捻转血矛线虫阿苯达唑耐药株给药前后比较转录组学分析
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摘要
为探明捻转血矛线虫阿苯达唑耐药株给药前后在转录组水平上的差异,本研究采用Illumina Hiseq4000对给药前后的捻转血矛线虫进行转录组测序,筛选得到差异表达基因并通过GO和KEGG数据库对其进行功能注释及富集性分析,并利用荧光定量PCR验证部分差异表达基因。结果显示,共筛选获得851个显著差异表达基因,其中包括584个上调基因和267个下调基因。通过GO功能富集分析显示,有458、418和367个基因分别注释到生物学过程、分子功能和细胞组分三大类;对差异表达基因进行KEGG富集分析显示,有173个差异表达基因参与到75条KEGG通路中,显著富集在核糖体合成、细胞凋亡、过氧化物酶增殖体激活受体(PPAR)等信号通路。本试验初步筛选了阿苯达唑耐药株给药前后的差异表达基因,为深入探索捻转血矛线虫耐药性分子机制、筛选对耐药性检测的分子标记及耐药性早期鉴别诊断方法的建立提供重要数据。
Aiming to find the transcription difference of albendazole resistant strain of Haemonchus contortus,before and after administration. In this study, Samples of bBZ(before albendazole administration) and aBZ(after albendazole administration) were analyzed using Illumina Hiseq4000 RNA sequencing platform and their transcriptome libraries were constructed. Screening for differentially expressed genes(DEGs) and functional annotation and enrichment analysis by GO and KEGG databases,at the same time,they were verified by real-time PCR. The results showed that: a total of 851 DEGs between aBZ and bBZ, of which 584 and 267 genes were up-and down-regulated, respectively. The DEGs were then searched against the GO and KEGG database for enrichment analysis. GO analysis showed that 458,418 and 367 DEGs were annotated into biological process, cellular component and molecular function. KEGG analysis showed that 173 differentially expressed genes were assigned to 75 KEGG pathways, and clustered significantly in ribosome synthesis, apoptosis-multiple species, PPAR signaling pathway, and so on. This experiment initially screened DEGs between bBZ and aBZ of Haemonchus contortus. The study provides a foundation for exploring the molecular mechanism of resistance to Haemonchus contortus, screening for molecular markers of drug resistance detection and the establishment of early differential diagnosis methods for drug resistance.
Aiming to find the transcription difference of albendazole resistant strain of Haemonchus contortus,before and after administration. In this study, Samples of bBZ(before albendazole administration) and aBZ(after albendazole administration) were analyzed using Illumina Hiseq4000 RNA sequencing platform and their transcriptome libraries were constructed. Screening for differentially expressed genes(DEGs) and functional annotation and enrichment analysis by GO and KEGG databases,at the same time,they were verified by real-time PCR. The results showed that: a total of 851 DEGs between aBZ and bBZ, of which 584 and 267 genes were up-and down-regulated, respectively. The DEGs were then searched against the GO and KEGG database for enrichment analysis. GO analysis showed that 458,418 and 367 DEGs were annotated into biological process, cellular component and molecular function. KEGG analysis showed that 173 differentially expressed genes were assigned to 75 KEGG pathways, and clustered significantly in ribosome synthesis, apoptosis-multiple species, PPAR signaling pathway, and so on. This experiment initially screened DEGs between bBZ and aBZ of Haemonchus contortus. The study provides a foundation for exploring the molecular mechanism of resistance to Haemonchus contortus, screening for molecular markers of drug resistance detection and the establishment of early differential diagnosis methods for drug resistance.
引文
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[4] MICKIEWICZ M, CZOPOWICZ M, GóRSKI P, et al. The first reported case of resistance of gastrointestinal nematodes to benzimidazole anthelmintic in goats in Poland[J]. Ann Parasitol, 2017, 63(4):317-322.
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[6] ZHANG Z Z, YANG X, AWAIS A A, et al. Development of a tetra-primer ARMS-PCR for detecting the E198A SNP in the isotype-1 β-tubulin gene of Haemonchus contortus populations in China[J]. Vet Parasitol, 2018, 252:127-130.
[7] 薄新文, 李祥瑞. 多重PCR检测捻转血矛线虫苯并咪唑类药物抗性等位基因[J]. 中国农业科学, 2005, 38(4):826-830.BO X W, LI X R. Multiplex PCR detection of allele on benzimidazole-resistance or -susceptibity in natural populations of Haemonchus contortus[J]. Scientia Agricultura Sinica, 2005, 38(4):826-830. (in Chinese)
[8] LAING R, KIKUCHI T, MARTINELLI A, et al. The genome and transcriptome of Haemonchus contortus, a key model parasite for drug and vaccine discovery[J]. Genome Biol, 2013, 14(8):R88.
[9] GASSER R B, SCHWARZ E M, KORHONEN P K, et al. Understanding Haemonchus contortus better through genomics and transcriptomics[J]. Adv Parasitol, 2016, 93:519-567.
[10] MARTIN M. Cutadapt removes adapter sequences from high-throughput sequencing reads[J]. EMBnet J, 2011, 17(1):10-12.
[11] LANGMEAD B, SALZBERG S L. Fast gapped-read alignment with Bowtie 2[J]. Nat Methods, 2012, 9(4):357-359.
[12] KIM D, PERTEA G, TRAPNELL C, et al. TopHat2:accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions[J]. Genome Biol, 2013, 14(4):R36.
[13] PERTEA M, PERTEA G M, ANTONESCU C M, et al. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads[J]. Nat Biotechnol, 2015, 33(3):290-295.
[14] KANDIL O M, GAMIL I S, HENDAWY S H M, et al. Efficacy of glutathione-S-transferase purified antigen of the gastro-intestinal nematode Haemonchus contortus in diagnosis of sheep haemonchosis[J]. J Parasit Dis, 2017, 41(4):968-975.
[15] WILLIAMSON S M, WOLSTENHOLME A J. P-glycoproteins of Haemonchus contortus:development of real-time PCR assays for gene expression studies[J]. J Helminthol, 2012, 86(2):202-208.
[16] MARTIS M M, TARBIAT B, TYDéN E, et al. RNA-Seq de novo assembly and differential transcriptome analysis of the nematode Ascaridia galli in relation to in vivo exposure to flubendazole[J]. PLoS One, 2017, 12(11):e0185182.
[17] ZHOU C, YANG H, WANG Z, et al. Comparative transcriptome analysis of Sogatella furcifera (Horváth) exposed to different insecticides[J]. Sci Rep, 2018, 8:8773.
[18] KAUR P, GARG M, HOMBACH-BARRIGAH A, et al. MAPK1 of Leishmania donovani interacts and phosphorylates HSP70 and HSP90 subunits of foldosome complex[J]. Sci Rep, 2017, 7:10202.
[2] MUCHIUT S M, FERNáNDEZ A S, STEFFAN P E, et al. Anthelmintic resistance:management of parasite refugia for Haemonchus contortus through the replacement of resistant with susceptible populations[J]. Vet Parasitol, 2018, 254:43-48.
[3] BARRERE V, FALZON L C, SHAKYA K P, et al. Assessment of benzimidazole resistance in Haemonchus contortus in sheep flocks in Ontario, Canada:comparison of detection methods for drug resistance[J]. Vet Parasitol, 2013, 198(1-2):159-165.
[4] MICKIEWICZ M, CZOPOWICZ M, GóRSKI P, et al. The first reported case of resistance of gastrointestinal nematodes to benzimidazole anthelmintic in goats in Poland[J]. Ann Parasitol, 2017, 63(4):317-322.
[5] BARRèRE V, ALVAREZ L, SUAREZ G, et al. Relationship between increased albendazole systemic exposure and changes in single nucleotide polymorphisms on the β-tubulin isotype 1 encoding gene in Haemonchus contortus[J]. Vet Parasitol, 2012, 186(3-4):344-349.
[6] ZHANG Z Z, YANG X, AWAIS A A, et al. Development of a tetra-primer ARMS-PCR for detecting the E198A SNP in the isotype-1 β-tubulin gene of Haemonchus contortus populations in China[J]. Vet Parasitol, 2018, 252:127-130.
[7] 薄新文, 李祥瑞. 多重PCR检测捻转血矛线虫苯并咪唑类药物抗性等位基因[J]. 中国农业科学, 2005, 38(4):826-830.BO X W, LI X R. Multiplex PCR detection of allele on benzimidazole-resistance or -susceptibity in natural populations of Haemonchus contortus[J]. Scientia Agricultura Sinica, 2005, 38(4):826-830. (in Chinese)
[8] LAING R, KIKUCHI T, MARTINELLI A, et al. The genome and transcriptome of Haemonchus contortus, a key model parasite for drug and vaccine discovery[J]. Genome Biol, 2013, 14(8):R88.
[9] GASSER R B, SCHWARZ E M, KORHONEN P K, et al. Understanding Haemonchus contortus better through genomics and transcriptomics[J]. Adv Parasitol, 2016, 93:519-567.
[10] MARTIN M. Cutadapt removes adapter sequences from high-throughput sequencing reads[J]. EMBnet J, 2011, 17(1):10-12.
[11] LANGMEAD B, SALZBERG S L. Fast gapped-read alignment with Bowtie 2[J]. Nat Methods, 2012, 9(4):357-359.
[12] KIM D, PERTEA G, TRAPNELL C, et al. TopHat2:accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions[J]. Genome Biol, 2013, 14(4):R36.
[13] PERTEA M, PERTEA G M, ANTONESCU C M, et al. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads[J]. Nat Biotechnol, 2015, 33(3):290-295.
[14] KANDIL O M, GAMIL I S, HENDAWY S H M, et al. Efficacy of glutathione-S-transferase purified antigen of the gastro-intestinal nematode Haemonchus contortus in diagnosis of sheep haemonchosis[J]. J Parasit Dis, 2017, 41(4):968-975.
[15] WILLIAMSON S M, WOLSTENHOLME A J. P-glycoproteins of Haemonchus contortus:development of real-time PCR assays for gene expression studies[J]. J Helminthol, 2012, 86(2):202-208.
[16] MARTIS M M, TARBIAT B, TYDéN E, et al. RNA-Seq de novo assembly and differential transcriptome analysis of the nematode Ascaridia galli in relation to in vivo exposure to flubendazole[J]. PLoS One, 2017, 12(11):e0185182.
[17] ZHOU C, YANG H, WANG Z, et al. Comparative transcriptome analysis of Sogatella furcifera (Horváth) exposed to different insecticides[J]. Sci Rep, 2018, 8:8773.
[18] KAUR P, GARG M, HOMBACH-BARRIGAH A, et al. MAPK1 of Leishmania donovani interacts and phosphorylates HSP70 and HSP90 subunits of foldosome complex[J]. Sci Rep, 2017, 7:10202.