F17大肠杆菌在湖羊羔羊个体脾脏中LncRNA表达谱变化

doi:10.3864/j.issn.0578-1752.2019.07.015

中国农业科学 ›› 2019, Vol. 52 ›› Issue (7): 1282-1294.doi: 10.3864/j.issn.0578-1752.2019.07.015

• 畜牧·兽医·资源昆虫 • 上一篇

F17大肠杆菌在湖羊羔羊个体脾脏中LncRNA表达谱变化

黄赛男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,孙伟^1,⁵()

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 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:2018-12-03 出版日期:2019-04-01 发布日期:2019-04-04
通讯作者: 孙伟
作者简介:黄赛男,E-mail： 1481258911@qq.com。|金澄艳,E-mail： 1342315339@qq.com。
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）项目(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(CX（18）2003);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;扬州大学研究生创新工程项目(XKYCX17_060);扬州大学研究生创新工程项目(SJCX18_0804)

Changes of LncRNA Expression Profile in Spleen of Diarrhea and Non-diarrhea Individuals in F17 of Hu Sheep Lamb

HUANG SaiNan¹,JIN ChengYan¹,BAO JianJun²,WANG Yue¹,CHEN WeiHao¹,WU TianYi¹,WANG LiHong¹,LÜ XiaoYang¹,GAO Wen¹,WANG BuZhong³,ZHU GuoQiang⁴,DAI GuoJun¹,SUN Wei^1,⁵()

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 Source Ecological Agriculture Co., Ltd., Taizhou 225300, Jiangsu
4 College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, Jiangsu
5 Joint Laboratory of International Cooperation in Agriculture and Agricultural Products Safety of the Ministry of Education, Yangzhou University, Yangzhou 225009, Jiangsu

Received:2018-09-18 Accepted:2018-12-03 Online:2019-04-01 Published:2019-04-04
Contact: Wei SUN

摘要/Abstract

摘要：

【目的】通过筛选对大肠杆菌（E.coli）F17菌毛非腹泻型与腹泻型的绵羊脾脏中差异表达的lncRNA,来探究lncRNA对绵羊抗腹泻的作用。【方法】本研究通过对湖羊羔羊口服E.coli F17菌株获得非腹泻和腹泻型个体,利用羔羊肠道细菌计数、病理组织切片验证攻毒成功性;构建非腹泻组和腹泻组羔羊脾脏的cDNA文库,使用Illumina HiSeq 2500平台进行配对测序;通过Gene Ontology（GO）和KEGG Pathway富集分析对差异表达转录本功能描述和细胞通路分析,利用FPKM法估计lncRNA和mRNA转录物的表达水平,并用高通量测序技术RNA-seq筛选出非腹泻和腹泻个体脾脏中的差异表达lncRNA;然后利用荧光定量PCR技术检测了非腹泻组和腹泻组羔羊脾脏组织中DE lncRNA和DE mRNA的表达水平,来验证筛选的DE lncRNA在非腹泻组过程中发挥作用。【结果】羔羊口服E.coli F17菌株后,出现非腹泻和腹泻两种表型,腹泻组羔羊肠道中的细菌数量显著高于非腹泻组（P<0.05）,同时腹泻组羔羊空肠黏膜组织出现不同程度的损伤,色泽暗沉,小肠绒毛部分脱落。笔者利用RNA-seq在非腹泻和腹泻羔羊脾脏中筛选出34个差异表达的（DE）lncRNA,703个的DE mRNA,随机选择一共12个DE lncRNA和DE mRNA,用q-PCR验证它们在非腹泻型和腹泻型羔羊体内的相对表达水平,发现与RNA-seq结果一致。通过Gene Ontology（GO）和KEGG Pathway富集分析,将DE lncRNA与GO 数据库进行比对的结果表明一共有34条lncRNA被注释和分类到302个功能亚类中,绵羊蛋白质结合（GO：0005515）,细胞核（GO：0005634）,poly（A）RNA结合（GO：0044822）,细胞质（GO：0005737）,组织重塑（GO：0048771）,内肽酶活性的调节（GO：0052548））,6-磷酸果糖-2-激酶/果糖-2,6-双磷酸酶复合物（GO：0043540）,磷脂酰肌醇磷酸化（GO：0046854）,果糖-2,6-二磷酸2-磷酸酶活性（GO：0004331）,钙依赖性磷脂酶C活性（GO：0050429）等10 个功能亚类的lncRNA较多,而其余的功能亚类的lncRNA分布较少。将DE lncRNA与KEGG 通路数据库进行比对的结果表明,一共有34条lncRNA被注释和归类到149个KEGG 通路中,绵羊甲状腺激素信号通路（路径：ko04919）,Spliceosome（路径：ko03040）,白细胞跨内皮迁移（路径：ko04670）,神经营养因子信号通路（路径：ko04722）,溶酶体（路径：ko04142）,MAPK信号通路 - 途径（路径： ko04011）,鞘脂信号通路（路径：ko04071）,吞噬体（路径：ko04145）,氧化磷酸化（路径：ko00190）等9 个KEGG 通路的lncRNA较多,而其余的KEGG 通路的lncRNA分布较少。通过lncRNA-mRNA相互作用网络分析,发现6个共表达基因：MYO1G、TIMM29、CARM1、ADGRB1、SEPT4、DESI2。【结论】探究了对于腹泻产生非腹泻和腹泻型羔羊脾脏中lncRNA的表达谱,发现了非腹泻和腹泻羔羊脾脏中差异表达的lncRNA,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, lncRNA, 湖羊羔羊

Abstract:

【Objective】The objective of this study was to investigate the effect of lncRNA on anti-diarrhea in sheep by screening lncRNA differentially expressed in E. coli F17 fimbriae non-diarrhea and diarrhea sheep spleen. 【Method】 In this study, individuals with non-diarrhea and diarrhea were obtained by oral administration of E. coli F17 strain to Lake Lamb. The success of the challenge was verified by using intestinal counts and pathological sections of the lambs. A cDNA library of spleen from lambs in non-diarrhea group and diarrhea group was constructed and sequenced by using Illumina HiSeq 2500 platform. Functional description and cell pathway analysis of differentially expressed transcripts were performed by Gene Ontology (GO) and KEGG Pathway enrichment analysis by using FPKM method. The expression levels of lncRNA and mRNA transcripts were screened by high-throughput sequencing technology RNA-seq for differential expression of lncRNA in spleens of non-diarrhea and diarrhea individuals; then, Quantitative PCR was used to detect spleen tissues in non-diarrhea and diarrhea lambs. The expression levels of differentially expressed (DE) lncRNA and DE mRNA were used to verify the role of screened DE lncRNA in the non-diarrhea group. 【Result】 After oral administration of E. coli F17 strain, there were two phenotypes of non-diarrhea and diarrhea. The number of bacteria in the intestine of the diarrhea group was significantly higher than that in the non-diarrhea group (P<0.05), and the jejunal mucosa of the diarrhea group appeared different degrees of damage, dull color, part of the small intestine villi off. We used RNA-seq to screen 34 DE lncRNAs and 703 DE mRNAs in non-diarrhea and diarrhea lamb spleens. A total of 12 DE lncRNA and DE mRNA were randomly selected and verified by q-PCR. Relative expression levels in the diarrhea and non-diarrhea lambs were found to be consistent with RNA-seq results. The comparison between DE lncRNA and GO database by GO and KEGG pathway enrichment analysis indicated that a total of 34 lncRNAs were annotated and classified into 302 functional subclasses. There were more than one functional subclass of lncRNA, such as sheep protein binding (GO: 0005515), nuclear (GO: 0005634), poly (A) RNA binding (GO: 0044822), cytoplasm (GO: 0005737), tissue remodeling (GO: 0048771), regulation of endopeptidase activity (GO: 0052548) ), 6-phosphate fructose-2-kinase/fructose-2,6-bisphosphatase complex (GO: 0043540), phosphatidylinositol phosphorylation (GO: 0848654), fructose-2, 6.2-phosphite 2-phosphatase activity (GO: 0004331) and calcium-dependent phospholipase C activity (GO: 0050429), while the remaining functional subclasses had less lncRNA distribution. The alignment of DE lncRNA with the KEGG pathway database indicated that a total of 34 lncRNAs were annotated and classified into 149 KEGG pathways, the sheep thyroid hormone signaling pathway (path: ko04919), Spliceosome (path: ko03040), white blood cell cross Endothelial migration (path: ko04670), neurotrophin signaling pathway (path: ko04722), lysosome (path: ko04142), MAPK signaling pathway-pathway (path: ko04011), sphingolipid signaling pathway (path: ko04071), phagocytosis the body (path: ko04145), oxidative phosphorylation (path: ko00190) and other 9 KEGG pathways had more lncRNAs, while the remaining KEGG pathways had less lncRNA distribution. Through lncRNA-mRNA interaction network analysis, we found six co-expressed genes: MYO1G, TIMM29, CARM1, ADGRB1, SEPT4, and DESI2. 【Conclusion】 This study explored the expression profile of lncRNA in the spleen of non-diarrhea and diarrhea lambs for diarrhea. It was found that lncRNA differentially expressed in the spleen of non-diarrhea and diarrhea lambs, which helped to find out how lambs resist diarrhea and provided a scientific basis for lambs to resist diarrhea.

Key words: E. coli F17, lncRNA, Lake lamb

黄赛男,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,孙伟. F17大肠杆菌在湖羊羔羊个体脾脏中LncRNA表达谱变化[J]. 中国农业科学, 2019, 52(7): 1282-1294.

HUANG SaiNan,JIN ChengYan,BAO JianJun,WANG Yue,CHEN WeiHao,WU TianYi,WANG LiHong,LÜ XiaoYang,GAO Wen,WANG BuZhong,ZHU GuoQiang,DAI GuoJun,SUN Wei. Changes of LncRNA Expression Profile in Spleen of Diarrhea and Non-diarrhea Individuals in F17 of Hu Sheep Lamb[J]. Scientia Agricultura Sinica, 2019, 52(7): 1282-1294.

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形态 Form	描述 Describe
第1种形态 Type 1	分散的干球便,如坚果,很难排出 Separate hard lumps, like nuts
第2种形态Type 2	香肠状,多块的 Sausage-shaped but lumpy
第3种形态 Type 3	腊肠样,但表面有裂缝Like a sausage or snake but with cracks on its surface
第4种形态Type 4	腊肠状或蛇状,表面光滑 Like a sausage or snake, smooth and soft
第5种形态Type 5	柔软成块,边缘清楚,容易排出Soft blobs with clear-cut edges
第6种形态Type 6	软片状,边缘毛糙或是糊状便 Fluffy pieces with ragged edges, a mushy stool
第7种形态 Type 7	水样便,无团状成分 Watery, no solid pieces

	基因符号Gene symbol	引物序列Primer sequence	产物长度Product length (bp)
10个DE mRNA的引物Primers of ten DE mRNA	XM_015103189.1	F:AGCACTTCCTCCTGTCCG	132
	XM_015103189.1	R:CAGCACAGAAGGCAAAGTC	132
	XM_012106626.2	F:CCGAGTTTGCAGGTACCCAAC	121
	XM_012106626.2	R:TTTTGGCGCATGTATACCTG	121
	XM_012095567.2	F:TGAGACTCTACTTCGCTGC	102
	XM_012095567.2	R:TTGCCCATCCTTAATAGCTG	102
	XM_012181407.2	F:ACGACGGTGGTTAAATACTC	125
	XM_012181407.2	R:AGTTGCCCATAGTCACTGGTC	125
	XM_012181919.2	F:TCAACCATATGCTGACGGAC	119
	XM_012181919.2	R:ATGCCGCCTATCAAGGTC	119
	XM_012152506.2	F:CCACCTGCGGTTCAAGTTAC	157
	XM_012152506.2	R:TGCCTGAATCACCTTGTC	157
10个DE lncRNA的引物 Primers of ten DE lncRNA	TCONS_00053949	F:CCTGGCTATATCCTTACATCAC	100
	TCONS_00053949	R:AAGTTCAAACTCCGCTGCAC	100
	TCONS_00059692	F:AATTTCTTCTCGTTCCAAGGC	131
	TCONS_00059692	R:CCAACAGGGAGCCAACTTC	131
	TCONS_00089751	F:AGAAGGCTTTGACCGAAC	153
	TCONS_00089751	R:TCAATGCCCTCCACGAGAC	153
	TCONS_00099925	F:AGTGCCACATGTACCTAGCAG	146
	TCONS_00099925	R:ACGACAGGCATTTTAACCCATG	146
	TCONS_00107469	F:TAGTACAGCCCATATTTATCG	115
	TCONS_00107469	R:ATTTTCTTTCCACAGGGACG	115
	TCONS_00099739	F:CCGACGCTGTCATGATGC	159
	TCONS_00099739	R:TCCGTCTCCAGAACCAAGGC	159
GAPDH的引物 Primers of GAPDH	GAPDH-F	F:GTTCCACGGCACAGTCAAGG	127
GAPDH的引物 Primers of GAPDH	GAPDH-F	R:ACTCAGCACCAGCATCACCC	127

组别 Group	肠道 Intestinal tract	稀释倍数 Dilution multiple				细菌数量 Count of bacterial (CFU/mL)
组别 Group	肠道 Intestinal tract	10⁵	10⁶	10⁷	10⁸	细菌数量 Count of bacterial (CFU/mL)
非腹泻组 Non-diarrhea group	十二指肠 Duodenum	70	6	NG	NG	6.0×10⁶
	空肠 Jejunum	51	NG	NG	NG	5.1×10⁶
	回肠 Ileum	>500	170	9	NG	9.0×10⁷
腹泻组 Diarrhea group	十二指肠 Duodenum	>1000	>500	128	13	1.3×10⁹
	空肠 Jejunum	>1000	272	47	NG	4.7×10⁸
	回肠 Ileum	>1000	>500	176	19	1.9×10⁹

lncRNA	表达趋势 Regulation	mRNA	基因 Gene	表达趋势 Regulation	染色体编号 Chrom	Pearson P值 P value	Pearson 相关系数Correlation	顺距 cis distance
TCONS_00053949	Down	XM_012176963.2	MYO1G	Up	NC_019461.2	0.034	-0.845	1670
TCONS_00059692	Up	XM_015095635.1	TIMM29	Down	NC_019462.2	0.01	-0.916	16037
TCONS_00059692	Up	XM_015095835.1	CARM1	Down	NC_019462.2	0.033	-0.848	22513
TCONS_00089751	Up	XM_015097644.1	ADGRB1	Up	NC_019466.2	0.0027	0.957	8560
TCONS_00099925	Down	XM_015098457.1	SEPT4	Up	NC_019468.2	0.049	-0.812	98788
TCONS_00107469	Up	XM_012187185.1	DESI2	Up	NC_019469.2	0.0026	0.958	94714

F17大肠杆菌在湖羊羔羊个体脾脏中LncRNA表达谱变化

Changes of LncRNA Expression Profile in Spleen of Diarrhea and Non-diarrhea Individuals in F17 of Hu Sheep Lamb

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