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

Abstract

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

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

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

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