关键词: 捻转血矛线虫, 转录组测序, 长链非编码RNA, 耐药性, 阿苯达唑

Abstract:

To understand the expression profile of long non-coding RNAs (lncRNAs) associated with albendazole resistance in Haemonchus contortus, screen differentially expressed lncRNAs and analyze lncRNAs that may be involved in drug resistance. The total RNA of sensitive and drug-resistant strains was extracted by the TRIzol method, and the cDNA library was constructed. Double-terminal sequencing was carried out on the Illumina HiSeq^TM4000 platform. The filtered data of fastp software were compared to the reference genome of H. contortus (GenBank accession number GCA_000469685.2) by HISAT2 software. The differentially expressed lncRNA was screened. The cis-regulation mode of significant differential lncRNA target genes was predicted and the differential genes were classified by gene ontology (GO) and enriched by Kyoto encyclopedia of gene and genomes (KEGG). Real-time fluorescence quantitative PCR (qRT-PCR) was used to verify the differentially expressed lncRNA. The results of sequence alignment analysis showed that the reads of the exon region, the intron region and the intergenic region of sensitive strains were 41 344 932 (80.2%), 5 641 886 (10.9%) and 4 550 203 (8.8%), respectively. The reads of exon region, intron region and intergenic region of drug-resistant strains were 36 894 046 (79.4%), 5 644 097 (12.1%) and 3 886 628 (8.3%), respectively. There were 640 differentially expressed lncRNAs detected between the sensitive and the drug-resistant strains. The differential expressions of 246 lncRNA were 32 up-regulated and 214 down-regulated, respectively. The prediction results of the cis-regulation location showed that 76 significant differential lncRNA were screened from 547 differential mRNA, and 81 significant differential mRNA with cis-regulation function were predicted, forming 91 groups of targeted cis-regulatory relationships. The result of GO function analysis showed that these genes were mainly involved in single tissue process, cell process, metabolism, cell composition, binding and catalytic activity. KEGG analysis showed that the dominant enriched signal pathways were fluid shear stress and atherosclerosis, pentose and glucuronate interconversions, drug metabolism-cytochrome P450, neomycin, kanamycin and gentamicin biosynthesis, glycolysis/gluconeogenesis, C-type lectin receptor signal pathway. The results of qRT-PCR showed the relative expression levels of three down-regulated expressions of lncRNA MSTRG.3773.1, MSTRG.7803.1 and MSTRG.6845.2 were 0.74 ± 0.1, 0.33 ± 0.05 and 0.24 ± 0.07, respectively. The relative expression levels of the two up-regulated expressions of MSTRG.3773.2 and MSTRG.7878.1 were 1.33 ± 0.27 and 3.00 ± 0.55, respectively, which was consistent with the sequencing results. There were significant differences in lncRNA expression profiles between albendazole-sensitive and drug-resistant strains of H. contortus. lncRNA such as MSTRG.3151.1, MSTRG.8532.1, MSTRG.7561.2 and MSTRG.4538.1 may be involved in the production of drug resistance.

Key words: Haemonchus contortus, RNA sequencing, lncRNA, Drug resistance, Albendazole