Tag-based Digital Gene Expression Research Articles

De novo transcriptional analysis of the response to starvation stress in the white ridgetail prawn, Exopalaemon carinicauda

To study the mechanism of the biomolecular response in Exopalaemon carinicauda to starvation stress, we subjected muscle tissue RNA samples from four stress points, including 0 d(control group), 10 d, 20 d, and 30 d, to starvation stress on white ridgetail prawn with a body weight of 1.41 + 0.42 g, aquaculture water temperature of 23–25 °C, salinity of 26, dissolved oxygen ≥5 mg/L, and pH 8–8.5, Then performed de novo transcriptome assembly and gene expression analysis using BGISEQ-500 with a tag-based digital gene expression (DGE) system. By de novo assembling at the four times, we obtained 28,167, 21,115, 24,497, and 27,080 reads, respectively. The results showed that the stress at 10 d led to no significant difference in the expressed genes, while the stress at 20 d and 30 d showed a significant increase (or decrease) in the expression of 97 (276) and 143 (410) genes, respectively, which were involved in 8 different metabolic pathways. In addition, we detected 2647 unigene transcription factors. Eleven upregulated and sixteen downregulated genes from the different starvation stress groups were choose to verify the reliability of the transcriptome data, and the results showed that the expression trends of these genes were consistent with the results shown by the transcriptome. The analysis of the experimental data and our discussion of the response mechanism of white ridgetail prawn under starvation stress provides a foundation for further screening of the key genes of starvation stress and may help to elucidate their functions.

Exploring gene expression changes in the amphioxus gill after poly(I:C) challenge using digital expression profiling

Amphioxus, a cephalochordate, is a key model animal for studying the evolution of vertebrate immunity. Recently, studies have revealed that microRNA (miRNA) expression profiles change significantly in the amphioxus gill after immune stimulation, but it remains largely unknown how gene expression responds to immune stress. Elucidating gene expression changes in the amphioxus gill will provide a deeper understanding of the evolution of gill immunity in vertebrates. Here, we used high-throughput RNA sequencing technology (RNA-seq) to conduct tag-based digital gene expression profiling (DGE) analyses of the gills of control Branchiostoma belcheri and of those exposed to the viral mimic, poly(I:C) (pIC). Six libraries were created for the control and treatment groups including three biological replicates per group. A total of 1999 differently expressed genes (DEGs) were obtained, with 571 and 1428 DEGs showing up- or down-regulation, respectively, in the treatment group. Enrichment analysis of gene ontology (GO) terms and pathways revealed that the DEGs were primarily related to immune and defense response, apoptosis, human disease, cancer, protein metabolism, enzyme activity, and regulatory processes. In addition, eight DEGs were randomly selected to validate the RNA-seq data using real-time quantitative PCR (qRT-PCR), and the results confirmed the accuracy of the RNA-seq approach. Next, we screened eight key responding genes to examine the dynamic changes in expression levels at different time points in more detail. The results indicated that expressions of TRADD, MARCH, RNF31, NF-κb, CYP450, TNFRSF6B, IFI and LECT1 were induced to participate in the antiviral response against pIC. This study provides a valuable resource for understanding the role of the amphioxus gill in antiviral immunity and the evolution of gill immunity in vertebrates.

Transcriptome profiling and digital gene expression analysis of the skin of Dybowski's frog (Rana dybowskii) exposed to Aeromonas hydrophila.

Recently, populations of Rana dybowskii, an important amphibian species in Northeast China, have decreased, mainly owing to the disease caused by Aeromonas hydrophila. However, effective control methods have not yet been developed. In order to explore the immune responses of R. dybowskii upon exposure to A. hydrophila infection, Illumina high-throughput transcriptome sequencing and digital gene expression (DGE) technology were employed to investigate transcriptomic changes in the skin of R. dybowskii exposed to A. hydrophila. In this work, a total of 26,244,446 transcriptome sequencing reads were obtained and assembled into 109,089 unique unigenes using de novo assembly, and a total of 37,105 unigenes (34.0%) were functionally annotated against the non-redundant (Nr), Swiss-Prot, Cluster of Orthologous Groups of Proteins (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) databases. Gene expression changes in the skin tissue of R. dybowskii exposed to A. hydrophila were investigated by a tag-based DGE system, and a total of 1435 significantly differentially expressed genes were identified, including 460 that were up-regulated and 975 that were down-regulated, indicating a large change in the host transcriptome profile exposed to A. hydrophila. Among these, 478 genes were associated with immune-relevant pathways, metabolic pathways, cellular components, growth, migration, and muscle and hormone signaling pathways. We confirmed the differential expression of 106 immune-relevant genes associated with innate and adaptive immune responses. Our data provide a fairly comprehensive molecular biology background for the deeper understanding of the amphibian immune system following A. hydrophila infection.

Differential gene expression profile of Buyanghuanwu decoction in rats with ventricular remodeling post-myocardial infarction

ObjectiveTo investigate the effect of Buyanghuanwu decoction (BYHWD) on gene expression in ventricular remodeling post-myocardial infarction in rats. MethodsAnimal models of myocardial infarction were established by permanent ligation of the left anterior descending coronary artery. Echocardiography measurements were performed after the treatment of BYHWD (18 g·kg−1·dΔ1) for 90 days. Myocardial collagen was observed by mallory trichrome staining. Capillary density was quantified by using Factor VIII immunohistochemical staining. Differentially expressed genes were explored by a short-read sequencing technology combined with a tag-based digital gene expression profiling (DGE) system. Real-time quantitative polymerase chain reaction detecting system (qPCR) was used to validate the sequencing results. After assembling the gene information from Sham, model and BYHWD groups, we constructed three DGE libraries based on each group. The sequencing of three libraries generated 66 000-73 000 unique tags, which were mapped to reference sequences for annotation of expressed genes. ResultsAmong them, 511 and 352 differentially expressed genes were found in comparison with sham/model and model/BYHWD, respectively. Fifty-five genes exhibited reversed direction of gene expression differences between Sham/Model and Model/BYHWD groups. We found that transforming growth factor beta receptor-1, junctophilin-2, monocyte chemotactic protein 1, neuropeptide Y, arachidonate 5-Lipoxygenase, arachidonate 15-Lipoxygenase were significantly modulated, which suggested the involvement of these genes in BYHWD treatment. ConclusionThe DGE profiling data provide comprehensive gene expression information at the transcriptional level that could facilitate our understanding of the pharmacological mechanisms of BYHWD in ventricular remodeling post-myocardial infarction.

Characterization of transcriptome in the Indian meal moth Plodia interpunctella (Lepidoptera: Pyralidae) and gene expression analysis during developmental stages

The Indian meal moth Plodia interpunctella (Lepidoptera: Pyralidae) is a worldwide pest that causes serious damage to stored foods. Although many efforts have been conducted on this species due to its economic importance, the study of genetic basis of development, behavior and insecticide resistance has been greatly hampered due to lack of genomic information. In this study, we used high throughput sequencing platform to perform a de novo transcriptome assembly and tag-based digital gene expression profiling (DGE) analyses across four different developmental stages of P. interpunctella (egg, third-instar larvae, pupae and adult). We obtained approximate 9gigabyte (GB) of clean data and recovered 84,938 unigenes, including 37,602 clusters and 47,336 singletons. These unigenes were annotated using BLAST against the non-redundant protein databases and then functionally classified based on Gene Ontology (GO), Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes databases (KEGG). A large number of differentially expressed genes were identified by pairwise comparisons among different developmental stages. Gene expression profiles dramatically changed between developmental stage transitions. Some of these differentially expressed genes were related to digestion and cuticularization. Quantitative real-time PCR results of six randomly selected genes conformed the findings in the DGEs. Furthermore, we identified over 8000 microsatellite markers and 97,648 single nucleotide polymorphisms which will be useful for population genetics studies of P. interpunctella. This transcriptomic information provided insight into the developmental basis of P. interpunctella and will be helpful for establishing integrated management strategies and developing new targets of insecticides for this serious pest.

Transcriptome Analysis of Capsicum Chlorosis Virus-Induced Hypersensitive Resistance Response in Bell Capsicum.

BackgroundCapsicum chlorosis virus (CaCV) is an emerging pathogen of capsicum, tomato and peanut crops in Australia and South-East Asia. Commercial capsicum cultivars with CaCV resistance are not yet available, but CaCV resistance identified in Capsicum chinense is being introgressed into commercial Bell capsicum. However, our knowledge of the molecular mechanisms leading to the resistance response to CaCV infection is limited. Therefore, transcriptome and expression profiling data provide an important resource to better understand CaCV resistance mechanisms.Methodology/Principal FindingsWe assembled capsicum transcriptomes and analysed gene expression using Illumina HiSeq platform combined with a tag-based digital gene expression system. Total RNA extracted from CaCV/mock inoculated CaCV resistant (R) and susceptible (S) capsicum at the time point when R line showed a strong hypersensitive response to CaCV infection was used in transcriptome assembly. Gene expression profiles of R and S capsicum in CaCV- and buffer-inoculated conditions were compared. None of the genes were differentially expressed (DE) between R and S cultivars when mock-inoculated, while 2484 genes were DE when inoculated with CaCV. Functional classification revealed that the most highly up-regulated DE genes in R capsicum included pathogenesis-related genes, cell death-associated genes, genes associated with hormone-mediated signalling pathways and genes encoding enzymes involved in synthesis of defense-related secondary metabolites. We selected 15 genes to confirm DE expression levels by real-time quantitative PCR.Conclusion/SignificanceDE transcript profiling data provided comprehensive gene expression information to gain an understanding of the underlying CaCV resistance mechanisms. Further, we identified candidate CaCV resistance genes in the CaCV-resistant C. annuum x C. chinense breeding line. This knowledge will be useful in future for fine mapping of the CaCV resistance locus and potential genetic engineering of resistance into CaCV-susceptible crops.

Decoding regulatory landscape of somatic embryogenesis reveals differential regulatory networks between japonica and indica rice subspecies.

Somatic embryogenesis is a unique process in plants and has considerable interest for biotechnological application. Compare to japonica, indica rice has been less responsive to in vitro culture. We used Illumina Hiseq 2000 sequencing platform for comparative transcriptome analysis between two rice subspecies at six different developmental stages combined with a tag-based digital gene expression profiling. Global gene expression among different samples showed greater complexity in japonica rice compared to indica which may be due to polyphyletic origin of two rice subspecies. Expression pattern in initial stage indicate major differences in proembryogenic callus induction phase that may serve as key regulator to observe differences between both subspecies. Our data suggests that phytohormone signaling pathways consist of elaborate networks with frequent crosstalk, thereby allowing plants to regulate somatic embryogenesis pathway. However, this crosstalk varies between the two rice subspecies. Down regulation of positive regulators of meristem development (i.e. KNOX, OsARF5) and up regulation of its counterparts (OsRRs, MYB, GA20ox1/GA3ox2) in japonica may be responsible for its better regeneration and differentiation of somatic embryos. Comprehensive gene expression information in the present experiment may also facilitate to understand the monocot specific meristem regulation for dedifferentiation of somatic cell to embryogenic cells.

Identification of genes regulated by histone acetylation during root development in Populus trichocarpa

BackgroundHistone deacetylases (HDACs) are key enzymes catalyzing the removal of acetyl groups from histones. HDACs act in concert with histone acetyltransferases (HATs) to regulate histone acetylation status, which modifies chromatin structure, affecting gene transcription and thus regulating multiple biological processes such as plant growth and development. Over a decade, certain HDACs in herbaceous plants have been deeply studied. However, functions of HDACs in woody plants are not well understood.ResultsHistone deacetylase specific inhibitor trichostatin A (TSA) was used to investigate the role of HDACs in organogenesis of roots and root development in Populus trochocarpa. The adventitious roots were regenerated and grown on medium supplemented with 0, 1, and 2.5 μM TSA. TSA treatment delayed root regeneration and inhibited primary root growth. To examine the genes modified by TSA in the regenerated roots, tag-based digital gene expression (DGE) analysis was performed using Illumina HiSeqTM 2000. Approximately 4.5 million total clean tags were mapped per library. The distinct clean tags for the three libraries corresponding to 0, 1 and 2.5 μM TSA treatment were 166167, 143103 and 153507, from which 38.45 %, 31.84 % and 38.88 % were mapped unambiguously to the unigene database, respectively. Most of the tags were expressed at similar levels, showing a < 5-fold difference after 1 μM and 2.5 μM TSA treatments and the maximum fold-change of the tag copy number was around 20. The expression levels of many genes in roots were significantly altered by TSA. A total of 36 genes were up-regulated and 1368 genes were down-regulated after 1 μM TSA treatment, while 166 genes were up-regulated and 397 genes were down-regulated after 2.5 μM TSA treatment. Gene ontology (GO) and pathway analyses indicated that the differentially expressed genes were related to many kinds of molecular functions and biological processes. The genes encoding key enzymes catalyzing gibberellin biosynthesis were significantly down-regulated in the roots exposed to 2.5 μM TSA and their expression changes were validated by using real-time PCR.ConclusionsHDACs were required for de novo organogenesis and normal growth of populus roots. DGE data provides the gene profiles in roots probably regulated by histone acetylation during root growth and development, which will lead to a better understanding of the mechanism controlling root development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2407-x) contains supplementary material, which is available to authorized users.

Activation of the TOR Signalling Pathway by Glutamine Regulates Insect Fecundity.

The target of rapamycin (TOR) positively controls cell growth in response to nutrients such as amino acids. However, research on the specific nutrients sensed by TOR is limited. Glutamine (Gln), a particularly important amino acid involved in metabolism in organisms, is synthesised and catalysed exclusively by glutamine synthetase (GS), and our previous studies have shown that Gln may regulate fecundity in vivo levels of the brown planthopper (BPH) Nilaparvata lugens. Until now, it has remained unclear whether Gln activates or inhibits the TOR signalling pathway. Here, we performed the combined analyses of iTRAQ (isobaric tags for relative and absolute quantification) and DGE (tag-based digital gene expression) data in N.lugens at the protein and transcript levels after GS RNAi, and we found that 52 pathways overlap, including the TOR pathway. We further experimentally demonstrate that Gln activates the TOR pathway by promoting the serine/threonine protein kinase AKT and inhibiting the 5′AMP-activated protein kinase AMPK phosphorylation activity in the pest. Furthermore, TOR regulates the fecundity of N. lugens probably by mediating vitellogenin (Vg) expression. This work is the first report that Gln activates the TOR pathway in vivo.

Transcriptomic Analysis of Drought Stress Responses in Ammopiptanthus mongolicus Leaves Using the RNA-Seq Technique.

Ammopiptanthus mongolicus (Maxim. Ex Kom.) Cheng f., a relic tree of the Tertiary period, plays a critical role in maintaining desert ecosystems in the Mid-Asia region. Genome-scale gene expression profiling studies will provide deep insight into the molecular mechanism underlying the drought tolerance of A. mongolicus. In the present study, we investigated the transcriptional changes induced by drought treatment in A. mongolicus leaves by establishing a comprehensive transcriptome database and then performing a Digital Gene Expression (DGE) analysis using Solexa sequencing technology. A comprehensive transcriptome database was obtained by assembling the Illumina unigenes with expressed sequence tags (EST) available publicly, and other high throughput sequencing data. To analyze the dynamic and complicated gene regulation network during PEG6000-induced drought treatment in leaves of A. mongolicus, a time-course gene expression analysis was performed using tag-based DGE technology, which identified 437, 1,247 and 802 differentially expressed transcripts in 1, 24 and 72 h drought stress libraries, respectively. GO and KEGG analyses revealed hormone signal transduction and phenylpropanoid biosynthesis were enriched during drought treatment. A batch of drought-regulated transcription factor transcripts were identified, including the subsets of HD-ZIP, bZIP, WRKY, AP2/ERF and bHLH family members, which may play roles in drought response in A. mongolicus. The sequence collection assembled in the present study represents one of the most comprehensive transcriptome databases for A. mongolicus currently. The differentially expressed transcripts identified in our study provide a good start for identifying the key genes in stress response and performing functional analysis to reveal their roles in stress adaptation in planta.

Transcriptome analysis of the Capra hircus ovary.

Background Capra hircus is an important economic livestock animal, and therefore, it is necessary to discover transcriptome information about their reproductive performance. In this study, we performed de novo transcriptome sequencing to produce the first transcriptome dataset for the goat ovary using high-throughput sequencing technologies. The result will contribute to research on goat reproductive performance.Method and ResultsRNA-seq analysis generated more than 38.8 million clean paired end (PE) reads, which were assembled into 80,069 unigenes (mean size = 619 bp). Based on sequence similarity searches, 64,824 (60.6%) genes were identified, among which 29,444 and 11,271 unigenes were assigned to Gene Ontology (GO) categories and Clusters of Orthologous Groups (COG), respectively. Searches in the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG) showed that 27,766 (63.4%) unigenes were mapped to 258 KEGG pathways. Furthermore, we investigated the transcriptome differences of goat ovaries at two different ages using a tag-based digital gene expression system. We obtained a sequencing depth of over 5.6 million and 5.8 million tags for the two ages and identified a large number of genes associated with reproductive hormones, ovulatory cycle and follicle. Moreover, many antisense transcripts and novel transcripts were found; clusters with similar differential expression patterns, enriched GO terms and metabolic pathways were revealed for the first time with regard to the differentially expressed genes.ConclusionsThe transcriptome provides invaluable new data for a functional genomic resource and future biological research in Capra hircus, and it is essential for the in-depth study of candidate genes in breeding programs.

Digital gene expression profiling (DGE) of cadmium-treated Drosophila melanogaster

Cadmium is highly toxic and can cause oxidative damage, metabolic disorders, and reduced lifespan and fertility in animals. In this study, we investigated the effects of cadmium in Drosophila melanogaster, performing transcriptome analysis by using tag-based digital gene expression (DGE) profiling. Among 1970 candidate genes, 1443 were up-regulated and 527 were down-regulated following cadmium exposure. Using Gene Ontology analysis, we found that cadmium stress affects three processes: transferase activity, stress response, and the cell cycle. Furthermore, we identified five differentially expressed genes (confirmed by real-time PCR) involved in all three processes: Ald, Cdc2, skpA, tefu, and Pvr. Pathway analysis revealed that these genes were involved in the cell cycle pathway and fat digestion and absorption pathway. This study reveals the gene expression response to cadmium stress in Drosophila, it provides insights into the mechanisms of this response, and it could contribute to our understanding of cadmium toxicity in humans.

Transcriptome characterization and differential expression analysis of cold-responsive genes in young spikes of common wheat

With the frequent occurrence of climatic anomalies, spring frost has become a significant limiting factor on wheat production, especially during the reproductive growth stage. A high-throughput sequencing technology was applied and a total of 54 million clean reads that corresponded to 7.44Gb of total nucleotides were generated. These reads were then de novo assembled into 120,715 unigenes with an average length of 627bp. Functional annotations were then obtained by aligning all unigenes with public protein databases. In total, 9657 potential EST-SSRs were identified, and 6310 primer pairs for 1329 SSRs were obtained. Meanwhile, a comparison of four tag-based digital gene expression libraries, which was built from the control and cold-treated young spikes were performed. Overall, 526 up-regulated and 489 down-regulated genes were identified, and GO and KEGG pathway analyses of those genes were further conducted. Based on these results, a series of candidate genes involved in cold response pathways were identified, and 12 of them were confirmed by qRT-PCR. The combination of RNA-Seq and digital gene expression analysis in this study provides a powerful approach for investigating the transcriptional changes and obtained a large number of unigenes annotated to public databases.

Gene Expression Profiling in Winged and Wingless Cotton Aphids, Aphis gossypii (Hemiptera: Aphididae)

While trade-offs between flight capability and reproduction is a common phenomenon in wing dimorphic insects, the molecular basis is largely unknown. In this study, we examined the transcriptomic differences between winged and wingless morphs of cotton aphids, Aphis gossypii, using a tag-based digital gene expression (DGE) approach. Ultra high-throughput Illumina sequencing generated 5.30 and 5.39 million raw tags, respectively, from winged and wingless A. gossypii DGE libraries. We identified 1,663 differentially expressed transcripts, among which 58 were highly expressed in the winged A. gossypii, whereas 1,605 expressed significantly higher in the wingless morphs. Bioinformatics tools, including Gene Ontology, Cluster of Orthologous Groups, euKaryotic Orthologous Groups and Kyoto Encyclopedia of Genes and Genomes pathways, were used to functionally annotate these transcripts. In addition, 20 differentially expressed transcripts detected by DGE were validated by the quantitative real-time PCR. Comparative transcriptomic analysis of sedentary (wingless) and migratory (winged) A. gossyii not only advances our understanding of the trade-offs in wing dimorphic insects, but also provides a candidate molecular target for the genetic control of this agricultural insect pest.

Transcriptome profiling of fruit development and maturation in Chinese white pear (Pyrus bretschneideri Rehd)

BackgroundPear (Pyrus spp) is an important fruit species worldwide; however, its genetics and genomic information is limited. Combining the Solexa/Illumina RNA-seq high-throughput sequencing approach (RNA-seq) with Digital Gene Expression (DGE) analysis would be a powerful tool for transcriptomic study. This paper reports the transcriptome profiling analysis of Chinese white pear (P. bretschneideri) using RNA-seq and DGE to better understand the molecular mechanisms in fruit development and maturation of Chinese white pear.ResultsDe novo transcriptome assembly and gene expression analysis of Chinese white pear were performed in an unprecedented depth (5.47 gigabase pairs) using high-throughput Illumina RNA-seq combined with a tag-based Digital Gene Expression (DGE) system. Approximately, 60.77 million reads were sequenced, trimmed, and assembled into 90,227 unigenes. These unigenes comprised 17,619 contigs and 72,608 singletons with an average length of 508 bp and had an N50 of 635 bp. Sequence similarity analyses against six public databases (Uniprot, NR, and COGs at NCBI, Pfam, InterPro, and KEGG) found that 61,636 unigenes can be annotated with gene descriptions, conserved protein domains, or gene ontology terms. By BLASTing all 61,636 unigenes in KEGG, a total of 31,215 unigenes were annotated into 121 known metabolic or signaling pathways in which a few primary, intermediate, and secondary metabolic pathways are directly related to pear fruit quality. DGE libraries were constructed for each of the five fruit developmental stages. Variations in gene expression among all developmental stages of pear fruit were significantly different in a large amount of unigenes.ConclusionExtensive transcriptome and DGE profiling data at five fruit developmental stages of Chinese white pear have been obtained from a deep sequencing, which provides comprehensive gene expression information at the transcriptional level. This could facilitate understanding of the molecular mechanisms in fruit development and maturation. Such a database can also be used as a public information platform for research on molecular biology and functional genomics in pear and other related species.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-14-823) contains supplementary material, which is available to authorized users.

Mining Genes Involved in Insecticide Resistance of Liposcelis bostrychophila Badonnel by Transcriptome and Expression Profile Analysis

BackgroundRecent studies indicate that infestations of psocids pose a new risk for global food security. Among the psocids species, Liposcelis bostrychophila Badonnel has gained recognition in importance because of its parthenogenic reproduction, rapid adaptation, and increased worldwide distribution. To date, the molecular data available for L. bostrychophila is largely limited to genes identified through homology. Also, no transcriptome data relevant to psocids infection is available.Methodology and Principal FindingsIn this study, we generated de novo assembly of L. bostrychophila transcriptome performed through the short read sequencing technology (Illumina). In a single run, we obtained more than 51 million sequencing reads that were assembled into 60,012 unigenes (mean size = 711 bp) by Trinity. The transcriptome sequences from different developmental stages of L. bostrychophila including egg, nymph and adult were annotated with non-redundant (Nr) protein database, gene ontology (GO), cluster of orthologous groups of proteins (COG), and KEGG orthology (KO). The analysis revealed three major enzyme families involved in insecticide metabolism as differentially expressed in the L. bostrychophila transcriptome. A total of 49 P450-, 31 GST- and 21 CES-specific genes representing the three enzyme families were identified. Besides, 16 transcripts were identified to contain target site sequences of resistance genes. Furthermore, we profiled gene expression patterns upon insecticide (malathion and deltamethrin) exposure using the tag-based digital gene expression (DGE) method.ConclusionThe L. bostrychophila transcriptome and DGE data provide gene expression data that would further our understanding of molecular mechanisms in psocids. In particular, the findings of this investigation will facilitate identification of genes involved in insecticide resistance and designing of new compounds for control of psocids.

Transcriptome Analysis of Chlorantraniliprole Resistance Development in the Diamondback Moth Plutella xylostella

BackgroundThe diamondback moth Plutella xyllostella has developed a high level of resistance to the latest insecticide chlorantraniliprole. A better understanding of P. xylostella’s resistance mechanism to chlorantraniliprole is needed to develop effective approaches for insecticide resistance management.Principal FindingsTo provide a comprehensive insight into the resistance mechanisms of P. xylostella to chlorantraniliprole, transcriptome assembly and tag-based digital gene expression (DGE) system were performed using Illumina HiSeq™ 2000. The transcriptome analysis of the susceptible strain (SS) provided 45,231 unigenes (with the size ranging from 200 bp to 13,799 bp), which would be efficient for analyzing the differences in different chlorantraniliprole-resistant P. xylostella stains. DGE analysis indicated that a total of 1215 genes (189 up-regulated and 1026 down-regulated) were gradient differentially expressed among the susceptible strain (SS) and different chlorantraniliprole-resistant P. xylostella strains, including low-level resistance (GXA), moderate resistance (LZA) and high resistance strains (HZA). A detailed analysis of gradient differentially expressed genes elucidated the existence of a phase-dependent divergence of biological investment at the molecular level. The genes related to insecticide resistance, such as P450, GST, the ryanodine receptor, and connectin, had different expression profiles in the different chlorantraniliprole-resistant DGE libraries, suggesting that the genes related to insecticide resistance are involved in P. xylostella resistance development against chlorantraniliprole. To confirm the results from the DGE, the expressional profiles of 4 genes related to insecticide resistance were further validated by qRT-PCR analysis.ConclusionsThe obtained transcriptome information provides large gene resources available for further studying the resistance development of P. xylostella to pesticides. The DGE data provide comprehensive insights into the gene expression profiles of the different chlorantraniliprole-resistant stains. These genes are specifically related to insecticide resistance, with different expressional profiles facilitating the study of the role of each gene in chlorantraniliprole resistance development.

De novo characterization of Larix gmelinii (Rupr.) Rupr. transcriptome and analysis of its gene expression induced by jasmonates

BackgroundLarix gmelinii is a dominant tree species in China’s boreal forests and plays an important role in the coniferous ecosystem. It is also one of the most economically important tree species in the Chinese timber industry due to excellent water resistance and anti-corrosion of its wood products. Unfortunately, in Northeast China, L. gmelinii often suffers from serious attacks by diseases and insects. The application of exogenous volatile semiochemicals may induce and enhance its resistance against insect or disease attacks; however, little is known regarding the genes and molecular mechanisms related to induced resistance.ResultsWe performed de novo sequencing and assembly of the L. gmelinii transcriptome using a short read sequencing technology (Illumina). Chemical defenses of L. gmelinii seedlings were induced with jasmonic acid (JA) or methyl jasmonate (MeJA) for 6 hours. Transcriptomes were compared between seedlings induced by JA, MeJA and untreated controls using a tag-based digital gene expression profiling system. In a single run, 25,977,782 short reads were produced and 51,157 unigenes were obtained with a mean length of 517 nt. We sequenced 3 digital gene expression libraries and generated between 3.5 and 5.9 million raw tags, and obtained 52,040 reliable reference genes after removing redundancy. The expression of disease/insect-resistance genes (e.g., phenylalanine ammonialyase, coumarate 3-hydroxylase, lipoxygenase, allene oxide synthase and allene oxide cyclase) was up-regulated. The expression profiles of some abundant genes under different elicitor treatment were studied by using real-time qRT-PCR.The results showed that the expression levels of disease/insect-resistance genes in the seedling samples induced by JA and MeJA were higher than those in the control group. The seedlings induced with MeJA elicited the strongest increases in disease/insect-resistance genes.ConclusionsBoth JA and MeJA induced seedlings of L. gmelinii showed significantly increased expression of disease/insect-resistance genes. MeJA seemed to have a stronger induction effect than JA on expression of disease/insect-resistance related genes. This study provides sequence resources for L. gmelinii research and will help us to better understand the functions of disease/insect-resistance genes and the molecular mechanisms of secondary metabolisms in L. gmelinii.

Global transcriptome analysis of orange wheat blossom midge, Sitodiplosis mosellana (Gehin) (Diptera: Cecidomyiidae) to identify candidate transcripts regulating diapause.

BackgroundMany insects enter a developmental arrest (diapause) that allows them to survive harsh seasonal conditions. Despite the well-established ecological significance of diapause, the molecular basis of this crucial adaptation remains largely unresolved. Sitodiplosis mosellana (Gehin), the orange wheat blossom midge (OWBM), causes serious damage to wheat throughout the northern hemisphere, and sporadic outbreaks occur in the world. Traits related to diapause appear to be important factors contributing to their rapid spread and outbreak. To better understand the diapause mechanisms of OWBM, we sequenced the transcriptome and determined the gene expression profile of this species.Methodology/Principal FindingsIn this study, we performed de novo transcriptome analysis using short-read sequencing technology (Illumina) and gene expression analysis with a tag-based digital gene expression (DGE) system. The sequencing results generated 89,117 contigs, and 45,713 unigenes. These unigenes were annotated by Blastx alignment against the NCBI non-redundant (nr), Clusters of orthologous groups (COG), gene orthology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. 20,802 unigenes (45.5% of the total) matched with protein in the NCBI nr database. Two digital gene expression (DGE) libraries were constructed to determine differences in gene expression profiles during diapause and non-diapause developmental stages. Genes related to diapause were analyzed in detail and in addition, nine diapause-related genes were analyzed by real time PCR.Conclusions/SignificanceThe OWBM transcriptome greatly improves our genetic understanding and provides a platform for functional genomics research of this species. The DGE profiling data provides comprehensive information at the transcriptional level that facilitates our understanding of the molecular mechanisms of various physiological aspects including development and diapause stages in OWBM. From this study it is evident that various genes coding metabolic enzymes are crucial for diapause and metamorphosis.

Analysis of gene expression and enzyme activities related to starch metabolism in Lycoris sprengeri bulbs of different sizes

Differentially expressed genes related to starch metabolism in mature and juvenile bulbs of Lycoris sprengeri were screened via tag-based digital gene expression profiling. The relative expression levels of these genes were further verified by quantitative real-time polymerase chain reaction. Related enzyme activities and carbohydrate concentrations were determined to study the molecular mechanism of starch accumulation during bulb development. Four starch metabolic genes (LsBAM1, LsAMY1, LsAMY3 and LsSS1) were differentially expressed between the mature and juvenile bulbs. LsSS1 is utilised during starch synthesis and the three other genes are utilised for starch degradation. The expressions of the four genes were significantly higher in juvenile bulbs than in mature bulbs. Along with bulb development (seven sizes, from bulblets to mature bulbs), β-amylase (BAM) activity significantly increased whereas α-amylase (AMY) activity slightly fluctuated. The expression levels of LsBAM1, LsAMY1 and LsAMY3 were either negatively correlated or not correlated with BAM and AMY activities. Soluble starch synthase (SSS) activity increased with decreased LsSS1 expression whereas granule-bound starch synthase (GBSS) activity decreased, indicating a positive correlation with the decreased expression level of LsSS1. During bulb development, the carbohydrate concentrations increased. In the largest mature bulbs, the highest concentration of total soluble sugar coincided with the highest β-amylase activity. However, no evidence was found to confirm the relationship between this phenomenon and related gene expression. The results revealed preliminary characteristics related to starch metabolism during the bulb development of L. sprengeri.