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Electron. j. biotechnol ; 48: 23-28, nov. 2020. tab, ilus
Article in English | LILACS | ID: biblio-1254683


BACKGROUND: Meretrix petechialis is one of the commercially important marine bivalves. In this study, we selected six highly polymorphic EST-derived microsatellite markers to assess the genetic diversity and population differentiation on nine wild populations of Meretrix petechialis. RESULTS: The number of alleles detected per-locus ranged from 4 to 30 (mean NA = 27.5) with a total of 165 alleles. The mean value of observed and expected heterozygosities varied from 0.717 to 0.861 and from 0.797 to 0.856, respectively. Meanwhile, the result of Neighbor-joining and overall FST = 0.214 (P < 0.01) reveled that M. petechialis populations from GX are the farthest populations, a certain degree of genetic variation among individuals in each population and the genetic differentiation is significant. CONCLUSIONS: GX population has high genetic diversity among individual, and there are certain differences in genetic characteristics among different populations. This study will provide a basis for the domestication and cultivation of genetic diversity of M. petechialis population and the protection of clam germplasm resources.

Animals , Genetic Variation , Bivalvia/genetics , Microsatellite Repeats , Expressed Sequence Tags , Population , Alleles
Electron. j. biotechnol ; 39: 30-41, may. 2019. tab, ilus
Article in English | LILACS | ID: biblio-1051590


BACKGROUND: A total of 62,591 cowpea expressed sequence tags (ESTs) were BLAST aligned to the whole-genome sequence of barrel medic (Medicago truncatula) to develop conserved intron scanning primers (CISPs). The efficacy of the primers was tested across 10 different legumes and on different varieties of cowpea, chickpea, and pigeon pea. Genetic diversity was assessed using the same primers on different cowpea genotypes. Singlenucleotide polymorphisms (SNPs) were detected, which were later converted to length polymorphism markers for easy genotyping. CISPs developed in this study were used in tagging resistance to bacterial leaf blight disease in cowpea. RESULTS: A total of 1262 CISPs were designed. The single-copy amplification success rates using these primers on 10 different legumes and on different varieties of cowpea, chickpea, and pigeon pea were approximately 60% in most of the legumes except soybean (47%) and peanut (37%). Genetic diversity analysis of 35 cowpea genotypes using 179 CISPs revealed 123 polymorphic markers with PIC values ranging from 0.05 to 0.59. Potential SNPs identified in cowpea, chickpea, and pigeon pea were converted to PCR primers of various sizes for easy genotyping. Using the markers developed in this study, a genetic linkage map was constructed with 11 linkage groups in cowpea. QTL mapping with 194 F3 progeny families derived from the cross C-152 × V-16 resulted in the identification of three QTLs for resistance to bacterial leaf blight disease. Conclusions: CISPs were proved to be efficient markers to identify various other marker classes like SNPs through comparative genomic studies in lesser studied crops and to aid in systematic sampling of the entire genome for well-distributed markers at low cost

Genome, Plant , Genomics/methods , Medicago truncatula/genetics , Polymerase Chain Reaction , Chromosome Mapping , Expressed Sequence Tags , Polymorphism, Single Nucleotide , Genomics , Quantitative Trait Loci , Fabaceae/genetics
IJB-Iranian Journal of Biotechnology. 2017; 15 (1): 22-32
in English | IMEMR | ID: emr-192438


Background: Micro RNAs [miRNAs] are a pivotal part of non-protein-coding endogenous small RNA molecules that regulate the genes involved in plant growth and development, and respond to biotic and abiotic environmental stresses posttranscriptionally

Objective: In the present study, we report the results of a systemic search for identification of new miRNAs in B. rapa using homology-based ESTs [Expressed Sequence Tags] analysis and considering a series of filtration criteria

Materials and Methods: Plant mature miRNA sequences were searched in non-protein coding ESTs registered in NCBI EST database. Zuker RNA folding algorithm was used to generate the secondary structures of the ESTs. Potential sequences were candidate as miRNA genes and characterized evolutionarily only and if only they fit some described criteria. Also, the web tool psRNATarget was applied to predict candidate B. rapa miRNA targets

Results: In this study, 10 novel miRNAs from B. rapa belonging to 6 miRNA families were identified using EST-based homology analysis by considering a series of filtration criteria. All potent miRNAs appropriate fold back structure. Several potential targets with known/unknown functions for these novel miRNAs were identified. The target genes mainly encode transcription factors, enzymes, DNA binding proteins, disease resistance proteins, carrier proteins and other biological processes

Conclusions: MicroRNA having diverse functions in plant species growth, development and evolution by posttranscriptionally regulating the levels of specific transcriptome so by effecting on their translation products. Research in miRNA led to the identification of many miRNAs and their regulating genes from diverse plant species

MicroRNAs , Expressed Sequence Tags , Research
Mem. Inst. Oswaldo Cruz ; 110(5): 687-690, Aug. 2015. ilus
Article in English | LILACS | ID: lil-755906


The functional characterisation of thousands of Trypanosoma cruzi genes remains a challenge. Reverse genetics approaches compatible with high-throughput cloning strategies can provide the tool needed to tackle this challenge. We previously published the pTcGW platform, composed by plasmid vectors carrying different options of N-terminal fusion tags based on Gateway® technology. Here, we present an improved 1.1 version of pTcGW vectors, which is characterised by a fully flexible structure allowing an easy customisation of each element of the vectors in a single cloning step. Additionally, both N and C-terminal fusions are available with new tag options for protein complexes purification. Three of the newly created vectors were successfully used to determine the cellular localisation of four T. cruzi proteins. The 1.1 version of pTcGW platform can be used in a variety of assays, such as protein overexpression, identification of protein-protein interaction and protein localisation. This powerful and versatile tool allows adding valuable functional information to T. cruzigenes and is freely available for scientific community.


Genetic Vectors/genetics , Trypanosoma cruzi/genetics , Chromatography, Affinity , Cloning, Molecular , Expressed Sequence Tags/metabolism , Gene Expression/genetics , Plasmids
Article in English | WPRIM | ID: wpr-85013


PURPOSE: Cockroach feces are known to be rich in IgE-reactive components. Various protease allergens were identified by proteomic analysis of German cockroach fecal extract in a previous study. In this study, we characterized a novel allergen, a chymotrypsin-like serine protease. METHODS: A cDNA sequence homologous to chymotrypsin was obtained by analysis of German cockroach expressed sequence tag (EST) clones. The recombinant chymotrypsins from the German cockroach and house dust mite (Der f 6) were expressed in Escherichia coli using the pEXP5NT/TOPO vector system, and their allergenicity was investigated by ELISA. RESULTS: The deduced amino acid sequence of German cockroach chymotrypsin showed 32.7 to 43.1% identity with mite group 3 (trypsin) and group 6 (chymotrypsin) allergens. Sera from 8 of 28 German cockroach allergy subjects (28.6%) showed IgE binding to the recombinant protein. IgE binding to the recombinant cockroach chymotrypsin was inhibited by house dust mite chymotrypsin Der f 6, while it minimally inhibited the German cockroach whole body extract. CONCLUSIONS: A novel allergen homologous to chymotrypsin was identified from the German cockroach and was cross-reactive with Der f 6.

Allergens , Amino Acid Sequence , Blattellidae , Chymotrypsin , Clone Cells , Cockroaches , DNA, Complementary , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Expressed Sequence Tags , Feces , Hypersensitivity , Immunoglobulin E , Mites , Pyroglyphidae , Sequence Homology , Serine Proteases
Mem. Inst. Oswaldo Cruz ; 109(8): 1081-1085, 12/2014. graf
Article in English | LILACS | ID: lil-732602


We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).

Gene Expression/genetics , Genetic Vectors/genetics , Plasmids , Restriction Mapping/methods , Trypanosoma cruzi/genetics , Blotting, Western , Expressed Sequence Tags/metabolism , Green Fluorescent Proteins , Life Cycle Stages/genetics , Mutagenesis, Insertional , Tetracycline/pharmacology , Trypanosoma cruzi/drug effects
Rev. biol. trop ; 62(4): 1637-1648, oct.-dic. 2014. graf, tab
Article in English | LILACS | ID: lil-753716


Eichhornia crassipes is an aquatic plant native to the Amazon River Basin. It has become a serious weed in freshwater habitats in rivers, lakes and reservoirs both in tropical and warm temperate areas worldwide. Some research has stated that it can be used for water phytoremediation, due to its strong assimilation of nitro- gen and phosphorus, and the accumulation of heavy metals, and its growth and spread may play an important role in environmental ecology. In order to explore the molecular mechanism of E. crassipes to responses to nitrogen deficiency, we constructed forward and reversed subtracted cDNA libraries for E. crassipes roots under nitrogen deficient condition using a suppressive subtractive hybridization (SSH) method. The forward subtraction included 2 100 clones, and the reversed included 2 650 clones. One thousand clones were randomly selected from each library for sequencing. About 737 (527 unigenes) clones from the forward library and 757 (483 unigenes) clones from the reversed library were informative. Sequence BlastX analysis showed that there were more transporters and adenosylhomocysteinase-like proteins in E. crassipes cultured in nitrogen deficient medium; while, those cultured in nitrogen replete medium had more proteins such as UBR4-like e3 ubiquitin- protein ligase and fasciclin-like arabinogalactan protein 8-like, as well as more cytoskeletal proteins, including actin and tubulin. Cluster of Orthologous Group (COG) analysis also demonstrated that in the forward library, the most ESTs were involved in coenzyme transportation and metabolism. In the reversed library, cytoskeletal ESTs were the most abundant. Gene Ontology (GO) analysis categories demonstrated that unigenes involved in binding, cellular process and electron carrier were the most differentially expressed unigenes between the forward and reversed libraries. All these results suggest that E. crassipes can respond to different nitrogen status by efficiently regulating and controlling some transporter gene expressions, certain metabolism processes, specific signal transduction pathways and cytoskeletal construction.

Se ha convertido en una maleza importante en hábitats de agua dulce en ríos, lagos y embalses, tanto en zonas tropicales como templadas de todo el mundo. Algunas investigaciones han indicado que se puede utilizar para la fitorremediación de agua, debido a su fuerte asimilación de nitrógeno y fósforo, y la acumulación de metales pesados, su crecimiento y propagación puede desempeñar un papel importante en la ecología ambiental. Con el fin de explorar el mecanismo molecular de respuesta a la deficiencia de nitrógeno en E. crassipes, se construyeron bibliotecas de cDNA mediante síntesis adelantada y retrasada para raíces de E. crassipes en condiciones de deficiencia de nitrógeno mediante el método de hibridación supresiva sustractiva (SSH). Para este estudio se utilizaron 2 100 clones de síntesis adelantada y 2 650 de síntesis retrasada. De la biblioteca se escogieron al azar mil clones, 737 (527 unigenes) de síntesis adelanta- da y 757 (483 unigenes) de síntesis retrasada que fueron informativos. El análisis BLASTX mostró que había más transportadores y proteínas adenosilhomocisteinasa en E. crassipes cultivadas en un medio deficiente de nitrógeno; mientras que las cultivadas en un medio repleto de nitróge- no tenían más proteínas como UBR4 e3 ubiquitina-proteína ligasa y la proteína arabinogalactano 8 tipo fasciclina, así como otras proteínas del citoesqueleto, incluyendo la actina y la tubulina. Clúster del Grupo Ortológico (COG) también demostró que en la biblioteca de síntesis adelan- tada, la mayoría de los marcadores de secuencia expresada (ESTs) estaban involucrados en el transporte de coenzimas y el metabolismo.

Environmental Restoration and Remediation , Expressed Sequence Tags , Eichhornia/genetics , Genes, Plant , Nitrogen/metabolism , Phosphorus/metabolism , Eichhornia/classification , Eichhornia/metabolism , Gene Library , Nitrogen/deficiency , Polymerase Chain Reaction
Indian J Exp Biol ; 2014 Feb; 52(2): 181-188
Article in English | IMSEAR | ID: sea-150348


Double haploid technique is not routinely used in legume breeding programs, though recent publications report haploid plants via anther culture in chickpea (Cicer arietinum L.). The focus of this study was to develop an efficient and reproducible protocol for the production of double haploids with the application of multiple stress pre-treatments such as centrifugation and osmotic shock for genotypes of interest in chickpea for their direct use in breeding programs. Four genotypes, ICC 4958, WR315, ICCV 95423 and Arearti were tested for anther culture experiments. The yield was shown to be consistent with 3-5 nucleate microspores and 2-7 celled structures with no further growth. To gain a further insight into the molecular mechanism underlying the switch from microsporogenesis to androgenesis, bioinformatics tools were employed. The challenges on the roles of such genes were reviewed while an attempt was made to find putative candidates for androgenesis using Expressed Sequenced Tags (EST) and interolog based protein interaction analyses.

Breeding , Cicer/genetics , Computational Biology , Expressed Sequence Tags , Fabaceae/genetics , Genotype , Haploidy , Plant Roots/genetics , Plant Roots/metabolism , Protein Interaction Maps/genetics , Stress, Physiological
Article in Korean | WPRIM | ID: wpr-194019


The thymus is the central lymphoid organ for the development of bone marrow-derived precursor cells into mature T-cells. Understanding the molecular mechanism of thymic involution and regeneration is critical to develop methods to normalize or improve host immunity from the decreased immune function caused by thymic involution. In this study, the regenerating thymus cDNA library was constructed in the rat from a model of thymic involution and regeneration induced by cyclophosphamide. Expressed sequence tags (ESTs) were obtained by partial sequencing of 700 randomly selected insert-containing clones. A total of 630 ESTs were analyzed, of which 486 ESTs (78%) matched to known genes and 125 ESTs (19%) matched to other ESTs (unknown genes). The 19 ESTs (3%) did not match with any known sequences. The ESTs were grouped into six main functional categories: metabolism (44%), signaling components (20%), membrane transport (7%), cytoskeleton (2%), cell division (2%) and defense (2%). As a result of RT-PCR analysis, expression of putative gene 01, putative E2IG2 gene, musculin and osteoactivin significantly increased in rat thymus during regeneration. The putative gene 01 showed complete homology with mitochondrial ribosomal protein S4 by homology search and multiple alignment of amino acid. These results provide the extensive molecular information on thymus regeneration and will be useful source to identify various genes which may play an important role in the thymus regeneration as well as to clone novel genes. Furthermore, the availability of these data will serve as a basis for further research to understand the molecular mechanism of thymus regeneration.

Animals , Cell Division , Clone Cells , Cyclophosphamide , Cytoskeleton , Expressed Sequence Tags , Gene Expression , Gene Library , Membranes , Metabolism , Rats , Regeneration , Ribosomal Proteins , T-Lymphocytes , Thymus Gland
Mem. Inst. Oswaldo Cruz ; 108(6): 707-717, set. 2013. tab, graf
Article in English | LILACS | ID: lil-685497


Schistosomiasis is a major neglected tropical disease caused by trematodes from the genus Schistosoma. Because schistosomes exhibit a complex life cycle and numerous mechanisms for regulating gene expression, it is believed that spliced leader (SL) trans-splicing could play an important role in the biology of these parasites. The purpose of this study was to investigate the function of trans-splicing in Schistosoma mansoni through analysis of genes that may be regulated by this mechanism and via silencing SL-containing transcripts through RNA interference. Here, we report our analysis of SL transcript-enriched cDNA libraries from different S. mansoni life stages. Our results show that the trans-splicing mechanism is apparently not associated with specific genes, subcellular localisations or life stages. In cross-species comparisons, even though the sets of genes that are subject to SL trans-splicing regulation appear to differ between organisms, several commonly shared orthologues were observed. Knockdown of trans-spliced transcripts in sporocysts resulted in a systemic reduction of the expression levels of all tested trans-spliced transcripts; however, the only phenotypic effect observed was diminished larval size. Further studies involving the findings from this work will provide new insights into the role of trans-splicing in the biology of S. mansoni and other organisms. All Expressed Sequence Tags generated in this study were submitted to dbEST as five different libraries. The accessions for each library and for the individual sequences are as follows: (i) adult worms of mixed sexes (LIBEST_027999: JZ139310 - JZ139779), (ii) female adult worms (LIBEST_028000: JZ139780 - JZ140379), (iii) male adult worms (LIBEST_028001: JZ140380 - JZ141002), (iv) eggs (LIBEST_028002: JZ141003 - JZ141497) and (v) schistosomula (LIBEST_028003: JZ141498 - JZ141974).

Animals , Female , Male , Gene Knockdown Techniques , RNA Precursors/isolation & purification , RNA, Spliced Leader/genetics , Schistosoma mansoni/genetics , Trans-Splicing/physiology , Expressed Sequence Tags , Gene Library , Gene Expression Regulation/genetics , Larva , Life Cycle Stages/genetics , Phenotype , Real-Time Polymerase Chain Reaction , RNA Precursors/genetics , RNA, Double-Stranded , RNA, Small Interfering/metabolism , Schistosoma mansoni/growth & development , Trans-Splicing/genetics
Indian J Exp Biol ; 2013 Jul; 51(7): 522-530
Article in English | IMSEAR | ID: sea-147623


Chinese cabbage (Brassica rapa) is widely recognized for its economic importance and contribution to human nutrition but abiotic and biotic stresses are main obstacle for its quality, nutritional status and production. In this study, 3,429 Express Sequence Tag (EST) sequences were generated from B. rapa cv. Osome cDNA library and the unique transcripts were classified functionally using a gene ontology (GO) hierarchy, Kyoto encyclopedia of genes and genomes (KEGG). KEGG orthology and the structural domain data were obtained from the biological database for stress related genes (SRG). EST datasets provided a wide outlook of functional characterization of B. rapa cv. Osome. In silico analysis revealed % 83 of ESTs to be well annotated towards reeds one dimensional concept. Clustering of ESTs returned 333 contigs and 2,446 singlets, giving a total of 3,284 putative unigene sequences. This dataset contained 1,017 EST sequences functionally annotated to stress responses and from which expression of randomly selected SRGs were analyzed against cold, salt, drought, ABA, water and PEG stresses. Most of the SRGs showed differentially expression against these stresses. Thus, the EST dataset is very important for discovering the potential genes related to stress resistance in chinese cabbage, and can be of useful resources for genetic engineering of Brassica sp.

Brassica/drug effects , Brassica/genetics , Brassica/growth & development , Databases, Genetic , Expressed Sequence Tags/metabolism , Gene Expression Profiling , Gene Library , Gene Regulatory Networks , Genes, Plant/genetics , Genome, Plant , Humans , Molecular Sequence Annotation , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Sodium Chloride/pharmacology , Stress, Physiological/genetics
Article in English | WPRIM | ID: wpr-124615


Long intergenic non-coding RNAs (lincRNAs) have historically been ignored in cancer biology. However, thousands of lincRNAs have been identified in mammals using recently developed genomic tools, including microarray and high-throughput RNA sequencing (RNA-seq). Several of the lincRNAs identified have been well characterized for their functions in carcinogenesis. Here we performed RNA-seq experiments comparing gastric cancer with normal tissues to find differentially expressed transcripts in intergenic regions. By analyzing our own RNA-seq and public microarray data, we identified 31 transcripts, including a known expressed sequence tag, BM742401. BM742401 was downregulated in cancer, and its downregulation was associated with poor survival in gastric cancer patients. Ectopic overexpression of BM742401 inhibited metastasis-related phenotypes and decreased the concentration of extracellular MMP9. These results suggest that BM742401 is a potential lincRNA marker and therapeutic target.

Animals , DNA, Intergenic/genetics , Expressed Sequence Tags/metabolism , Extracellular Space/metabolism , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genetic Predisposition to Disease , Humans , Male , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Inbred C57BL , Multivariate Analysis , Neoplasm Metastasis , Neoplasm Staging , Phenotype , Proportional Hazards Models , RNA, Long Noncoding/genetics , RNA, Messenger/genetics , Reproducibility of Results , Stomach Neoplasms/genetics , Survival Analysis
Article in English | WPRIM | ID: wpr-819978


OBJECTIVE@#To predict structure and function of translationally controlled tumor protein (TCTP) from Spirometra mansoni by bioinformatics technology, and to provide a theoretical basis for further study.@*METHODS@#Open reading frame (ORF) of EST sequence from Spirometra mansoni was obtained by ORF finder and was translated into amino acid residue by DNAclub. The structure domain was analyzed by Blast. By the method of online analysis tools: Protparam, InterProScan, protscale, SignalP-3.0, PSORT II, BepiPred, TMHMM, VectorNTI Suite 9 packages and Phyre2, the structure and function of the protein were predicted and analyzed.@*RESULTS@#The results showed that the EST sequence was Sm TCTP with 173 amino acid residues, theoretical molecular weight was 19 872.0 Da. The protein has the closest evolutionary status with Clonorchis sinensis, Schistosoma mansoni, and Schistosoma japonicum. Then it had no signal peptide site and transmembrane domain. Secondary structure of TCTP contained two α -helices and eight β -strands.@*CONCLUSIONS@#Sm TCTP was a variety of biological functions of protein that may be used as a vaccine candidate molecule and drug target.

Amino Acid Sequence , Animals , Base Sequence , Biomarkers, Tumor , Chemistry , Genetics , Metabolism , Cestode Infections , Parasitology , Computational Biology , Dog Diseases , Dogs , Expressed Sequence Tags , Helminth Proteins , Chemistry , Genetics , Metabolism , Helminths , Chemistry , Classification , Genetics , Open Reading Frames , Phylogeny , Protein Structure, Secondary , Protein Structure, Tertiary , Spirometra , Chemistry , Genetics , Metabolism , Parasitology
Article in Chinese | WPRIM | ID: wpr-346849


<p><b>OBJECTIVE</b>Taxus species are highly valued for the production of taxol. Based on high-throughput sequenceing, EST-SSRs were explored and studied in the transcriptome of Taxus cuspidata.</p><p><b>METHOD</b>T cuspidata leaf cDNA was extracted and sequenced by 454 GS FLX Titanium. High-quality sequences were assembled using Newbler Assembler Software, which produced unique sequences. SSRs motif was explored using simple sequence repeat identification tool (Perl Script). Primers were designed using PRIMER3.</p><p><b>RESULT</b>A total of 81 148 high-quality reads from the needles of T. cuspidata were produced using the Roche GS FLX Titanium system. A total of 20 557 unique sequences were obtained. There were 753 simple sequence repeat motifs identified. Primers of PCR were obtained for 519 EST-SSRs, randomly selected cloning sequencing revealed that 87.5% of ESTs were the same as the results of Sanger sequencing.</p><p><b>CONCLUSION</b>The results provide the first EST-SSRs collection in Taxus and are essential for future efforts of gene discovery, functional genomics, and genome annotation in related species.</p>

DNA Primers , Genetics , Expressed Sequence Tags , Gene Expression Profiling , Methods , Gene Expression Regulation, Plant , High-Throughput Nucleotide Sequencing , Methods , Microsatellite Repeats , Genetics , Taxus , Genetics , Transcriptome , Genetics
Article in English | WPRIM | ID: wpr-155521


Recently, the technologies of DNA sequence variation and gene expression profiling have been used widely as approaches in the expertise of genome biology and genetics. The application to genome study has been particularly developed with the introduction of the next-generation DNA sequencer (NGS) Roche/454 and Illumina/Solexa systems, along with bioinformation analysis technologies of whole-genome de novo assembly, expression profiling, DNA variation discovery, and genotyping. Both massive whole-genome shotgun paired-end sequencing and mate paired-end sequencing data are important steps for constructing de novo assembly of novel genome sequencing data. It is necessary to have DNA sequence information from a multiplatform NGS with at least 2x and 30x depth sequence of genome coverage using Roche/454 and Illumina/Solexa, respectively, for effective an way of de novo assembly. Massive short-length reading data from the Illumina/Solexa system is enough to discover DNA variation, resulting in reducing the cost of DNA sequencing. Whole-genome expression profile data are useful to approach genome system biology with quantification of expressed RNAs from a whole-genome transcriptome, depending on the tissue samples. The hybrid mRNA sequences from Rohce/454 and Illumina/Solexa are more powerful to find novel genes through de novo assembly in any whole-genome sequenced species. The 20x and 50x coverage of the estimated transcriptome sequences using Roche/454 and Illumina/Solexa, respectively, is effective to create novel expressed reference sequences. However, only an average 30x coverage of a transcriptome with short read sequences of Illumina/Solexa is enough to check expression quantification, compared to the reference expressed sequence tag sequence.

Base Sequence , Biology , Chimera , DNA , DNA Fingerprinting , Expressed Sequence Tags , Gene Expression Profiling , Genome , RNA , RNA, Messenger , Sequence Analysis, DNA , Transcriptome
Acta Pharmaceutica Sinica ; (12): 803-810, 2012.
Article in Chinese | WPRIM | ID: wpr-276240


Simple and effective methods are needed for the identification of Chinese medicinal material species and their variety. Lonicera japonica Thunb. is one of Chinese herbal medicines widely demanded. A total of 3 705 EST-SSRs of L. japonica and 2 818 EST-SSRs of L. japonica var. chinensis Thunb. were identified from EST database in our lab. In average, there was one EST-SSR per 4.05 kb in L. japonica ESTs and per 7.49 kb in L. japonica var. chinensis ESTs, separately. The identified SSRs in L. japonica were consisted of 51.98% dinucleotide and 34.61% trinucleotide repeats, while SSRs in L. japonica var. chinensis had 57.45% dinucleotide and 30.09% trinucleotide. The results reviewed that the classes AG/TC and GAG/TCT were predominant in the dinucleotide motifs and the trinucleotide motifs, respectively. Total 87 EST-SSRs were identified of significant difference between L. japonica and L. japonica var. chinensis. PCR products were obtained from 52 L. japonica samples in 13 out of 15 SSR markers tested. The polymorphism in L. japonica, L. japonica var. chinensis and other honeysuckles could be distinguished by three markers (jp.ssr4, jp.ssr64 and jp.ssr65) tested.

Dinucleotide Repeats , Expressed Sequence Tags , Flowers , Genetics , Lonicera , Classification , Genetics , Microsatellite Repeats , Plants, Medicinal , Classification , Genetics , Polymerase Chain Reaction , Polymorphism, Genetic , Trinucleotide Repeats
Acta Pharmaceutica Sinica ; (12): 1070-1078, 2012.
Article in Chinese | WPRIM | ID: wpr-276198


Traditional Chinese medicine (TCM) genomics and TCM synthetic biology are two hot fields in the TCM modernization. TCM genomics, including transcriptomics, structural genomics, genomic markers and functional genomics, aims to elucidate the biosynthetic pathways of TCM bioactive compounds and mine the related genes encoding enzymes involved in these pathways by analyzing genetic information on the original species of TCM, thus promoting the development of TCM synthetic biology, genome-assisted molecular identification and molecular breeding, and elucidation of the genetic mechanism underlying "Daodi". Genomics and related research provide us much deeper understanding of life process and improve our ability to create new life or modify the present organisms. Based on TCM genomics, TCM synthetic biology sets up a series of procedures to realize the production of TCM pharmacological active compounds in microorganism, including screening and modification of parts and devices, establishment of standardized part and device libraries, and reconstruction and modification of the biosynthetic pathway of TCM pharmacological active compounds in microorganism. TCM synthetic biology will provide a new resource of TCM pharmacological active compounds for the pharmacological study and research & development of new drugs, thus enhancing the core competitiveness of our pharmaceutical industry in the international markets.

Expressed Sequence Tags , Genomics , Medicine, Chinese Traditional , Methods , Plants, Medicinal , Genetics , Synthetic Biology , Transcriptome , Genetics
Acta Pharmaceutica Sinica ; (12): 1079-1084, 2012.
Article in Chinese | WPRIM | ID: wpr-276197


Squalene synthase (SQS) is a key enzyme in plant terpenoid biosynthetic pathway. This study focused on cloning and analysis of Huperzia serrata SQS (HsSQS1) gene. After searching the transcriptome dataset of H serrata, one unique sequence encoding SQS was discovered. The primers were designed according to the transcript sequence of HsSQS1 from the H. serrata transcriptome dataset. The open reading frame of HsSQS1 was cloned using RT-PCR strategy. The bioinformatic analysis of this gene and its corresponding protein were performed. The cDNA (named as HsSQS1) contains a 1263 bp open reading frame and encodes a predicted protein of 420 amino acids. The GenBank accession number for this gene is JQ004938. HsSQS1 contains two transmembrane regions, without signal peptide. The conserved domain of squalene synthase was presented in HsSQS1. HsSQS1 was more abundant in H. serrata root than in leaf and stem. This study cloned and analyzed squalene synthase gene from H. serrata for the first time. The result will provide a foundation for exploring the mechanism ofterpenoid biosynthesis in H. serrata plants.

Amino Acid Sequence , Biosynthetic Pathways , Cloning, Molecular , DNA, Complementary , Genetics , Expressed Sequence Tags , Farnesyl-Diphosphate Farnesyltransferase , Genetics , Metabolism , Genes, Plant , Genetics , Huperzia , Genetics , Molecular Sequence Data , Open Reading Frames , Phylogeny , Plant Leaves , Plant Roots , Plant Stems , Plants, Medicinal , Genetics , Triterpenes , Chemistry
Acta Pharmaceutica Sinica ; (12): 1106-1110, 2012.
Article in Chinese | WPRIM | ID: wpr-276193


Chinese agarwood is formed in the aromatic resinous wood formed in Aquilaria sinensis (Lour.) Gilg (botanical family: Thymelaeaceae). Only when suffering stress of wound, etc, can A. sinensis produce sesquiterpenes etc. compounds of agarwood around wounds. However, little is known about how wound induced the biosynthesis pathway of sesquiterpenes. To reveal the molecular mechanism of wound-induced agarwood formation, RNA sequencing (RNA-seq) technology was used to investigate the profile of gene expression in A. sinensis treated by mechanical wounding and elucidate its functional gene. A total of 40,295 ESTs with an average read length of 305 bp were generated and 22 095 unigenes were formed by initial gene splicing. 61.6% of these unigenes (13 611) were annotated using BLAST searches against the SwissProt, KEGG, Nr and Nt databases. Twenty-six unigenes (encoding 7 enzymes) were found to be involved in sesquiterpene of agarwood biosynthesis by bioinformatic tools of Gene Ontology and KEGG. Novel genes that are potentially involved in sesquiterpenes biosynthesis were identified in A. sinensis, providing data for further sesquiterpenes biosynthesis pathway by molecular methods and the EST data establish a foundation for future studies in the molecular mechanisms of wound-induce agarwood formation in A. sinensis.

Drugs, Chinese Herbal , Chemistry , Metabolism , Expressed Sequence Tags , Genes, Plant , Genetics , Plants, Medicinal , Chemistry , Genetics , Sequence Analysis, RNA , Sesquiterpenes , Chemistry , Metabolism , Stress, Physiological , Genetics , Thymelaeaceae , Chemistry , Genetics , Transcriptome , Genetics , Wood , Genetics , Metabolism
Article in Chinese | WPRIM | ID: wpr-274302


<p><b>OBJECTIVE</b>To study the distribution frequency and characteristics of nucleotide repeat of 94 923 ESTs for developing microsatellite primers, providing a theoretical basis and technical support for appropriate conservation and application of sweet wormwood (Artemisia annua).</p><p><b>METHOD</b>EST-SSR detection was performed using Perl program MISA. Gene Ontology (GO) annotations were formatted for input into the GOSlim program and the output was parsed to count the occurrence of each GO category. Primer 3 software was used to design 18 pairs primers, amplified products were separated on a 6% denaturing polyacrylamide gel using silver staining.</p><p><b>RESULT</b>By searching with Active Perl, totally 2 110 SSRs were detected, accounting for 8.6%. The frequency of occurrence of dinucleotide and trinucleotide was 28% and 50.4%, respectively. The most common repeat motifs of trinucleotide were ACC/GGT, accounting for 9.8%. Three hundred and twelve SSR-ESTs were annotated using GO terms. The suitable PCR system of 15 pairs primers was established, and revealed microsatellite polymorphism in 36 individuals.</p><p><b>CONCLUSION</b>There are a variety of motifs at EST-SSR locus in sweet wormwood, and more effective amplification and polymorphism in 18 pairs detected primers. Therefore, EST resource is an effective and feasible approach to develop SSR markers, and EST-SSRs will a powerful tool for studies of sweet wormwood genetic resources.</p>

Artemisia annua , Genetics , Expressed Sequence Tags , Microsatellite Repeats , Polymorphism, Genetic