RNA sequencing analysis of gene expression in Dermatophagoides farinae post-exposure to horseradish and the underlying mechanisms

doi:10.12140/j.issn.1000-7423.2025.01.017

Abstract

Abstract:

Objective To identify differentially expressed genes (DEGs) in Dermatophagoides farinae post-treatment with horseradish using RNA sequencing, and to unravel the underlying mechanisms. Methods Ninety fumigation bottles were randomly divided into the experimental and control groups. In each bottle, 80 D. farinaes were placed back down on the adhesive plates of the fumigation bottles. D. farinaes were exposed to 25 μl of 0.25 ml/L horseradish dilutions in the experimental group, and paraffin in the control group for 24-hour fumigation. Then 2 000 survival D. farinae were collected from each group. All experiments were repeated 6 times, with triplicates for sequencing and others for real-time fluorescent quantitative PCR (qPCR) verification. RNA was extracted from D. farinae and reversely transcribed into cDNA, and a library was constructed for RNA sequencing. Following quality control and assembly, the sequencing data were aligned with non-redundant protein sequence (NR), gene ontology (GO), and Kyoto encyclopedia of genes and genomes (KEGG) databases, and the GO and KEGG functional annotations and enrichment analysis of DEGs were performed with the edge R software. In addition, DEGs identified in D. farinaes post-treatment with horseradish were sampled for qPCR assay, the qPCR data were subjected to analysis of variance with the R software. Results RNA sequencing yielded 56 625 transcripts with a total length of 72 418 610 nt, and a total of 39 510 UniGene clusters were yielded, with the highest number of clusters aligned to the NR database (25 450 clusters), followed by the evolutionary genealogy of genes: non-supervised orthologous groups (eggNOG） database (17 643 clusters), Swiss-prot database (13 338 clusters), GO database (13 234 clusters), and KEGG database (9 445 clusters), respectively. A total of 2 719 DEGs were identified in D. farinae post-treatment with horseradish, including 1 185 up-regulated genes and 1 534 down-regulated genes. GO annotations showed that the DEGs were mainly related to cellular processes, binding and catalytic activities, and GO enrichment analysis showed that the up-regulated genes were mainly enriched in ATP-binding cassette transporter G (ABCG) subfamily, cytochrome P450 (CYP450), heat shock proteins, and acetylcholine receptor and the down-regulated genes were mainly enriched in calmodulin and RNA polymerase. KEGG annotations showed that DEGs were mainly involved in the processes of transport and catabolism, signal transduction, and translation, and KEGG enrichment analysis revealed that the DEGs were significantly enriched in 30 pathways, which were mainly enriched in ribosome, microbial metabolism across diverse environments, and biosynthesis of secondary metabolites. qPCR results show that the relative transcription levels of UDP-glucuronosyltransferase (UGT) and multidrug resistance-associated protein 1 (MRP1) in the experimental group were 1.18 ± 0.22 and 6.22 ± 0.42, respectively, which were significantly upregulated compared to the control group (0.76 ± 0.30 and 2.52 ± 1.75) (F = 11.22, 16.83, both P < 0.05). And the relative transcription level of mitochondrial ribosome-associated GTPase (MTG) was 0.05 ± 0.04, which was significantly downregulated compared to the control group (1.23 ± 0.62) (F = 13.95, P < 0.05), which was consistent with RNA sequencing results. Conclusion There is differential expression of multiple genes in D. farinae post-treatment with horseradish, and UGT, CYP450, MRP1 and ABCG genes play important roles in the development of resistance to horseradish or detoxification metabolism of horseradish.

Key words: Dermatophagoides farinae, Horseradish, Transcriptome, Differentially expressed gene

CLC Number:

R384.429

XU Xinjie, LI Jing, WU Yike, YANG Feng, SHAN Wenxin, ZHANG Zhiding, YE Changjiang, SUN Entao. RNA sequencing analysis of gene expression in Dermatophagoides farinae post-exposure to horseradish and the underlying mechanisms[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2025, 43(1): 112-118.

Figures/Tables 3

Fig. 1

Table 1

GO enrichment analysis results of differentially expressed genes of D. farinae after horseradish action

上调 UP	下调 Down
ABC转运蛋白G（ABCG3、ABCG-33）	线粒体核糖体相关GTP酶
ABC transporter G（ABCG3, ABCG-33）	Mitochondrial Ribosome-associated GTPase
细胞色素P450（CYP450-525A1）	钙调蛋白、类钙调蛋白、钙调蛋白调节的收缩蛋白相关蛋白2样异构体X2
Cytochrome P450（CYP450-525A1）	Calmodulin, calmodulin-like, calmodulin-regulated spectrin-associated protein 2-like isoform X2
UDP-葡萄糖醛酸转移酶（UGT-1、UGT1-6）	Toll样受体
UDP-glucuronidase（UGT-1, UGT1-6）	Toll-like receptor
多重耐药相关蛋白（MRP1、MRP1B、MRP7）	G蛋白偶联受体激酶1样
Multidrug resistance proteins（MRP1, MRP1B, MRP7）	G protein-coupled receptor kinase 1-like
60S核糖体蛋白（l11-a样、l8样、17样、L30、L27e、L24a、L16B、L35、L25、L5、L23、L8B、L11、L4-B、L26-1、L10、L13、L20）	ATP酶（蛋白酶体ATPase相关因子1样、V型质子ATP酶催化亚基a样、质膜钙转运ATP酶2样异构体X3）
60S ribosomal protein（l11-a-like, l8-like, 17-like, L30, L27e, L24a, L16B, L35, L25, L5, L23, L8B, L11, L4-B, L26-1, L10, L13, L20）	ATPase（proteasome ATpase-related factor 1, V-type proton-ATPase catalytic subunit a, plasma membrane calcium transport ATPase 2-type isomer X3）
热休克蛋白（I型HSP40共伴蛋白、HSP70伴侣蛋白、HSP12、HSP40、HSP70）	RNA聚合酶（Ⅱ转录亚基26b介质、Ⅱ转录亚基16样蛋白）、Poly（A）RNA聚合酶 RNA polymerase（Ⅱtranscription subunit 26b medium, Ⅱtranscriptional subunit 16-like protein）
Heat shock proteins（TypeⅠ HSP40 coprotein, HSP70 chaperone, HSP12, HSP40, HSP70）	Poly（A）RNA polymerase
神经元乙酰胆碱受体亚基α-5样	GTPase-3样蛋白、adp-核糖化因子gtase-激活蛋白2样、gtpase激活蛋白和VPS9结构域1样蛋白
Neuronal acetylcholine receptor subunit α-5	GTPase 3-like protein, adp-ribosylation factor, GTASe-activating protein 2 samples, gtpase activating protein and VPS9 domain 1-like protein
表皮蛋白10、几丁质酶	NADH-细胞色素b5还原酶（3样异构体X1）
Epidermal protein 10, chitinases	NADH-cytochrome b5 reductase（3-like isomer X1）

Table 1

Fig. 2

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