Impact of sublethal chlorantraniliprole on epidermis of Bombyx mori during prepupal-pupal transition.

The silkworm Bombyx mori, an economically important insect with a long domestication history, exhibits high sensitivity to chemical pesticides. Extensive application of chlorantraniliprole (CAP) in control of pests of agricultural crops and mulberry plants causes residue toxicity to silkworm. We have demonstrated that sublethal concentration of CAP exposure causes defects in the formation of new epidermis and incomplete shedding of old epidermis during prepupal-pupal transition of B. mori. However, the underlying mechanism still remains unclear. Here, we investigated the transcriptional responses of the epidermis of B. mori on day 2 at prepupal stage to sublethal CAP exposure using digital gene expression (DGE) profiling sequencing. We identified 5823 differentially expressed genes (DEGs), with 4830 genes up-regulated and 993 genes down-regulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that CAP exposure induced disruption of energy homeostasis, oxidative stress, autophagy and apoptosis in the epidermis of B. mori. Meanwhile, trehalose content was increased while most of the genes involved in trehalose metabolism were down-regulated. In addition, chitin contents in CAP-exposed silkworms were decreased. Taken together, these results reveal that sublethal concentration of CAP probably targets trehalose metabolism to impair chitin synthesis, leading to perturbation of pupation metamorphosis in B. mori.

Transcriptomic responses of Caco-2 cells to Lactobacillus rhamnosus GG and Lactobacillus plantarum J26 against oxidative stress.

Oxidative stress is the basic reason for aging and age-related diseases. In this study, we investigated the protective effect of 2 strains of lactic acid bacteria (LAB), Lactobacillus rhamnosus GG and L. plantarum J26, against oxidative stress in Caco-2 cells, and gave an overview of the mechanisms of lactic acid bacteria antioxidant activity using digital gene expression profiling. The 2 LAB strains provided significant protection against hydrogen peroxide (H2O2)-induced reduction in superoxide dismutase activity and increase in glutathione peroxidase activity in Caco-2 cells. However, inactive bacteria had little effect on alleviating oxidation stress in Caco-2 cells. Eight genes related to oxidative stress-FOSB, TNF, PPP1R15A, NUAK2, ATF3, TNFAIP3, EGR2, and FBN2-were significantly upregulated in H2O2-induced Caco-2 cells compared with untreated Caco-2 cells. After incubation of the H2O2-induced Caco-2 cells with L. rhamnosus GG and L. plantarum J26, 5 genes (TNF, EGR2, NUAK2, FBN2, and TNFAIP3) and 2 genes (NUAK2 and FBN2) were downregulated, respectively. In addition, the Kyoto Encyclopedia of Genes and Genomes indicated that some signaling pathways associated with inflammation, immune response, and apoptosis, such as Janus kinase/signal transducers and activators of transcription (Jak-STAT), mitogen-activated protein kinase (MAPK), nuclear factor-κB, and tumor necrosis factor, were all negatively modulated by the 2 strains, especially L. rhamnosus GG. In this paper, we reveal the mechanism of LAB in relieving oxidative stress and provide a theoretical basis for the rapid screening and evaluation of new LAB resources.

Transcriptome Analysis of Male Drosophila melanogaster Exposed to Ethylparaben Using Digital Gene Expression Profiling.

Ethylparaben (EP) has been shown to have estrogenic effects and can affect the normal development, longevity, and reproductive system of some animals. In this study, we investigated the effects of EP in male Drosophila melanogaster using transcriptome analysis or digital gene expression profiling. We then screened differentially expressed genes (DEGs) between the two groups (EP-treated and control group) of Drosophila, and performed clustering analysis, gene ontology (GO) function annotation, kyoto encyclopedia of gene and genomes metabolic pathway analysis. We found that EP enriched GO in three processes: cellular component, molecular function, and biological process. Consequently, we detected 13,959 genes and among them, 18 genes were identified to be significantly expressed between the EP-treated and control samples. Of these, seven genes were down-regulated, and eleven genes were up-regulated in EP-treated samples. Furthermore, four DEGs including two down-regulated genes (CG9465, CG9468) and two up-regulated genes (TotA, Sqz) were verified by real-time quantitative PCR. This study revealed the impact of EP on gene expression in fruit fly and provided new insight into the mechanisms of this response, which is helpful for understanding EP toxicity to humans.

Gene transcript profiles in the desert plant Nitraria tangutorum during fruit development and ripening.

Nitraria tangutorum Bobr., a valuable wild shrub distributed in Northwest China, produces edible and medicinal berries. However, little is known about the molecular mechanisms of its fruit development and ripening. We performed de novo transcriptome sequencing of N. tangutorum fruit using the Illumina HiSeq™ 2000 sequencing platform. More than 62.94 million reads were obtained and assembled into 69,306 unigenes (average length, 587 bp). These unigenes were annotated by querying against five databases (Nr, Swiss-Prot, GO, COG, and KEGG); 42,929 and 26,809 unigenes were found in the Nr and Swiss-Prot databases, respectively. In ortholog analyses, 33,363 unigenes were assigned with one or more GO terms, 15,537 hits were aligned to 25 COG classes, and 24,592 unigenes were classified into 128 KEGG pathways. Digital gene expression analyses were conducted on N. tangutorum fruit at the green (S1), yellow (S2), and red (S3) developmental stages. In total, 8240, 5985, and 4994 differentially expressed genes (DEGs) were detected for S1 vs. S2, S1 vs. S3, and S2 vs. S3, respectively. Cluster analyses showed that a large proportion of DEGs related to plant hormones and transcription factors (TFs) showed high expression in S1, down-regulated expression in S2, and up-regulated expression in S3. We analyzed the expression patterns of 23 genes encoding 12 putative enzymes involved in flavonoid biosynthesis. The expression profiles of 10 DEGs involved in flavonoid biosynthesis were validated by Q-PCR analysis. The assembled and annotated transcriptome sequences and gene expression profile analyses provide valuable genetic resources for research on N. tangutorum.

Identification of differentially expressed genes associated with differential body size in mandarin fish (Siniperca chuatsi).

Body size is an obvious and important characteristic of fish. Mandarin fish Siniperca chuatsi (Basilewsky) is one of the most valuable perciform species widely cultured in China. Individual differences in body size are common in mandarin fish and significantly influence the aquaculture production. However, little is currently known about its genetic control. In this study, digital gene expression profiling and transcriptome sequencing were performed in mandarin fish with differential body size at 30 and 180 days post-hatch (dph), respectively. Body weight, total length and body length of fish with big-size were significantly higher than those with small-size at both 30 and 180 dph (P < 0.05). 2171 and 2014 differentially expressed genes were identified between small-size and big-size fish at 30 and 180 dph, respectively. RT quantitative PCR (qPCR) analysis showed that the differential expression of 10 selected genes in mandarin fish that went through the same training procedure. The genes were involved in the growth hormone-insulin-like growth factor axis, cell proliferation and differentiation, appetite control, glucose metabolism, reproduction and sexual size dimorphism pathways. This study will help toward a comprehensive understanding of the complexity of regulation of body size in mandarin fish individuals and provide valuable information for future research.

Glyphosate effects on the gene expression of the apical bud in soybean (Glycine max).

Glyphosate is a broad spectrum, non-selective herbicide which has been widely used for weed control. Much work has focused on elucidating the high accumulation of glyphosate in shoot apical bud (shoot apex). However, to date little is known about the molecular mechanisms of the sensitivity of shoot apical bud to glyphosate. Global gene expression profiling of the soybean apical bud response to glyphosate treatment was performed in this study. The results revealed that the glyphosate inhibited tryptophan biosynthesis of the shikimic acid pathway in the soybean apical bud, which was the target site of glyphosate. Glyphosate inhibited the expression of most of the target herbicide site genes. The promoter sequence analysis of key target genes revealed that light responsive elements were important regulators in glyphosate induction. These results will facilitate further studies of cloning genes and molecular mechanisms of glyphosate on soybean shoot apical bud.

Novel insights into the immune regulatory effects of Megalobrama amblycephala intelectin on the phagocytosis and killing activity of macrophages.

Previous studies have found that the expression level of Megalobrama amblycephala intelectin (MaINTL) increased significantly post Aeromonas hydrophila infection, and recombinant MaINTL (rMaINTL) protein could activate macrophages and enhance the phagocytosis and killing activity of macrophages. In order to reveal the immune regulatory mechanisms of MaINTL, primary M. amblycephala macrophages were treated with endotoxin-removed rMaINTL and GST-tag proteins, then total RNA were extracted and used for comparative Digital Gene Expression Profiling (DGE). 1247 differentially expressed genes were identified by comparing rMaINTL and GST-tag treated macrophage groups, including 482 up-regulated unigenes and 765 down-regulated unigenes. In addition, eleven randomly selected differentially expressed genes were verified by qRT-PCR, and most of them shared the similar expression patterns as that of DGE results. GO enrichment revealed that the differentially expressed genes were mainly concentrated in the membrane part and cytoskeleton of cellular component, the binding and signal transducer activity of molecular function, the cellular process, regulation of biological process, signaling and localization of biological process, most of which might related with the phagocytosis and killing activity of macrophages. KEGG analysis revealed the activation and involvement of differentially expressed genes in immune related pathways, such as Tumor necrosis factor (TNF) signaling pathway, Interleukin 17 (IL-17) signaling pathway, Toll-like receptor signaling pathway, and NOD like receptor signaling pathway, etc. In these pathways, TNF-ɑ, Activator protein-1 (AP-1), Myeloid differentiation primary response protein MyD88 (MyD88), NF-kappa-B inhibitor alpha (ikBɑ) and other key signaling factors were significantly up-regulated. These results will be helpful to clarify the immune regulatory mechanisms of fish intelectin on macrophages, thus providing a theoretical basis for the prevention and control of fish bacterial diseases.

Loss of GINS2 inhibits cell proliferation and tumorigenesis in human gliomas.

AIMS: In this study, we examined the expression of GINS2 in glioma and determined its role in glioma development. METHODS: The protein expression of GINS2 was assessed in 120 human glioma samples via immunohistochemistry. Then, we suppressed the expression of GINS2 in glioma cell strains U87 and U251 using a short hairpin RNA lentiviral vector. In addition, RNA sequencing and bioinformatics analysis were performed on glioma cells before and after GINS2 knockdown. Subsequent co-immunoprecipitation and western blot experiments indicated possible downstream regulatory molecules. RESULTS: The present results showed that GINS2 can accelerate the growth of glioma cells, whereas the suppression of GINS2 expression decreased the proliferation and tumorigenicity of glioma cells. Mechanism research experiments proved that GINS2 can block the cell cycle by regulating certain downstream molecules, such as MCM2, ATM, and CHEK2. CONCLUSION: GINS2 is closely related to the occurrence and development of glioma, and is likely to become a prognostic marker for glioma patients, as well as a potential therapeutic target in the treatment of glioma.

Bacillus thuringiensis Suppresses the Humoral Immune System to Overcome Defense Mechanism of Plutella xylostella.

Background: Plutella xylostella has become a notorious pest of cruciferous crops all over the world. Delta-endotoxins of Bacillus thuringiensis are widely used insecticidal proteins for controlling P. xylostella. However, the interaction mechanism of B. thuringiensis with the immune system of P. xylostella, at the genomic level, is still unclear. This study explored the immune response of P. xylostella to B. thuringiensis, at different time intervals, 6 h, 12 h, 18 h, 24 h, and 36 h, by using RNA-Sequencing (RNA-Seq) and RT-qPCR. Results: In total, 167 immunity-related genes were identified and placed into different families, including pattern recognition receptors (PRRs), signal modulators, immune pathways (Toll, IMD, and JAK/STAT), and immune effectors. It is worth mentioning that the analyses of the differentially expressed immunity-related genes revealed that most of the differentially expressed genes (DEGs) (87, 56, 76, 67, and 73 genes) were downregulated in P. xylostella following B. thuringiensis oral infection at 6 h, 12 h, 18 h, 24 h, and 36 h. Interestingly, our RNA-Seq analysis also revealed reduced expression of antimicrobial peptides, that play a vital role in the humoral immune system of P. xylostella. Conclusion: This study demonstrates that B. thuringiensis plays a novel role in controlling P. xylostella, by suppressing the immune system.

Identification of Putative Genes Involved in Limonoids Biosynthesis in Citrus by Comparative Transcriptomic Analysis.

Limonoids produced by citrus are a group of highly bioactive secondary metabolites which provide health benefits for humans. Currently there is a lack of information derived from research on the genetic mechanisms controlling the biosynthesis of limonoids, which has limited the improvement of citrus for high production of limonoids. In this study, the transcriptome sequences of leaves, phloems and seeds of pummelo (Citrus grandis (L.) Osbeck) at different development stages with variances in limonoids contents were used for digital gene expression profiling analysis in order to identify the genes corresponding to the biosynthesis of limonoids. Pair-wise comparison of transcriptional profiles between different tissues identified 924 differentially expressed genes commonly shared between them. Expression pattern analysis suggested that 382 genes from three conjunctive groups of K-means clustering could be possibly related to the biosynthesis of limonoids. Correlation analysis with the samples from different genotypes, and different developing tissues of the citrus revealed that the expression of 15 candidate genes were highly correlated with the contents of limonoids. Among them, the cytochrome P450s (CYP450s) and transcriptional factor MYB demonstrated significantly high correlation coefficients, which indicated the importance of those genes on the biosynthesis of limonoids. CiOSC gene encoding the critical enzyme oxidosqualene cyclase (OSC) for biosynthesis of the precursor of triterpene scaffolds was found positively corresponding to the accumulation of limonoids during the development of seeds. Suppressing the expression of CiOSC with VIGS (Virus-induced gene silencing) demonstrated that the level of gene silencing was significantly correlated to the reduction of limonoids contents. The results indicated that the CiOSC gene plays a pivotal role in biosynthesis of limonoids.

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.

Chemosensory Gene Families in Ectropis grisescens and Candidates for Detection of Type-II Sex Pheromones.

Tea grey geometrid (Ectropis grisescens), a devastating chewing pest in tea plantations throughout China, produces Type-II pheromone components. Little is known about the genes encoding proteins involved in the perception of Type-II sex pheromone components. To investigate the olfaction genes involved in E. grisescens sex pheromones and plant volatiles perception, we sequenced female and male antennae transcriptomes of E. grisescens. After assembly and annotation, we identified 153 candidate chemoreception genes in E. grisescens, including 40 odorant-binding proteins (OBPs), 30 chemosensory proteins (CSPs), 59 odorant receptors (ORs), and 24 ionotropic receptors (IRs). The results of phylogenetic, qPCR, and mRNA abundance analyses suggested that three candidate pheromone-binding proteins (EgriOBP2, 3, and 25), two candidate general odorant-binding proteins (EgriOBP1 and 29), six pheromone receptors (EgriOR24, 25, 28, 31, 37, and 44), and EgriCSP8 may be involved in the detection of Type-II sex pheromone components. Functional investigation by heterologous expression in Xenopus oocytes revealed that EgriOR31 was robustly tuned to the E. grisescens sex pheromone component (Z,Z,Z)-3,6,9-octadecatriene and weakly to the other sex pheromone component (Z,Z)-3,9-6,7-epoxyoctadecadiene. Our results represent a systematic functional analysis of the molecular mechanism of olfaction perception in E. grisescens with an emphasis on gene encoding proteins involved in perception of Type-II sex pheromones, and provide information that will be relevant to other Lepidoptera species.

Systematic analysis of O-methyltransferase gene family and identification of potential members involved in the formation of O-methylated flavonoids in Citrus.

The O-methylation of various secondary metabolites is mainly catalyzed by S-adenosyl-l-methionine (SAM)-dependent O-methyltransferase (OMT) proteins that are encoded by the O-methyltransferase gene family. Citrus fruits are a rich source of O-methylated flavonoids that have a broad spectrum of biological activities, including anti-inflammatory, anticarcinogenic, and antiatherogenic properties. However, little is known about this gene family and its members that are involved in the O-methylation of flavonoids and their regulation in Citrus. In this study, 58 OMT genes were identified from the entire Citrus sinensis genome and compared with those from 3 other representative dicot plants. A comprehensive analysis was performed, including functional/substrate predictions, identification of chromosomal locations, phylogenetic relationships, gene structures, and conserved motifs. Distribution mapping revealed that the 58 OMT genes were unevenly distributed on the 9 citrus chromosomes. Phylogenetic analysis of 164 OMT proteins from C.sinensis, Arabidopsis thaliana, Populus trichocarpa, and Vitis vinifera showed that these proteins were categorized into group I (COMT subfamily) and group II (CCoAOMT subfamily), which were further divided into 10 and 2 subgroups, respectively. Finally, digital gene expression and quantitative real-time polymerase chain reaction analyses revealed that citrus OMT genes had distinct temporal and spatial expression patterns in different tissues and developmental stages. Interestingly, 18 and 11 of the 27 genes predicted to be involved in O-methylation of flavonoids had higher expression in the peel and pulp during fruit development, respectively. The citrus OMT gene family identified in this study might help in the selection of appropriate candidate genes and facilitate functional studies in Citrus.

Transcriptome profiling and pathway analysis of hepatotoxicity induced by tris (2-ethylhexyl) trimellitate (TOTM) in mice.

Tris (2-ethylhexyl) trimellitate (TOTM) is commonly used as an alternative plasticizer for medical devices. But very little information was available on its biological effects. In this study, we investigated toxicity effects of TOTM on hepatic differential gene expression analyzed by using high-throughput sequencing analysis for over-represented functions and phenotypically anchored to complementary histopathologic, and biochemical data in the liver of mice. Among 1668 candidate genes, 694 genes were up-regulated and 974 genes were down-regulated after TOTM exposure. Using Gene Ontology analysis, TOTM affected three processes: the cell cycle, metabolic process and oxidative activity. Furthermore, 11 key genes involved in the above processes were validated by real time PCR. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these genes were involved in the cell cycle pathway, lipid metabolism and oxidative process. It revealed the transcriptome gene expression response to TOTM exposure in mouse, and these data could contribute to provide a clearer understanding of the molecular mechanisms of TOTM-induced hepatotoxicity in human.

Transcriptional Profiling of Rice Treated with MoHrip1 Reveal the Function of Protein Elicitor in Enhancement of Disease Resistance and Plant Growth.

MoHrip1 is a protein elicitor isolated from Magnaporthe oryzae and was found to induce blast-resistance in rice. To investigate the comprehensive functions of MoHrip1, next-generation sequencing (NGS)-based digital gene expression (DGE) profiling was performed to collect the transcriptional data of differentially expressed genes (DEGs) induced by MoHrip1. A total of 308 genes were identified with differential expression, and 80 genes were predicted to be induced specifically by MoHrip1. Among these 308 genes, a series of genes associated with the salicylic acid (SA) pathway, phytoalexin, transcription factors, and pathogen-related proteins were identified. Both the SA signaling pathway and the gibberellin (GA) pathway were activated, while the jasmonic acid (JA) signaling pathway was repressed. The contents of endogenous SA and GA and the morphological characteristics of the rice after treatment were measured to provide evidence supporting the predictions made based on the DGE data. The 80 genes mentioned above might be candidate genes for studying interactions with MoHrip1. The transcriptional data provided global effect information in rice induced by MoHrip1, and all the results demonstrated that MoHrip1 could induce pathogen resistance and promote plant growth by regulating the contents of SA and GA directly or indirectly.