基因本体论在福氏志贺菌转录组研究中的应用

利用RNA-seq技术进行环丙沙星敏感和耐药的福氏志贺菌2457T株的序列测定与组装,对其碱基序列借助基因本体(Gene Ontology,GO)在生物进程、细胞组分、分子功能三方面进行注释和生物学分析。研究经过耐药诱导的转录组差异表达情况,并探讨差异表达基因在耐药性产生过程中的作用及相互关系。结果表明,通过耐药诱导有3 641个基因差异表达,其中961个GO条目注释到39个功能类别。同时发现硫酸盐、硝酸盐氧化还原相关酶系,电子/H+传导基因,膜转运蛋白,ABC外排泵家族4类耐药相关的功能基因组分。

Analysis of the action mechanism of Fang Ji Huang Qi decoction in treating rheumatoid arthritis by network pharmacology

Objective:To explore the pharmacological action mechanism of Fang Ji Huang Qi decoction(FHD)in the treatment of rheumatoid arthritis(RA)by network pharmacology.Methods:The chemical compositions and functional targets of the TCM were retrieved using the systematic pharmacological analysis platform TCMSP,and the gene name of each target protein was obtained from the UniProtKB network platform.The targets of RA were queried through the CTD database.The protein–protein interaction network was constructed in the STRING database,and the network visualization analysis was performed in Cytoscape.The Gene Ontology and Kyoto Gene and Genomic Encyclopedia pathways enrichment analyses of key target proteins were performed using the DAVID data platform.Results:A total of 472 drug active ingredients were screened from the TCMSP database.Seventy-five disease targets from the CTD database were screened.The compound-target network map contained further screened out 98 components and corresponding 75 targets.The key compounds included quercetin and kaempferol.The key targets were prostaglandin G/H synthase 2 and nitric oxide synthase 2.The protein-protein interaction network consisted of 75 proteins,of which 37 were key proteins,including tumor protein 53,JUN and interleukin-6.There were 260 Gene Ontology entries,of which 246 were biological processes.Fifty-five Kyoto Gene and Genomic Encyclopedia pathways were enriched,mainly the cancer pathway,NOD-like receptor signaling pathway,and Toll-like receptor signaling pathway,which are involved in the action mechanism of FHD.Conclusion:The results of this study preliminarily verified the basic pharmacological action mechanism of FHD in the treatment of RA,laying a foundation for elucidating its mechanism of action.

Construction of a Normalized Full-Length cDNA Library of Cephalopod Amphioctopus fangsiao and Development of Microsatellite Markers

Amphioctopus fangsiao is one of the most economically important species and has been considered to be a candidate for aquaculture. In order to facilitate its fine-scale genetic analyses, we constructed a normalized full-length library successfully and developed a set of microsatellite markers in this study. The normalized full-length library had a storage capacity of 6.9×105 independent clones. The recombination efficiency was 95% and the average size of inserted fragments was longer than 1000 bp. A total of 3440 high quality ESTs were obtained, which were assembled into 1803 unigenes. Of these unigenes, 450(25%) were assigned into 33 Gene Ontology terms, 576(31.9%) into 153 Kyoto Encyclopedia of Genes and Genomes pathways, and 275(15.3%) into 22 Clusters of Orthologous Groups. Seventy-six polymorphic microsatellite markers were identified. The number of alleles per locus ranged from 4 to 17, and the observed and expected heterozygosities varied between 0.167 and 0.967 and between 0.326 and 0.944, respectively. Twelve loci were significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction and no linkage disequilibrium was found between different loci. This study provided not only a useful resource for the isolation of the functional genes, but also a set of informative microsatellites for the assessment of population structure and conservation genetics of A. fangsiao.

Integrating Gene Ontology and Blast to predict gene functions

A GoBlast system was built to predict gene function by integrating Blast search and Gene Ontology (GO) annotations together. The operation system was based on Debian Linux 3.1, with Apache as the web server and Mysql database as the data storage system. FASTA files with GO annotations were taken as the sequence source for blast alignment, which were formatted by wu-formatdb program. The GoBlast system includes three Bioperl modules in Perl:a data input module, a data process module and a data output module. A GoBlast query starts with an amino acid or nucleotide sequence. It ends with an output in an html page, presenting high scoring gene products which are of a high homology to the queried sequence and listing associated GO terms beside respective gene poducts. A simple click on a GO term leads to the detailed explanation of the specific gene function. This avails gene function prediction by Blast. GoBlast can be a very useful tool for functional genome research and is available for free at http://bioq.org/goblast.

Characterization of Chiton Ischnochiton hakodadensis Foot Based on Transcriptome Sequencing

Chiton(Ischnochiton hakodadensis) is one of marine mollusks well known for its eight separate shell plates. I. hakodadensis is important, which plays a vital role in the ecosystems it inhabits. So far, the genetic studies on the chiton are scarce due in part to insufficient genomic resources available for this species. In this study, we investigated the transcriptome of the chiton foot using Illumina sequencing technology. The reads were assembled and clustered into 256461 unigenes, of which 42247 were divided into diverse functional categories by Gene Ontology(GO) annotation terms, and 17256 mapped onto 365 pathways by KEGG pathway mapping. Meanwhile, a set of differentially expressed genes(DEGs) between distal and proximal muscles were identified as the foot adhesive locomotion associated, thus were useful for our future studies. Moreover, up to 679384 high-quality single nucleotide polymorphisms(SNPs) and 19814 simple sequence repeats(SSRs) were identified in this study, which are valuable for subsequent studies on genetic diversity and variation. The transcriptomic resource obtained in this study should aid to future genetic and genomic studies of chiton.

Prioritization of orphan disease-causing genes using topological feature and GO similarity between proteins in interaction networks

Identification of disease-causing genes among a large number of candidates is a fundamental challenge in human disease studies.However,it is still time-consuming and laborious to determine the real disease-causing genes by biological experiments.With the advances of the high-throughput techniques,a large number of protein-protein interactions have been produced.Therefore,to address this issue,several methods based on protein interaction network have been proposed.In this paper,we propose a shortest path-based algorithm,named SPranker,to prioritize disease-causing genes in protein interaction networks.Considering the fact that diseases with similar phenotypes are generally caused by functionally related genes,we further propose an improved algorithm SPGOranker by integrating the semantic similarity of gene ontology(GO)annotations.SPGOranker not only considers the topological similarity between protein pairs in a protein interaction network but also takes their functional similarity into account.The proposed algorithms SPranker and SPGOranker were applied to 1598 known orphan disease-causing genes from 172 orphan diseases and compared with three state-of-the-art approaches,ICN,VS and RWR.The experimental results show that SPranker and SPGOranker outperform ICN,VS,and RWR for the prioritization of orphan disease-causing genes.Importantly,for the case study of severe combined immunodeficiency,SPranker and SPGOranker predict several novel causal genes.

Regulatory effects of antitumor agent matrine on FOXO and PI3K-AKT pathway in castration-resistant prostate cancer cells

We previously demonstrated that matrine could inhibit the proliferating, migrating, as well as invading processes of both PC-3 and DU145 cells. However, the underlying molecular mechanisms have not yet been clearly defined. In this study, using various techniques such as high throughput sequencing technology, bioinformatics, quantitative real-time PCR, and immunoblot analysis,we aimed to understand whether matrine serves as a novel regulator of FOXO and PI3K-AKT signaling pathway. DU145 and PC-3 cell lines were cultured for 24 h in vitro. Cells were treated with either matrine or control serum for 48 h, followed by extraction of total RNA. The RNA was sequenced using HiSeq 2500 high-throughput sequencing platform (Illumina). A gene library was established and quality analysis of read data carried out. Integrated database from the website DAVID was used to analyze Gene Ontology (GO), and Kyoto encyclopedia of genes and genomes (KEGG) pathway of differential genes was used for pathway analysis, screening for fold differences of more than two times. The FOXO and PI3K-AKT signaling pathways were screened, and expression levels of mRNA and core protein detected by real-time PCR and immunoblotting, respectively. High throughput sequencing and GO analysis revealed that differentially expressed genes before and after treatment played an important role in cell metabolic process, growth process, anatomical structure formation, cellular component organization, and biological regulation. KEGG signal pathway analysis revealed that FOXO and PI3K-AKT signal pathways had a significant difference between before and after matrine-treated androgen-independent prostate cancer cells PC-3 and DU145. Real-time PCR showed that matrine treatment led to a significant increase in the expression levels of FOXO1A, FOXO3A, FOXO4, and FOXO6 in DU145 and PC-3 cells (P<0.01 or P<0.05), whereas the PI3K expression levels decreased (P<0.01). Similarly, immunoblotting revealed a significant increase (P<0.05) in the expression levels of FOXO1A FOXO3A, FOXO4, and FOXO6 in both PC-3 and DU145 cells, whereas PI3K expression levels decreased (P<0.05). Matrine had a broad regulating effect on the mRNA expression profiles of both PC-3 and DU145 cells. Matrine may inhibit cell proliferation, migration, as well as invasion, and induce apoptosis in both PC-3 and DU145 cells through FOXO and PI3K-AKT signaling pathways. Matrine could therefore be used as a complementary drug to present chemotherapeutic agents, for treating androgen-independent prostate cancer.

Insights into the Temporal Gene Expression Pattern in Lymantria dispar Larvae During the Baculovirus Induced Hyperactive Stage

Baculoviruses are effective biological control agents for many insect pests. They not only efficiently challenge the host immune system but also make them hyperactive for better virus dispersal. Some investigations have focused on the viral mechanisms for induction of such altered response from the host. However, there are no current studies monitoring changes in gene expression during this altered phenotype in infected larvae. The L. dispar multiple nucleopolyhedrovirus(Ld MNPV) induces hyperactivity in third instar L. dispar larvae at 3-days post infection(dpi), to continued till 6 dpi. The transcriptome profiles of the infected and uninfected larvae at these time points were analyzed to provide new clues on the response of the larvae towards infection during hyperactivity. Gene ontology enrichment analysis revealed, most of the differentially expressed genes(DEGs) were involved in proteolysis, extracellular region, and serine-type endopeptidase activity. Similarly, Kyoto Encyclopedia of Genes and Genome enrichment analysis showed maximum enrichment of 487 genes of the signal transduction category and neuroactive ligand–receptor interaction sub-category with 85 annotated genes. In addition, enrichment map visualization of gene set enrichment analysis showed the coordinated response of neuroactive ligand–receptor interaction genes with other functional gene sets, as an important signal transduction mechanism during the hyperactive stage. Interestingly all the DEGs in neuroactive ligand–receptor interactions were serine proteases, their differential expression during the hyperactive stage correlated with their conceivable involvement in disease progression and the resulting altered phenotype during this period. The outcome provides a basic understanding of L. dispar larval responses to Ld MNPV infection during the hyperactive stage and helps to determine the important host factors involved in this process.

Molecular-level effects of acupuncture on depression: a genome-wide transcriptome analysis of pituitary gland in rats exposed to chronic restraint stress

OBJECTIVE: To investigate the antidepressant mechanism of acupuncture at the molecular level in the pituitary gland of rats exposed to chronic restraint stress(CRS).METHODS: Forty-eight rats were randomly divided into a control group(C), model group(M), fluoxetine group(F), and acupuncture group(A). CRS was induced in the model group by restraining rats for 28 days. Groups F and A were given fluoxetine administration and acupuncture intervention, respectively, 1 h before the CRS procedures were implemented. On the 29 th day, rat pituitary glands were harvested for RNA extraction and RNA-sequencing analysis.RESULTS: Differentially expressed gene analysis identified 148, 87, and 179 response genes differen-tially expressed in group C vs group M, group F vs group M, and group A vs group M, respectively. Using Gene Ontology term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, we identified the up-regulation of gene sets involved in extracellular space,receptor binding, and monoamine neurotransmitter metabolism, and the down-regulation of gene sets involved in immune response and inflammatory response as the prevailing transcriptomic signatures in the pituitary gland of rats treated with CRS,fluoxetine, or acupuncture.CONCLUSION: Our findings suggest that acupuncture has a multitarget antidepressant effect at the molecular and behavioral levels.