mitochondrion gene and the identity was 100% which suggested EST-7 may be the part of BN/SsNHsdMCW mitochondrion gene. In contrast, other three fragments (EST-1, EST-3 and EST-5) had extremely low identity to any genes registered in GENBANK databases. Conclusions: BN/SsNHsdMCW mitochondrion gene was expressed in different periods of hepatocarcinogenesis. Moreover, EST-I, EST-3 and EST-5 were suggested to contribute to the development of rat hepatocarcinogenesis, and thus may be candidates of new targets of oncogenes or cancer suppressor genes.
文摘Molecular genetic maps were commonly constructed by analyzing the segregation of restriction fragment length polymorphisms (RFLPs). Here we described methodology-marker sequences in a new mapping based on recent documents. With the methods they were unique sequences detected by the polymerase chain reaction (PCR). Each of the methods had its Iimitations and the current trend was to integrate the maps produced by the different methods. Marker sequences contained mainly expressed sequence tags (ESTs),polymorphie sequence-tagged sites (STSs), randomly amplified polymorphic DNA (RAPDs), cIeaved amplified polymorphic sequences (CAPS), amplified fragment Iength pofymorphism (AFLPs), genorne sequence sampling (GSS) and sequence-tagged connectors (STCs) in this paper.