C型产气荚膜梭菌性腹泻仔猪脾脏转录组分析

doi:10.7668/hbnxb.20194102

摘要/Abstract

摘要：

旨在获得C型产气荚膜梭菌性腹泻仔猪血清炎症因子含量、脾脏病理变化及转录组表达谱特征,为其防治提供参考。选择健康状况良好、体质量相近的7日龄二元仔猪6头,随机分为2组,即对照组(CS)和处理组(TS)。TS组每头仔猪每天灌服1 mL菌液(1×10⁹ cfu/mL),连续5 d,CS组仔猪灌服不含菌液的培养液,感染后0,1,3,5 d采集血液分离血清,用ELISA方法检测IL-8、IFN-α和IFN-γ含量。试验结束后屠宰仔猪采集脾脏,进行HE染色和RNA-Seq测序,对测序获得的差异表达基因进行GO功能和KEGG信号通路富集分析,并采用RT-qPCR检测仔猪感染C型产气荚膜梭菌过程中关键差异表达基因的表达量。结果表明:TS组仔猪脾脏出现明显炎症,中性粒细胞浸润,伴有变性及坏死的实质细胞;第3,5天CS组血清中IL-8、IFN-α和IFN-γ含量极显著低于(P<0.01)TS组;TS组与CS组间有1 079个差异表达基因,上调583个,下调496个;差异表达基因富集的GO功能主要有细胞对β干扰素的反应、细胞对DNA损伤刺激的反应、炎症反应的负调控等,富集的KEGG信号通路主要有肺结核、乙型肝炎、B细胞受体等;抵抗C型产气荚膜梭菌感染的候选基因(如JAK3、TLR3、ILF2、CXXC1等)RNA-Seq结果与RT-qPCR结果一致。以上结果表明,JAK3、TLR3等基因通过关键信号通路可能在仔猪脾脏抵抗C型产气荚膜梭菌感染过程中发挥了重要功能。

关键词: 仔猪, 腹泻, 脾脏, C型产气荚膜梭菌, 转录组

Abstract:

The aim of this study was to obtain the serum inflammatory factor content,spleen pathological changes and transcriptome expression profile characteristics of diarrheic piglets with Clostridium perfringens type C,and to provide a reference for the diarrhea prevention and treatment.Six 7-day-old binary piglets with good health and similar body weight were randomly selected and divided into two groups,namely control group (CS) and treatment group (TS).Each piglet in the TS group was fed with 1 mL bacterial solution (1×10⁹ cfu/mL) every day for 5 consecutive days.Then,piglets in the CS group were fed with culture medium without the bacterial solution.Blood samples were collected and serum was separated on 0,1,3 and 5 days after infection.The serum IL-8,IFN-α and IFN-γ concentration were detected by ELISA.After the experiment,piglets were slaughtered to collect the spleen.The partial spleen was collected for HE staining to observe the pathological changes.Additionally,differentially expressed (DE) genes in the spleen were obtained using RNA-Seq.GO terms and KEGG signaling pathways enrichment analysis were performed.The key spleen genes during the infection of piglets with C.perfringens type C were verified by RT-qPCR.The spleen of piglets in the TS group displayed obvious inflammation,neutrophil infiltration,accompanied by degeneration and necrosis of parenchymal cells;on the 3rd and 5th day,the levels of IL-8,IFN-α,and IFN-γ in the CS group serum were extremely significantly lower than those in the TS group (P<0.01);a total of 1 079 DG genes was screened between TS group and CS group,including 583 up-regulated genes and 496 down-regulated genes.DG genes were mainly enriched in GO terms such as cell response to interferon-β,cell response to DNA damage stimulation,and negative regulation of inflammatory response.Differentially expressed gene were mainly enriched in KEGG signaling pathways such as tuberculosis,hepatitis B,and B cell receptors.These GO terms and KEGG signaling pathways were involved in piglet resistance to C.perfringens type C infection;the RNA-Seq results of the selected candidate genes (such as JAK3,TLR3,ILF2,CXXC1) resistant to C.perfringens type C infection were consistent with the RT-qPCR results.The above results indicated that JAK3,TLR3 and other genes played important roles in the process of piglet spleen resistance to C.perfringens type C infection through key signaling pathways.

Key words: Piglet, Diarrhea, Spleen, Clostridium perfringens type C, Transcriptome

闫尊强, 王鹏飞, 杨巧丽, 黄晓宇, 高小莉, 滚双宝. C型产气荚膜梭菌性腹泻仔猪脾脏转录组分析[J]. 华北农学报, 2023, 38(5): 188-196. doi: 10.7668/hbnxb.20194102.

YAN Zunqiang, WANG Pengfei, YANG Qiaoli, HUANG Xiaoyu, GAO Xiaoli, GUN Shuangbao. Transcriptomic Analysis of the Spleen in Diarrheic Piglet Caused by Clostridium perfringens Type C[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(5): 188-196. doi: 10.7668/hbnxb.20194102.

http://www.hbnxb.net/CN/Y2023/V38/I5/188

图/表 7

表1 引物信息

Tab.1 Primer information

基因 Gene	获取号 Accession number	序列(5'-3') Sequence(5'-3')	产物大小/bp Product size
JAK3	XM_005654907.3	F:TTCACAAGATCCCTGCCGAC	150
		R:TGTGCTTGGCATCCATCACT
TLR3	NM_001097444.1	F:CAACTGATGCTCCGAAGGGT	204
		R:AGGGTTTGCGTGTTTCCAGA
ILF2	XM_005663409.3	F:GCCGTTTGTGCCACATATCC	207
		R:CCTGGAGCCACGATCAAGTT
ILF3	XM_021083793.1	F:GAACGTAAAACAGCAGGGGC	151
		R:ATCCACCTCGACCTCCATGA
CXXC1	XM_005654427.3	F:ACAAATCCTCTCCACAGCCC	250
		R:GTCACTGGCGAGAGCCG
CSF1	XM_021088653.1	F:GGCCTGTGTCCGAACTTTCT	174
		R:TTGCAATCAGGCTTGGTCAC
AKT2	NM_001256779.1	F:CTGCTGAAGAGTGACGGCTC	196
		R:GGGAGTCCACATGGAAGGTC
CDK8	XM_021065497.1	F:AAAGTGAAGCTGAGCAGCGA	175
		R:ATGCCGACATAGAGATCCCG
β-actin	XM_003124280.5	F:AGGCGGACTGTTAGTTGCAT	187
		R:TGTCACCTTCACCGTTCCAG

图1 仔猪脾脏病理结果红箭头.中性粒细胞浸润;黑色箭头.实质细胞变性及坏死;黄色箭头.脾小梁增宽、增多;A-C.TS组;D-F.CS组。

Fig.1 Histological result of piglet spleen Red arrows.Infiltration with neutrophilic granulocytes;Black arrows.Parenchymal cells with degeneration and necrosis; Yellow arrows.Spleen trabecular widened and increased;A-C.TS group;D-F.CS group.

图2 血清细胞因子IL-8、IFN-α和IFN-γ含量不同大写字母表示差异极显著(P<0.01);不同小写字母表示差异显著(P<0.05)。图6同。

Fig.2 The serum content of the inflammatory cytokines IL-8,IFN-α and IFN-γ Different uppercase letter superscripts mean extremely significant difference (P< 0.01);different lowercase letter superscripts mean significant difference (P<0.05).The same as Fig.6.

图3 差异表达基因筛选及聚类分析 A.转录本表达水平;B.差异表达基因数量;C.差异表达基因热图(行表示基因,列表示样本)。

Fig.3 Screening and cluster analysis of DE gene A.Transcripts expression level;B.The number of DE gene;C.Heat map of DE gene(Rows indicate genes,columns represent samples).

图4 差异表达基因GO功能富集分析结果

Fig.4 GO term enrichment analyses of differentially expressed gene

图5 差异表达基因KEGG信号通路富集分析

Fig.5 KEGG signaling pathway enrichment analysis of differentially expressed gene

图6 测序结果的RT-qPCR验证

Fig.6 Validation of sequencing result by RT-qPCR

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