感染大肠杆菌F17湖羊羔羊脾脏中差异circRNA分析

doi:10.3864/j.issn.0578-1752.2019.06.012

中国农业科学 ›› 2019, Vol. 52 ›› Issue (6): 1090-1101.doi: 10.3864/j.issn.0578-1752.2019.06.012

• 畜牧·兽医·资源昆虫 • 上一篇下一篇

感染大肠杆菌F17湖羊羔羊脾脏中差异circRNA分析

邹双霞¹,金澄艳¹,鲍建军²,王悦¹,陈炜昊¹,吴天弋¹,王利宏¹,吕晓阳¹,高雯¹,王步忠³,朱国强⁴,戴国俊¹,师东方⁵,孙伟^1,⁶()

1 扬州大学动物科学与技术学院,江苏扬州225009
2 南京新九州农牧科技有限公司,南京210000
3 江苏西来原生态农业有限公司,江苏泰州225300
4 扬州大学兽医学院,江苏扬州225009
5 东北农业大学动物医学院,哈尔滨150030
6 扬州大学教育部农业与农产品安全国际合作联合实验室, 江苏扬州225009

收稿日期:2018-09-18 接受日期:2018-12-12 出版日期:2019-03-16 发布日期:2019-03-22
通讯作者: 孙伟
作者简介:邹双霞,E-mail： 1074432801@qq.com;Tel：18705271578。
基金资助:
国家自然科学基金(31872333);科技部家养动物平台项目;江苏省重点研发计划现代农业(BE2018354);江苏省农业重大新品种创制项目(PZCZ201739);江苏省农业科技自主创新项目(CX182003);江苏高校优势学科建设工程资助项目;江苏省高校自然科学研究重大项目(17KJA230001);江苏省六大高峰人才项目和扬州大学研究生创新工程项目(XKYCX17_060);江苏省六大高峰人才项目和扬州大学研究生创新工程项目(SJCX18_0804)

Differential circRNA Analysis in the Spleen of Hu-sheep Lambs Infected with F17 Escherichia coli

ZOU ShuangXia¹,JIN ChengYan¹,BAO JianJun²,WANG Yue¹,CHEN WeiHao¹,WU TianYi¹,WANG LiHong¹,LÜ XiaoYang¹,GAO Wen¹,WANG BuZhong³,ZHU GuoQiang⁴,DAI GuoJun¹,SHI DongFang⁵,SUN Wei^1,⁶()

1 College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, Jiangsu
2 Nanjing New Kyushu Agriculture and Animal Husbandry Technology Co., Ltd., Nanjing 210000
3 Jiangsu Xiyuan Ecological Agriculture Co., Ltd., Taizhou 225300, Jiangsu
4 College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, Jiangsu
5 College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030
6 Joint International Research Laboratory of Agriculture and Agri-product Safety of Ministry of Education of China, Yangzhou University, Yangzhou 225009, Jiangsu

Received:2018-09-18 Accepted:2018-12-12 Online:2019-03-16 Published:2019-03-22
Contact: Wei SUN

摘要/Abstract

摘要：

【背景】羊大肠杆菌病是一种以剧烈腹泻和败血症为特征的急性传染病,是规模化羊场最为常见高发的细菌性疾病之一,尤其是初生羔羊易被产肠毒素大肠杆菌（ETEC）感染,引起羔羊腹泻,又叫羔羊白痢,使养殖场遭受严重的经济损失。而传统的抗生素治疗方案存在诸多缺陷。【目的】本研究通过让湖羊羔羊口服大肠杆菌 F17菌株获得不腹泻和腹泻的羔羊个体,筛选出服用大肠杆菌F17菌毛后不腹泻与腹泻型个体中差异表达的circRNA,进而探究circRNA在绵羊抗腹泻中的作用,从而发现与抗大肠杆菌病性状相关的候选基因。从circRNA层面上,加深对绵羊拮抗大肠杆菌F17菌株的认识,确定绵羊拮抗大肠杆菌F17菌株的功能基因。【方法】用CIRI软件从头预测circRNA,利用RNA-seq技术,首次筛选出感染大肠杆菌F17菌株后不腹泻与腹泻型羔羊个体脾脏中差异表达的(DE)circRNA,对差异表达转录本进行GO富集分析,结合GO注释结果对其功能进行描述。统计每个GO条目中所包括的差异转录本个数,并用Fisher's exact test计算每个GO条目中差异转录本富集的显著性。然后随机选择6个DE circRNA,利用q-PCR分别验证这6个DE circRNA在不腹泻和腹泻组羔羊脾脏内的相对表达水平,进而利用Miranda软件来预测与miRNA结合的circRNA以及miRNA的靶基因,根据miRNA靶基因的功能注释来阐明此部分circRNA的功能,分析circRNA-miRNA-mRNA相互作用,最后用q-PCR验证circRNA在不腹泻组和腹泻组羔羊体内的相对表达水平。【结果】绘制参考序列后,鉴定出已知的7 730个circRNA,DE circRNA与GO 数据库进行比对,发现一共有60条circRNA被注释和分类到297个功能亚类中。利用RNA-seq在不腹泻和腹泻羔羊脾脏中筛选出60个差异表达的(DE) circRNA,其中31个上调和29个下调,用q-PCR验证随机选择的6个DE circRNA在不腹泻组和腹泻组羔羊体内的相对表达水平,发现与RNA-seq结果一致。利用Miranda分析circRNA-miRNA-mRNA相互作用,发现6个circRNA、5个miRNA和8个mRNA之间存在一定的靶标关系,用q-PCR验证mRNA在不腹泻组和腹泻组羔羊体内的相对表达水平,发现与RNA-seq结果一致。【结论】探究了对于不腹泻和腹泻羔羊脾脏中circRNA的表达谱,进一步了解其在绵羊抗病发生过程中的调控作用。发现了不腹泻和腹泻羔羊脾脏中差异表达的circRNA,有助于找出羔羊如何抵抗腹泻的发生机制,为羔羊抵抗腹泻提供科学的依据。

关键词: 大肠杆菌F17, circRNA, 湖羊, circRNA-miRNA-mRNA互作

Abstract:

【Background】Sheep colibacillosis is an acute infectious disease by E. coli that is characterized by severe diarrhea and sepsis, which is one of the most common bacterial diseases in large-scale sheep farms. Especially the newborn lamb is infected by enterotoxin-producing Escherichia coli (ETEC), causing lamb diarrhea, also known as lamb white diarrhea. Sheep colibacillosis let the farm to suffer serious economic losses, but the traditional antibiotic treatment program has many defects. 【Objective】In this study, we obtained oral diarrhea and diarrhea lambs by oral administration of E. coli F17 strain, and screened out circRNAs that were differentially expressed in individuals with diarrhea and diarrhea after taking E. coli F17 pili, and then explored circRNA for sheep in anti-diarrhea to find candidate genes associated with resistance to E. coli disease traits. From the circRNA level, the understanding of sheep antagonizing E. coli F17 fimbriae was deepened, and the functional genes of sheep antagonizing E. coli F17 fimbriae were determined.【Method】We used CIRI software to predict circRNA from scratch, and firstly screened differentially expressed (DE) circRNA in spleen of Escherichia coli F17 fimbriae without diarrhea and diarrhea by using RNA-seq technology. Then we performed GO on differentially expressed transcripts, and its function was described in combination with GO annotation results. The number of differential transcripts included in each GO entry was counted and the significance of differential transcript enrichment in each GO entry was calculated by using Fisher's exact test. Then, 6 DE circRNAs were randomly selected, and the relative expression levels of the 6 DE circRNAs in the spleen of lambs without diarrhea and diarrhea were verified by q-PCR, and the miRNAs and miRNA target genes were predicted by Miranda software. The function of this part of circRNA was elucidated based on the functional annotation of the miRNA target gene, and the circRNA-miRNA-mRNA interaction was analyzed. Finally, the relative expression level of mRNA in the non-diarrhea group and the diarrhea group lamb was verified by q-PCR.【Result】After mapping the reference sequence, we identified 7730 circRNAs, and DE circRNA was compared with the GO database. A total of 60 circRNAs were annotated and classified into 297 functional subclasses. Using RNA-seq to screen 60 DE circRNAs in spleens of diarrhea-free and diarrhea lambs, 31 of which were up-regulated, and 29 of which were down-regulated. Relative expression levels of randomly selected 6 DE circRNAs in non-diarrhea and diarrhea lambs by q-PCR and found to be consistent with RNA-seq results. Using Miranda to analyze circRNA-miRNA-mRNA interactions, we found a certain target relationship between 6 circRNAs, 5 miRNAs and 8 mRNAs. q-PCR was used to verify the relative expression of mRNA in lambs without diarrhea and diarrhea, and the results were found to be consistent with RNA-seq results.【Conclusion】 This study explored the expression profile of circRNA in the spleen of lambs without diarrhea and diarrhea, and further understood its regulation in the process of disease resistance in sheep. The discovery of circRNA, which was differentially expressed in the spleen of lambs without diarrhea and diarrhea, could help to find out how the lambs resist the mechanism of diarrhea, and to provide a scientific basis for improving the lamb's ability to resist diarrhea.

Key words: Escherichia coli F17, circRNA, Lake sheep, circRNA-miRNA-mRNA interaction

邹双霞,金澄艳,鲍建军,王悦,陈炜昊,吴天弋,王利宏,吕晓阳,高雯,王步忠,朱国强,戴国俊,师东方,孙伟. 感染大肠杆菌F17湖羊羔羊脾脏中差异circRNA分析[J]. 中国农业科学, 2019, 52(6): 1090-1101.

ZOU ShuangXia,JIN ChengYan,BAO JianJun,WANG Yue,CHEN WeiHao,WU TianYi,WANG LiHong,LÜ XiaoYang,GAO Wen,WANG BuZhong,ZHU GuoQiang,DAI GuoJun,SHI DongFang,SUN Wei. Differential circRNA Analysis in the Spleen of Hu-sheep Lambs Infected with F17 Escherichia coli[J]. Scientia Agricultura Sinica, 2019, 52(6): 1090-1101.

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	基因符号Gene symbol	引物序列Primer sequence	产物长度Length of product (bp)
差异表达的环状RNA的引物 Primers of DE circRNA	circRNA_2125	F:ATTGAATCACTTCTCTGTTGC	129
	circRNA_2125	R:TAGGTGCTCAAAATAGGAC	129
	circRNA_3832	F:AGCCTCTCATCTGTACAC	134
	circRNA_3832	R:CAGTAACTGCCTAGAGCA	134
	circRNA_7711	F:ACAAAGATTCCATTGACAG	101
	circRNA_7711	R:ACCAAGAGGCTAGCAAGAC	101
	circRNA_6710	F:CAGATTACAGCTATGGCGA	124
	circRNA_6710	R:CCCTCATGATCTCATAGG	124
	circRNA_6914	F:TTGGCTGTTACTATCATGAG	124
	circRNA_6914	R:CTGAACTCTTAACTTGCA	124
	circRNA_4030	F:TGATGCAGATATTAAACCTC	133
	circRNA_4030	R:CCAATCTCGGATAACTTCAC	133
差异表达的信使RNA的引物 Primers of DE mRNA	NEB	F:ATTACAGCTATCCACCCGAC	149
	NEB	R:TGCCTTTTCCATTTCTAAG	149
	UBE3B	F:TAAGATTGCCAGGAAACTGC	133
	UBE3B	R:AGCCAGGGACACGTACCAC	133
	ADGRF2	F:GGCGTTTACCTCTTTCTCG	103
	ADGRF2	R:CAAGCTGCAAATAGAAAC	103
	LAMA1	F:AAATGATCGAAAAGGCTAC	127
	LAMA1	R:AACCGCCTTTTCCGTAGGAC	127
	LTF	F:GAAAAGCGTATCCCAACCTG	103
	LTF	R:TTGAAGGCACCAGAATAAC	103
	MGAT5	F:CATCATCCACACCTACACG	111
	MGAT5	R:AACTGCAAGTCTCGTCCGC	111
	TLN2	F:ACGACGGTGGTTAAATAC	125
	TLN2	R:AGTTGCCCATAGTCACTGGTC	125
	ARHGAP30	F:TCTTCAACCTGGGTCGCTC	159
	ARHGAP30	R:GCAGCCCCTCTGGTTCATC	159
	SLC25A29	F:GCGTCCTGGCTCTCCACCT	125
	SLC25A29	R:CCCTGCCTCCCCGCGCTC	125
GAPDH引物 Primers of GAPDH	GAPDH-F	F:GTTCCACGGCACAGTCAAGG	127
GAPDH引物 Primers of GAPDH	GAPDH-F	R:ACTCAGCACCAGCATCACCC	127

环状RNA circRNA	最佳circRNA基因 Best gene of circRNA	P值 P vale	微小RNA miRNA	微小RNA的靶基因 Target gene of miRNA	靶标基因转录ID Transcription ID of target gene
circRNA_6577	LOC101111058 (Btnl 1)	0.000190825	oar-miR-381-5p
circRNA_7725		0.003873598	oar-miR-1193-5p	NEB	XM_012137591.2
				UBE3B	XM_004017436.3
circRNA_0309	LOC101108092 (GSTM1)	0.004205007	oar-miR-370-3p	ADGRF2	XM_004018870.3
circRNA_2125	LOC101115614 (NRAMP2)	0.004205007	oar-miR-370-3p	LAMA1	XM_012103553.2
circRNA_3832	B2M	0.004205007	oar-miR-370-3p	LTF	NM_001024862.1
circRNA_6577	LOC101111058 (Btnl 1)	0.004205007	oar-miR-370-3p	MGAT5	XM_012139230.2
circRNA_7711		0.004205007	oar-miR-370-3p	TLN2	XM_012181407.2
circRNA_6577	LOC101111058 (Btnl 1)	0.006550584	oar-miR-3956-3p	SLC25A29	XM_015102051.1
circRNA_6577	LOC101111058 (Btnl 1)	0.011765813	oar-miR-370-5p

感染大肠杆菌F17湖羊羔羊脾脏中差异circRNA分析

Differential circRNA Analysis in the Spleen of Hu-sheep Lambs Infected with F17 Escherichia coli

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