Peri-implantitis is a frequently occurring gum disease linked to multi-factorial traits with various environmental and genetic causalities and no known concrete pathogenesis. The varying severity of peri-implantitis among patients with relatively similar environments suggests a genetic aspect which needs to be investigated to understand and regulate the pathogenesis of the disease. Six unrelated individuals with multiple clusterization implant failure due to severe peri-implantitis were chosen for this study. These six individuals had relatively healthy lifestyles, with minimal environmental causalities affecting peri-implantitis. Research was undertaken to investigate pathogenic genes in peri-implantitis albeit with a small number of subjects and incomplete elimination of environmental causalities. Whole-exome sequencing was performed on collected saliva samples via self DNA collection kit. Common variants with minor allele frequencies (MAF) > = 0.05 from all control datasets were eliminated and variants having high and moderate impact and loss of function were used for comparison. Gene set enrichment analysis was performed to reveal functional groups associated with the genetic variants. 2,022 genes were left after filtering against dbSNP, the 1000 Genomes East Asian population, and healthy Korean randomized subsample data (GSK project). 175 (p-value <0.05) out of 927 gene sets were obtained via GSEA (DAVID). The top 10 was chosen (p-value <0.05) from cluster enrichment showing significance of cytoskeleton, cell adhesion, and metal ion binding. Network analysis was applied to find relationships between functional clusters. Among the functional groups, ion metal binding was located in the center of all clusters, indicating dysfunction of regulation in metal ion concentration might affect cell morphology or cell adhesion, resulting in implant failure. This result may demonstrate the feasibility of and provide pilot data for a larger research project aimed at discovering biomarkers for early diagnosis of peri-implantitis.
Network analysis is a novel method to understand the complex pathogenesis of inflammation-driven atherosclerosis. Using this approach, we attempted to identify key inflammatory genes and their core transcriptional regulators in coronary artery disease (CAD). Initially, we obtained 124 candidate genes associated with inflammation and CAD using Polysearch and CADgene database for which protein-protein interaction network was generated using STRING 9.0 (Search Tool for the Retrieval of Interacting Genes) and visualized using Cytoscape v 2.8.3. Based on betweenness centrality (BC) and node degree as key topological parameters, we identified interleukin-6 (IL-6), vascular endothelial growth factor A (VEGFA), interleukin-1 beta (IL-1B), tumor necrosis factor (TNF) and prostaglandin-endoperoxide synthase 2 (PTGS2) as hub nodes. The backbone network constructed with these five hub genes showed 111 nodes connected via 348 edges, with IL-6 having the largest degree and highest BC. Nuclear factor kappa B1 (NFKB1), signal transducer and activator of transcription 3 (STAT3) and JUN were identified as the three core transcription factors from the regulatory network derived using MatInspector. For the purpose of validation of the hub genes, 97 test networks were constructed, which revealed the accuracy of the backbone network to be 0.7763 while the frequency of the hub nodes remained largely unaltered. Pathway enrichment analysis with ClueGO, KEGG and REACTOME showed significant enrichment of six validated CAD pathways - smooth muscle cell proliferation, acute-phase response, calcidiol 1-monooxygenase activity, toll-like receptor signaling, NOD-like receptor signaling and adipocytokine signaling pathways. Experimental verification of the above findings in 64 cases and 64 controls showed increased expression of the five candidate genes and the three transcription factors in the cases relative to the controls (p<0.05). Thus, analysis of complex networks aid in the prioritization of genes and their transcriptional regulators in complex diseases.
Transcriptome experiments are performed to assess protein abundance through mRNA expression analysis. Expression levels of genes vary depending on the experimental conditions and the cell response. Transcriptome data must be diverse and yet comparable in reference to stably expressed genes, even if they are generated from different experiments on the same biological context from various laboratories. In this study, expression patterns of 9090 microarray samples grouped into 381 NCBI-GEO datasets were investigated to identify novel candidate reference genes using randomizations and Receiver Operating Characteristic (ROC) curves. The analysis demonstrated that cell type specific reference gene sets display less variability than a united set for all tissues. Therefore, constitutively and stably expressed, origin specific novel reference gene sets were identified based on their coefficient of variation and percentage of occurrence in all GEO datasets, which were classified using Medical Subject Headings (MeSH). A large number of MeSH grouped reference gene lists are presented as novel tissue specific reference gene lists. The most commonly observed 17 genes in these sets were compared for their expression in 8 hepatocellular, 5 breast and 3 colon carcinoma cells by RT-qPCR to verify tissue specificity. Indeed, commonly used housekeeping genes GAPDH, Actin and EEF2 had tissue specific variations, whereas several ribosomal genes were among the most stably expressed genes in vitro. Our results confirm that two or more reference genes should be used in combination for differential expression analysis of large-scale data obtained from microarray or next generation sequencing studies. Therefore context dependent reference gene sets, as presented in this study, are required for normalization of expression data from diverse technological backgrounds.
We recently demonstrated that TNF-related apoptosis-inducing ligand (TRAIL) is protective of diet-induced diabetes in mice. While TRAIL has been implicated in chronic kidney disease, its role in vivo in diabetic nephropathy is not clear. The present study investigated the role of TRAIL in the pathogenesis of diabetic nephropathy using TRAIL-/-ApoE-/- mice.
TRAIL-/-ApoE-/- and ApoE-/- mice were fed a high fat diet for 20 w. Plasma glucose and insulin levels were assessed over 0, 5, 8 and 20 w. At 20 w, markers of kidney function including creatinine, phosphate, calcium and cystatin C were measured. Changes in mRNA expression of MMPs, TIMP-1, IL-1β and IL-18 were assessed in the kidney. Functional and histological changes in kidneys were examined. Glucose and insulin tolerance tests were performed.
TRAIL-/-ApoE-/- mice had significantly increased urine protein, urine protein:creatinine ratio, plasma phosphorous, and plasma cystatin C, with accelerated nephropathy. Histologically, increased extracellular matrix, mesangial expansion and mesangial cell proliferation in the glomeruli were observed. Moreover, TRAIL-/-ApoE-/- kidneys displayed loss of the brush border and disorganisation of tubular epithelium, with increased fibrosis. TRAIL-deficient kidneys also had increased expression of MMPs, TIMP-1, PAI-1, IL-1β and IL-18, markers of renal injury and inflammation. Compared with ApoE-/- mice, TRAIL-/-ApoE-/- mice displayed insulin resistance and type-2 diabetic features with reduced renal insulin-receptor expression.
Here, we show that TRAIL-deficiency in ApoE-/- mice exacerbates nephropathy and insulin resistance. Understanding TRAIL signalling in kidney disease and diabetes, may therefore lead to novel strategies for the treatment of diabetic nephropathy.
Switchgrass (Panicum virgatum) has received a lot of attention as a forage and bioenergy crop during the past few years. Gene expression studies are in progress to improve new traits and develop new cultivars. Quantitative real time PCR (qRT-PCR) has emerged as an important technique to study gene expression analysis. For accurate and reliable results, normalization of data with reference genes is essential. In this work, we evaluate the stability of expression of genes to use as reference for qRT-PCR in the grass P. virgatum. Eleven candidate reference genes, including eEF-1α, UBQ6, ACT12, TUB6, eIF-4a, GAPDH, SAMDC, TUA6, CYP5, U2AF, and FTSH4, were validated for qRT-PCR normalization in different plant tissues and under different stress conditions. The expression stability of these genes was verified by the use of two distinct algorithms, geNorm and NormFinder. Differences were observed after comparison of the ranking of the candidate reference genes identified by both programs but eEF-1α, eIF-4a, CYP5 and U2AF are ranked as the most stable genes in the samples sets under study. Both programs discard the use of SAMDC and TUA6 for normalization. Validation of the reference genes proposed by geNorm and NormFinder were performed by normalization of transcript abundance of a group of target genes in different samples. Results show similar expression patterns when the best reference genes selected by both programs were used but differences were detected in the transcript abundance of the target genes. Based on the above research, we recommend the use of different statistical algorithms to identify the best reference genes for expression data normalization. The best genes selected in this study will help to improve the quality of gene expression data in a wide variety of samples in switchgrass.
Variegation in flower color is commonly observed in many plant species and also occurs on ornamental peaches (Prunus persica f. versicolor [Sieb.] Voss). Variegated plants are highly valuable in the floricultural market. To gain a global perspective on genes differentially expressed in variegated peach flowers, we performed large-scale transcriptome sequencing of white and red petals separately collected from a variegated peach tree.
A total of 1,556,597 high-quality reads were obtained, with an average read length of 445 bp. The ESTs were assembled into 16,530 contigs and 42,050 singletons. The resulting unigenes covered about 60% of total predicted genes in the peach genome. These unigenes were further subjected to functional annotation and biochemical pathway analysis. Digital expression analysis identified a total of 514 genes differentially expressed between red and white flower petals. Since peach flower coloration is determined by the expression and regulation of structural genes relevant to flavonoid biosynthesis, a detailed examination detected four key structural genes, including C4H, CHS, CHI and F3H, expressed at a significantly higher level in red than in white petal. Except for the structural genes, we also detected 11 differentially expressed regulatory genes relating to flavonoid biosynthesis. Using the differentially expressed structural genes as the test objects, we validated the digital expression results by using quantitative real-time PCR, and the differential expression of C4H, CHS and F3H were confirmed.
In this study, we generated a large EST collection from flower petals of a variegated peach. By digital expression analysis, we identified an informative list of candidate genes associated with variegation in peach flowers, which offered a unique opportunity to uncover the genetic mechanisms underlying flower color variegation.
The thermal stability and topology of non-canonical structures of G-quadruplexes and hairpins in template DNA were investigated, and the effect of non-canonical structures on transcription fidelity was evaluated quantitatively. We designed ten template DNAs: A linear sequence that does not have significant higher-order structure, three sequences that form hairpin structures, and six sequences that form G-quadruplex structures with different stabilities. Templates with non-canonical structures induced the production of an arrested, a slipped, and a full-length transcript, whereas the linear sequence produced only a full-length transcript. The efficiency of production for run-off transcripts (full-length and slipped transcripts) from templates that formed the non-canonical structures was lower than that from the linear. G-quadruplex structures were more effective inhibitors of full-length product formation than were hairpin structure even when the stability of the G-quadruplex in an aqueous solution was the same as that of the hairpin. We considered that intra-polymerase conditions may differentially affect the stability of non-canonical structures. The values of transcription efficiencies of run-off or arrest transcripts were correlated with stabilities of non-canonical structures in the intra-polymerase condition mimicked by 20 wt% polyethylene glycol (PEG). Transcriptional arrest was induced when the stability of the G-quadruplex structure (−ΔGo37) in the presence of 20 wt% PEG was more than 8.2 kcal mol−1. Thus, values of stability in the presence of 20 wt% PEG are an important indicator of transcription perturbation. Our results further our understanding of the impact of template structure on the transcription process and may guide logical design of transcription-regulating drugs.
Computational analyses of functions of gene sets obtained in microarray analyses or by topical database searches are increasingly important in biology. To understand their functions, the sets are usually mapped to Gene Ontology knowledge bases by means of over-representation analysis (ORA). Its result represents the specific knowledge of the functionality of the gene set. However, the specific ontology typically consists of many terms and relationships, hindering the understanding of the ‘main story’. We developed a methodology to identify a comprehensibly small number of GO terms as “headlines” of the specific ontology allowing to understand all central aspects of the roles of the involved genes. The Functional Abstraction method finds a set of headlines that is specific enough to cover all details of a specific ontology and is abstract enough for human comprehension. This method exceeds the classical approaches at ORA abstraction and by focusing on information rather than decorrelation of GO terms, it directly targets human comprehension. Functional abstraction provides, with a maximum of certainty, information value, coverage and conciseness, a representation of the biological functions in a gene set plays a role. This is the necessary means to interpret complex Gene Ontology results thus strengthening the role of functional genomics in biomarker and drug discovery.
Herbivore grazing is a multiple-component process that includes wounding, defoliation, and saliva deposition. Despite the extensive published research on mechanical wounding and defoliation, no analysis to identify the genes that specify defoliation and mechanical wounding has been performed. Moreover, the influence of the expression of these genes on plant regrowth after defoliation remains poorly understood.
Seven cDNA libraries for RNA samples collected from stubble tissues that had been mechanically wounded or defoliated at 2, 6 and 24 h along with the control were sequenced using the Illumina/Solexa platform. A comparative transcriptomic analysis of the sequencing data was conducted. In total, 1,836 and 3,238 genes were detected with significant differential expression levels after wounding and defoliation, respectively, during one day. GO, KOG and pathway-based enrichment analyses were performed to determine and further understand the biological functions of those differentially expressed genes (DEGs). The results demonstrated that both wounding and defoliation activated the systemic synthesis of jasmonate (JA). However, defoliation specifically reduced the expression levels of ribosomal protein genes, cell division or cell expansion-related genes, and lignin biosynthesis genes and may have negatively affected plant growth. Further analysis revealed that the regrowth of elongating leaves was significantly retarded after defoliation at 6 h through the following 7 days of measurement, suggesting that the gene expression pattern and phenotype are consistent. Fifteen genes were selected, and their expression levels were confirmed by quantitative RT-PCR (qRT-PCR). Thirteen of them exhibited expression patterns consistent with the digital gene expression (DGE) data.
These sequencing datasets allowed us to elucidate the common and distinct mechanisms of plant responses to defoliation and wounding. Additionally, the distinct DEGs represent a valuable resource for novel gene discovery that may improve plant resistance to defoliation from various processes.
Betula platyphylla Suk (birch) is a fast-growing woody species that is important in pulp industries and the biofuels. However, as an important pulp species, few studies had been performed on its wood formation. In the present study, we investigated the molecular responses of birch xylem to artificial bending and gravitational stimuli. After trunks of birch trees were subjected to bending for 8 weeks, the cellulose content was significantly greater in tension wood (TW) than in opposite wood (OW) or normal wood (NW), whereas the lignin content in TW was significantly lower than that in OW and NW. In addition, TW grew more rapidly than OW and generated TW-specific fibers with an additional G-layer. Three transcriptome libraries were constructed from TW, OW and NW of B. platyphylla, respectively, after the plants were subjected to artificial bending. Overall, 80,909 nonredundant unigenes with a mean size of 768 nt were assembled. Expression profiles were generated, and 9,684 genes were found to be significantly differentially expressed among the TW, OW and NW libraries. These included genes involved in secondary cell wall structure, wood composition, and cellulose or lignin biosynthesis. Our study showed that during TW formation, genes involved in cellulose synthesis were induced, while the expression of lignin synthesis-related genes decreased, resulting in increased cellulose content and decreased lignin levels in TW. In addition, fasciclin-like arabinogalactan proteins play important role in TW formation. These findings may provide important insights into wood formation at the molecular level.
The Chinese salamander (Hynobius chinensis), an endangered amphibian species of salamander endemic to China, has attracted much attention because of its value of studying paleontology evolutionary history and decreasing population size. Despite increasing interest in the Hynobius chinensis genome, genomic resources for the species are still very limited. A comprehensive transcriptome of Hynobius chinensis, which will provide a resource for genome annotation, candidate genes identification and molecular marker development should be generated to supplement it.
We performed a de novo assembly of Hynobius chinensis transcriptome by Illumina sequencing. A total of 148,510 nonredundant unigenes with an average length of approximately 580 bp were obtained. In all, 60,388 (40.66%) unigenes showed homologous matches in at least one database and 33,537 (22.58%) unigenes were annotated by all four databases. In total, 41,553 unigenes were categorized into 62 sub-categories by BLAST2GO search, and 19,468 transcripts were assigned to 140 KEGG pathways. A large number of unigenes involved in immune system, local adaptation, reproduction and sex determination were identified, as well as 31,982 simple sequence repeats (SSRs) and 460,923 putative single nucleotide polymorphisms (SNPs).
This dataset represents the first transcriptome analysis of the Chinese salamander (Hynobius chinensis), an endangered species, to be also the first time of hynobiidae. The transcriptome will provide valuable resource for further research in discovery of new genes, protection of population, adaptive evolution and survey of various pathways, as well as development of molecule markers in Chinese salamander; and reference information for closely related species.
RBR (RING1-IBR-RING2) proteins play an important role in protein ubiquitination and are involved in many cellular processes. Recent studies showed plant RBR genes were induced by abiotic and biotic stresses. However, detailed studies on RBR genes in the important oil crop, soybean (Glycine max (L.) Merr.), is still lacking. Here we performed a genome-wide search and identified 24 RBR domain-containing genes from the soybean genome sequence and cloned 11 of them. Most soybean RBR proteins contain a highly conserved RBR supra-domain. Phylogenetic analyses indicated all 24 soybean RBR proteins are most related to the RBR proteins from Phaseolus vulgaris, and could be classified into seven groups including Ariadne A, Ariadne B, ARA54, Plant IIA, Plant IIB, Plant IIC, and Helicase. Tandem duplication and block duplication were found among the Ariadne B and Plant IIC group of soybean RBR genes. Despite the conserved RBR supra-domain, there are extensive variations in the additional protein motifs and exon-intron structures between different groups, which indicate they might have diverse functions. Most soybean RBR proteins are predicted to localize in nucleus, and four of them were experimentally confirmed by GFP fusion proteins. Soybean RBR genes are broadly expressed in many tissue types with a little more abundant in the roots and flowers than leaves, stems, and seeds. The expression of GmRTRTP3 (Plant IIB) and GmRTRTP5 (Plant IIC) are induced by NaCl treatment, which suggests these RBR genes might be involved in soybean response to abiotic stresses.
One of the fundamental tasks in biology is to identify the functions of all proteins to reveal the primary machinery of a cell. Knowledge of the subcellular locations of proteins will provide key hints to reveal their functions and to understand the intricate pathways that regulate biological processes at the cellular level. Protein subcellular location prediction has been extensively studied in the past two decades. A lot of methods have been developed based on protein primary sequences as well as protein-protein interaction network. In this paper, we propose to use the protein-protein interaction network as an infrastructure to integrate existing sequence based predictors. When predicting the subcellular locations of a given protein, not only the protein itself, but also all its interacting partners were considered. Unlike existing methods, our method requires neither the comprehensive knowledge of the protein-protein interaction network nor the experimentally annotated subcellular locations of most proteins in the protein-protein interaction network. Besides, our method can be used as a framework to integrate multiple predictors. Our method achieved 56% on human proteome in absolute-true rate, which is higher than the state-of-the-art methods.
Genetic aberrations of DNA repair enzymes are known to be common events and to be associated with different cancer entities. Aim of the following study was to analyze the genetic association of rs1136410 (Val762Ala) in PARP1 gene with the risk of breast cancer using genotypic assays and insilico structural predictions. Genotypic analysis of individual locus showed statistically significant association of Val762Ala with increased susceptibility to breast cancer. Protein structural analysis was performed with Val762Ala variant allele and compared with the predicted native protein structure. Protein prediction analysis showed that this nsSNP may cause changes in the protein structure and it is associated with the disease. In addition to the native and mutant 3D structures of PARP1 were also analyzed using solvent accessibility models for further protein stability confirmation. Taken together, this the first study that confirmed Val762Ala variant has functional effect and structural impact on the PARP1 and may play an important role in breast cancer progression in Saudi population.
Detecting biclusters from expression data is useful, since biclusters are coexpressed genes under only part of all given experimental conditions. We present a software called SiBIC, which from a given expression dataset, first exhaustively enumerates biclusters, which are then merged into rather independent biclusters, which finally are used to generate gene set networks, in which a gene set assigned to one node has coexpressed genes. We evaluated each step of this procedure: 1) significance of the generated biclusters biologically and statistically, 2) biological quality of merged biclusters, and 3) biological significance of gene set networks. We emphasize that gene set networks, in which nodes are not genes but gene sets, can be more compact than usual gene networks, meaning that gene set networks are more comprehensible. SiBIC is available at http://utrecht.kuicr.kyoto-u.ac.jp:8080/miami/faces/index.jsp.
To explore the endocrine mechanisms of aldosterone-producing adenoma (APA) by using the microarray expression profiles of normal and APA samples.
The gene expression profile GSE8514 was downloaded from Gene Expression Omnibus database, including samples from normal adrenals (n = 5) and APAs (n = 10). The differentially expressed genes (DEGs) were identified by samr package and endocrine DEGs were obtained according to Clinical Genome Database. Then, functional enrichment analysis of screened DEGs was performed by DAVID (Database for Annotation, Visualization and Integrated Discovery). Finally, a regulatory network was constructed to screen endocrine genes related with adrenal dysfunction and pathway enrichment analysis for the constructed network was performed.
A total of 2149 DEGs were identified including 379 up- and 1770 down-regulated genes. And 26 endocrine genes were filtered from the DEGs. Furthermore, the down-regulated DEGs are mainly related to protein kinase cascade, response to molecule of bacterial origin, response to lipopolysaccharide, cellular macromolecule catabolic process and macromolecule catabolic process, while the up-regulated DEGs are related with regulation of ion transport. The target genes of VDR (vitamin D receptor), one of the three endocrine genes differentially expressed in the regulatory network, were endocrine genes including CYP24A1 (25-hydroxyvitamin D-24-hydroxylase) and PTH (parathyroid hormone). Three pathways may be associated with APA pathogenesis including cytokine-cytokine receptor interaction, pathways in cancer and autoimmune thyroid disease.
The VDR is the most significant transcription factor and related endocrine genes might play important roles in the endocrine mechanisms of APA.
Plasmodiophora brassicae, the causal agent of clubroot disease of the Brassica crops, is widespread in the world. Quantitative trait loci (QTLs) for partial resistance to 4 different isolates of P. brassicae (Pb2, Pb4, Pb7, and Pb10) were investigated using a BC1F1 population from a cross between two subspecies of Brassica rapa, i.e. Chinese cabbage inbred line C59-1 as a susceptible recurrent parent and turnip inbred line ECD04 as a resistant donor parent. The BC1F2 families were assessed for resistance under controlled conditions. A linkage map constructed with simple sequence repeats (SSR), unigene-derived microsatellite (UGMS) markers, and specific markers linked to published clubroot resistance (CR) genes of B. rapa was used to perform QTL mapping. A total of 6 QTLs residing in 5 CR QTL regions of the B. rapa chromosomes A01, A03, and A08 were identified to account for 12.2 to 35.2% of the phenotypic variance. Two QTL regions were found to be novel except for 3 QTLs in the respective regions of previously identified Crr1, Crr2, and Crr3. QTL mapping results indicated that 1 QTL region was common for partial resistance to the 2 isolates of Pb2 and Pb7, whereas the others were specific for each isolate. Additionally, synteny analysis between B. rapa and Arabidopsis thaliana revealed that all CR QTL regions were aligned to a single conserved crucifer blocks (U, F, and R) on 3 Arabidopsis chromosomes where 2 CR QTLs were detected in A. thaliana. These results suggest that some common ancestral genomic regions were involved in the evolution of CR genes in B. rapa.
To test, whether 10 genes, diagnostic of renal allograft rejection in blood, are able to diagnose and predict cardiac allograft rejection, we analyzed 250 blood samples from heart transplant recipients with and without acute rejection (AR) and with cytomegalovirus (CMV) infection by QPCR. A QPCR-based logistic regression model was built on 5 of these 10 genes (AR threshold composite score>37% = AR) and tested for AR prediction in an independent set of 109 samples, where it correctly diagnosed AR with 89% accuracy, with no misclassifications for AR ISHLT grade 1b. CMV infection did not confound the AR score. The genes correctly diagnosed AR in a blood sample within 6 months prior to biopsy diagnosis with 80% sensitivity and untreated grade 1b AR episodes had persistently elevated scores until 6 months after biopsy diagnosis. The gene score was also correlated with presence or absence of cardiac allograft vasculopathy (CAV) irrespective of rejection grade. In conclusion, there is a common transcriptional axis of immunological trafficking in peripheral blood in both renal and cardiac organ transplant rejection, across a diverse recipient age range. A common gene signature, initially identified in the setting of renal transplant rejection, can be utilized serially after cardiac transplantation, to diagnose and predict biopsy confirmed acute heart transplant rejection.
In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for β-caryophyllene (CPS), epi-cedrol (ECS) and β-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production.
Pre–messenger RNA (mRNA) 3′-end cleavage and subsequent polyadenylation strongly regulate gene expression. In comparison with the upstream or downstream motifs, relatively little is known about the feature differences of polyadenylation [poly(A)] sites among major kingdoms. We suspect that the precise poly(A) sites are very selective, and we therefore mapped mRNA poly(A) sites on complete and nearly complete genomes using mRNA sequences available in the National Center for Biotechnology Information (NCBI) Nucleotide database. In this paper, we describe the mRNA nucleotide [i.e., the poly(A) tail attachment position] that is directly in attachment with the poly(A) tail and the pre-mRNA nucleotide [i.e., the poly(A) tail starting position] that corresponds to the first adenosine of the poly(A) tail in the 29 most-mapped species (2 fungi, 2 protists, 18 animals, and 7 plants). The most representative pre-mRNA dinucleotides covering these two positions were UA, CA, and GA in 17, 10, and 2 of the species, respectively. The pre-mRNA nucleotide at the poly(A) tail starting position was typically an adenosine [i.e., A-type poly(A) sites], sometimes a uridine, and occasionally a cytidine or guanosine. The order was U>C>G at the attachment position but A>>U>C≥G at the starting position. However, in comparison with the mRNA nucleotide composition (base composition), the poly(A) tail attachment position selected C over U in plants and both C and G over U in animals, in both A-type and non-A-type poly(A) sites. Animals, dicot plants, and monocot plants had clear differences in C/G ratios at the poly(A) tail attachment position of the non-A-type poly(A) sites. This study of poly(A) site evolution indicated that the two positions within poly(A) sites had distinct nucleotide compositions and were different among kingdoms.
The Asia-Pacific Bioinformatics Network (APBioNet) held the first International Conference on Bioinformatics (InCoB) in Bangkok in 2002 to promote North-South networking. Commencing as a forum for Asia-Pacific researchers to interact with and learn from with scientists of developed countries, InCoB has become a major regional bioinformatics conference, with participants from the region as well as North America and Europe. Since 2006, InCoB has selected the best submissions for publication in BMC Bioinformatics. In response to the growth and maturation of data-driven approaches, InCoB added BMC Genomics in 2009 and with the introduction of this conference supplement, BMC Systems Biology to its journal choices for submitting authors. Co-hosting InCoB2013 with the second International Conference for Translational Bioinformatics (ICTBI) is in line with InCoB's support for the current trend in taking bioinformatics to the bedside, along with a systems approach to solving biological problems.
Aromatic essential oils extracted from fresh fruits of Litsea cubeba (Lour.) Pers., have diverse medical and economic values. The dominant components in these essential oils are monoterpenes and sesquiterpenes. Understanding the molecular mechanisms of terpenoid biosynthesis is essential for improving the yield and quality of terpenes. However, the 40 available L. cubeba nucleotide sequences in the public databases are insufficient for studying the molecular mechanisms. Thus, high-throughput transcriptome sequencing of L. cubeba is necessary to generate large quantities of transcript sequences for the purpose of gene discovery, especially terpenoid biosynthesis related genes.
Using Illumina paired-end sequencing, approximately 23.5 million high-quality reads were generated. De
novo assembly yielded 68,648 unigenes with an average length of 834 bp. A total of 38,439 (56%) unigenes were annotated for their functions, and 35,732 and 25,806 unigenes could be aligned to the GO and COG database, respectively. By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG), 16,130 unigenes were assigned to 297 KEGG pathways, and 61 unigenes, which contained the mevalonate and 2-C-methyl-D-erythritol 4-phosphate pathways, could be related to terpenoid backbone biosynthesis. Of the 12,963 unigenes, 285 were annotated to the terpenoid pathways using the PlantCyc database. Additionally, 14 terpene synthase genes were identified from the transcriptome. The expression patterns of the 16 genes related to terpenoid biosynthesis were analyzed by RT-qPCR to explore their putative functions.
RNA sequencing was effective in identifying a large quantity of sequence information. To our knowledge, this study is the first exploration of the L. cubeba transcriptome, and the substantial amount of transcripts obtained will accelerate the understanding of the molecular mechanisms of essential oils biosynthesis. The results may help improve future genetic and genomics studies on the molecular mechanisms behind the chemical composition of essential oils in L. cubeba fruits.
Advanced button mushroom cultivars that are less sensitive to mechanical bruising are required by the mushroom industry, where automated harvesting still cannot be used for the fresh mushroom market. The genetic variation in bruising sensitivity (BS) of Agaricus bisporus was studied through an incomplete set of diallel crosses to get insight in the heritability of BS and the combining ability of the parental lines used and, in this way, to estimate their breeding value. To this end nineteen homokaryotic lines recovered from wild strains and cultivars were inter-crossed in a diallel scheme. Fifty-one successful hybrids were grown under controlled conditions, and the BS of these hybrids was assessed. BS was shown to be a trait with a very high heritability. The results also showed that brown hybrids were generally less sensitive to bruising than white hybrids. The diallel scheme allowed to estimate the general combining ability (GCA) for each homokaryotic parental line and to estimate the specific combining ability (SCA) of each hybrid. The line with the lowest GCA is seen as the most attractive donor for improving resistance to bruising. The line gave rise to hybrids sensitive to bruising having the highest GCA value. The highest negative SCA possibly indicates heterosis effects for resistance to bruising. This study provides a foundation for estimating breeding value of parental lines to further study the genetic factors underlying bruising sensitivity and other quality-related traits, and to select potential parental lines for further heterosis breeding. The approach of studying combining ability in a diallel scheme was used for the first time in button mushroom breeding.
Athetis lepigone Möschler (Lepidoptera: Noctuidae) has recently become an important insect pest of maize (Zea mays) crops in China. In order to understand the characteristics of the different developmental stages of this pest, we used Illumina short-read sequences to perform de novo transcriptome assembly and gene expression analysis for egg, larva, pupa and adult developmental stages. We obtained 10.08 Gb of raw data from Illumina sequencing and recovered 81,356 unigenes longer than 100 bp through a de novo assembly. The total sequence length reached 49.75 Mb with 858 bp of N50 and an average unigene length of 612 bp. Annotation analysis of predicted proteins indicate that 33,736 unigenes (41.47% of total unigenes) are matches to genes in the Genbank Nr database. The unigene sequences were subjected to GO, COG and KEGG functional classification. A large number of differentially expressed genes were recovered by pairwise comparison of the four developmental stages. The most dramatic differences in gene expression were found in the transitions from one stage to another stage. Some of these differentially expressed genes are related to cuticle and wing formation as well as the growth and development. We identified more than 2,500 microsatellite markers that may be used for population studies of A.
lepigone. This study lays the foundation for further research on population genetics and gene function analysis in A. lepigone.