Digital gene expression profiling analysis of A549 cells cultured with PM10 in moxa smoke

Background:Moxibustion is a traditional Chinese medicine therapy to cure diseases by fumigating meridians or affected parts via burning of moxa floss.Moxa smoke(MS)is one of the key factors in moxibustion.In this study,we adopted digital gene expression profiling,a next-generation gene sequencing technology,to investigate the effect of MS,inhalable particulate matter(PM10),on human lung adenocarcinoma A549 cells.Methods:The effects of MS PM10 on A549 cells,over different treatment durations were investigated in different groups:the 4-h group(4-h MS group and 4-h control group)and the 20-h group(20-h MS group and 20-h control group).Samples collected from the four groups were stored at
80C for subsequent digital gene expression analysis.The differentially expressed genes(DEGs),identified after PM10 treatment,were screened,and their expression patterns analyzed by cluster analysis,Gene Ontology term enrichment,and Kyoto Encyclopedia of Genes and Genomes pathway analysis.Results:Compared with two control groups,1109 DEGs were identified after 4 h of MS intervention and 3565 DEGs were found after 20 h of MS intervention,respectively.Compared with that after 4-h intervention,2149 DEGs were identified after 20-h intervention.Cluster analysis demonstrated that PM10 can significantly inhibit cell cycle process with the prolongation of intervention time.Significant pathway enrichment analysis showed that MS PM10 can inhibit A549 cell cycle process at all phases.When MS PM10 exposure time prolongs,the inhibitory effect on cell cycle process becomes more obvious.Conclusion:MS PM10 has many biological activities,and may cause differential expression of genes involved in various biological processes.Nevertheless,further research on MS is warranted for better understanding of the mechanistic details.

Insight into the possible mechanism of the summer diapause of Delia antiqua （Diptera： Anthomyiidae） through digital gene expression analysis

The onion fly, Delia antiqua, is a major underground agricultural pest that can enter pupal diapause in the summer and winter seasons. However, little is known about its molecular regulation due to the lack of genomic resources. To gain insight into the possible mechanism of summer diapause （SD）, high-throughput RNA-Seq data were generated from non-diapause （ND） and SD （initial, maintenance and quiescence phase） pupae. Three pair-wise comparisons were performed and identified, 1380, 1471 and 435, and were significantly regulated transcripts. Further analysis revealed that the enrichment of several functional terms related to juvenile hormone regulation, cell cycle, carbon hydrate and lipid metabolism, innate immune and stress responses, various signalling transductions, ubiquitin-dependent proteosome, and variation in cuticular and cytoskeleton components were found between ND and SD and between different phases of SD. Global characterization oftranscriptome profiling between SD and ND contributes to the in-depth elucidation of the molecular mechanism of SD. Our results also offer insights into the evolution of insect diapause and support the importance of using the onion fly as a model to compare the molecular regulation events of summer and winter diapauses.

Constructing protein-protein interaction network of hypertension with blood stasis syndrome via digital gene expression sequencing and database mining

OBJECTIVE： To construct a protein-protein interaction（PPI） network in hypertension patients with blood-stasis syndrome（BSS） by using digital gene expression（DGE） sequencing and database mining techniques.METHODS： DGE analysis based on the Solexa Genome Analyzer platform was performed on vascular endothelial cells incubated with serum of hypertension patients with BSS. The differentially expressed genes were f iltered by comparing the expression levels between the different experimental groups. Then functional categories and e nriched pathways of the unique genes for BSS were analyzed using Database for Annotation, Visualization and Integrated Discovery（DAVID） to select those in the enrichment pathways. I nterologous Interaction Database（I2D） was used to construct PPI networks with the selected genes for hypertension patients with BSS. The potential candidate genes related to BSS were identif ied by comparing the number of relationships among genes. Confi rmed by quantitative reverse transcription-polymerase chain reaction（q RTPCR）, gene ontology（GO） analysis was used to infer the functional annotations of the potential candidate genes for BSS.RESULTS： With gene enrichment analysis using DAVID, a list of 58 genes was chosen from the unique genes. The selected 58 genes were analyzed using I2 D, and a PPI network was constructed. Based on the network analysis results, candidate genes for BSS were identifi ed：DDIT3, JUN, HSPA8, NFIL3, HSPA5, HIST2H2 BE, H3F3 B, CEBPB, SAT1 and GADD45 A. Verif ied through qRT-PCR and analyzed by GO, the functional annotations of the potential candidate genes were explored.CONCLUSION： Compared with previous methodologies reported in the literature, the present DGE analysis and data mining method have shown a great improvement in analyzing BSS.

Comparative transcriptome analyses reveal candidate genes regulating wood quality in Japanese larch(Larix kaempferi)

We studied the molecular mechanism of the quality traits of wood formation in larch.We used the immature latewood cells of two Japanese larch(Larix kaempferi)clones with significant differences in density and in microfibrillar angle(MFA)as materials to analyze their gene expression profiles.A total of 1735 differentially expressed genes were detected in immature latewood cells of the two clones,among which,971 were up-regulated and 764 were down-regulated.Digital gene expression profiling analysis revealed that genes encoding transcription factor members NAC66 and R2R3-MYB4,microtubule-associated protein,actin-related protein,cell wall protein members,arabinogalactan protein,Fasciclin-like arabinogalactan protein and glycine-rich protein,and several cell-wall-synthesis genes affected wood density and MFA by regulating latewood formation at transcriptional level.Our study results represent a basis for selection of quality traits and genetic improvement of larch wood.

Genome-Wide Expression Profile of Maize Root Response to Phosphorus Deficiency Revealed by Deep Sequencing

Phosphorus （P） is one of the three primary macronutrients that are required in large amounts for plant growth and development. To better understand molecular mechanism of maize and identify relevant genes in response to phosphorus deficiency, we used Solexa/Illumina＇s digital gene expression （DGE） technology to investigate six genome-wide expression profiles of seedling roots of the low-P tolerant maize inbred line 178. DGE studies were conducted at 6, 24 and 72 h under both phosphorus deficient and sufficient conditions. Approximately 3.93 million raw reads for each sample were sequenced and 6 816 genes exhibited significant levels of differential expressions in at least one of three time points in response to P starvation. The number of genes with increased expression increased over time from 6 to 24 h, whereas genes with decreased expression were more abundant at 72 h, suggesting a gradual response process for P deficiency at different stages. Gene annotations illustrated that most of differentially expressed genes （DEGs） are involved in different cellular and molecular processes such as environmental adaptation and carbohydrate metabolism. The expression of some known genes identified in other plants, such as those involved in root architecture, P metabolism and transport were found to be altered at least two folds, indicating that the mechanisms of molecular and morphological adaptation to P starvation are conserved in plants. This study provides insight into the general molecular mechanisms underlying plant adaptation to low-P stress and thus may facilitate molecular breeding for improving P utilization in maize.

Genome-Wide Identification of ALDH Gene Family under Salt and Drought Stress in Phaseolus vulgaris

Background:Aldehyde dehydrogenase(ALDH)genes constitute an important family of supergenes that play key roles in synthesizing various biomolecules and maintaining cellular homeostasis by catalyzing the oxidation of aldehyde products.With climate change increasing the exposure of plants to abiotic stresses such as salt and drought,ALDH genes have been identified as important contributors to stress tolerance.In particular,they help to reduce stress-induced lipid peroxidation.Objectives:This study aims to identify and characterize members of the ALDH supergene family in Phaseolus vulgaris through a genome-wide bioinformatic analysis and investigate their role in response to abiotic stressors such as drought and salt stress.Methods:Genome-wide identification of 26 ALDH genes in P.vulgaris was performed using bioinformatics tools.The identified ALDH proteins were ana-lyzed for molecular weight,amino acid number,and exon number.Phylogenetic analysis was performed to clas-sify P.vulgaris,Arabidopsis thaliana,and Glycine max ALDH proteins into different groups.Strong links between these genes and functions related to growth,development,stress responses,and hormone signaling were identified by cis-element analysis in promoter regions.In silico expression,analysis was performed to assess gene expression levels in different plant tissues.Results:RT-qPCR results showed that the expression of ALDH genes was signif-icantly altered under drought and salt stress in beans.This study provides a comprehensive characterization of the ALDH supergene family in P.vulgaris,highlighting their potential role in abiotic stress tolerance.Conclusion:Thesefindings provide a basis for future research on the functional roles of ALDH genes in enhancing plant resis-tance to environmental stressors.

Comparison of transcriptomes undergoing waterlogging at the seedling stage between tolerant and sensitive varieties of Brassica napus L.

RNA sequencing of the sensitive GH01 variety of Brassica napus L. seedling roots under 12 h of waterlogging was compared with previously published data of the ZS9 tolerant variety to unravel genetic mechanisms of waterlogging tolerance beyond natural variation. A total of 2 977 genes with similar expression patterns and 17 genes with opposite expression patterns were identiifed in the transcription proifles of ZS9 and GH01. An additional 1 438 genes in ZS9 and 1 861 genes in GH01 showed strain speciifc regulation. Analysis of the overlapped genes between ZS9 and GH01 revealed that waterlogging tolerance is determined by ability to regulate genes with similar expression patterns. Moreover, differences in both gene expression proifles and abscisic acid (ABA) contents between the two varieties suggest that ABA may play some role in waterlogging tolerance. This study identiifes a subset of candidate genes for further functional analysis.

Why mosaic? Gene expression profiling of African cassava mosaic virus-infected cassava reveals the effect of chlorophyll degradation on symptom development

Cassava mosaic disease, caused by cassava bego- moviruses, is the most serious disease for cassava in Africa. However, the pathogenesis of this disease is poorly under- stood. We employed high throughput digital gene expression profiling based on the Illumina Solexa sequencing technology to investigate the global transcriptional response of cassava to African cassava mosaic virus infection. We found that 3,21o genes were differentially expressed in virus-infected cassava leaves. Gene ontology term and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that genes implicated in photosynthesis were most affected, consistent with the chlorotic symptoms observed in infected leaves. The upregu- lation of chlorophyll degradation genes, including the genes encoding chlorophyUase, pheophytinase, and pheophorbide a oxygenase, and downregulation of genes encoding the major apoproteins in light-harvesting complex II were confirmed by qRT-PCR. These findings, together with the reduction of chlorophyll b content and fewer grana stacks in the infected leaf cells, reveal that the degradation of chlorophyll plays an important role in A^rican cassava mosaic virus symptom development. This study will provide a road map for future investigations into viral pathogenesis.

Transcriptomic response to ammonia-N stress in the hepatopancreas of swimming crab Portunus trituberculatus

The swimming crab Portunus trituberculatus is an important farmed species in China.Ammonia-N represents a major physiological challenge for crab culture and the hepatopancreas plays a major role in physiological adaptation processes.To better understand the molecular mechanism of the crab in response to ammonia-N stress,we performed a transcriptome analysis in the hepatopancreas of P.trituberculatus challenged with ammonia-N stress(5 mg/L),using the high-throughput sequencing technology.In total,52,280 unigenes were obtained from the hepatopancreas of P.trituberculatus,with an average length of 678 bp.Functional categorization and pathways showed some diiferentially expressed genes were involved in amino acid and nucleobase metabolism,energy metabolism,antioxidation,immune response,reproduction,moulting and material transport.The differential expression patterns of eight randomly selected annotated genes were validated by quantitative real-time PCR(qPCR).Results revealed a substantial number of genes modified by ammonia-N stress and a few significant ammonia acclimation pathways,which will serve as an invaluable resource for revealing the molecular basis of physiological adaptation mechanism in P.trituberculatus.