MT和FGF5调控辽宁绒山羊绒毛生长相关LncRNA的筛选及鉴定

doi:10.3864/j.issn.0578-1752.2019.04.014

Abstract

Abstract:

【Objective】 The aim of this study was to screen out the LncRNA associated with villus growth in Liaoning cashmere goat skin fibroblasts, and provide basic data for the study of the function and mechanism of LncRNA related to villus growth. 【Method】 The total RNA of MT and FGF5 treated Liaoning cashmere goat skin fibroblasts was extracted, and the total RNA extracted was detected by total RNA electrophoresis detection, sequencing data quality evaluation, mapping comparison and inter-sample correlation test. The differentially expressed LncRNA was screened and its target gene was predicted. The LncRNA related to villus growth was screened by GO and KEGG enrichment analysis, and the target LncRNA was verified by Real-time PCR. 【Result】 (1) The total RNA quality of the sample showed that the RNA was in good integrity, the GC content was relatively high, the sequence was stable, and the expression level between samples was high, which met the sequencing requirements.(2) Screening of differentially expressed LncRNA showed that there were 32 differentially expressed LncRNA in 1.0g·L ^-1 24h group, 4 of which were up-regulated and 28 of which were down-regulated. There were 10 differentially expressed LncRNA in 0.2g·L ^-1 24h group, 4 of which were up-regulated and 6 were down-regulated. There were 113 differentially expressed LncRNA in the 0.2g·L ^-1 72h group, of which 5 were up-regulated and 108 were down-regulated. There were 164 differentially expressed LncRNA in the 10 ^-4g·L ^-1 24 h group, of which 70 were up-regulated and 94 were down-regulated. There were 189 differentially expressed LncRNA in the10 ^-4g·L ^-1 72 h group, of which 78 were up-regulated and 111 were down-regulated. There were 123 LncRNA differentially expressed in the 10 ^-6g·L ^-1 24 h group, among which 27 up and 96 down.(3) Target gene GO enrichment analysis showed that the 1.0g·L ^-1 24h group differentially expressed LncRNA target gene enrichment in GO's negative regulation of transcription from RNA polymerase II promoter; 0.2g·L ^-1 24h group did not differentially express LncRNA target gene enriched GO term;0.2g·L ^-1 72h group Differentially expressed LncRNA target gene enrichment in GO's cellular metabolic process biological_process, binding molecular_function, FGF5 treatment group only10 ^-4g·L ^-1 72 h group differentially expressed LncRNA target gene enriched in cell cellular_component, cell part cellular_component, intracellular cellular_component, binding molecular_function and other six items. (4) Target gene KEGG enrichment analysis showed that the differential expression of LncRNA target gene in 1.0g·L ^-1 24h group was enriched in Steroid biosynthesis pathway; in 0.2g·L ^-1 24h group, there was no differential expression of LncRNA target gene enrichment Pathway term; 0.2g·L ^-1 72h group differentially expressed LncRNA target gene enrichment in Cell cycle, DNA replication, Steroid biosynthesis, TNF, Nod-like receptor, NF-kappa B and other signaling pathways, in which TNF and NF-kappa B signaling pathways are involved in villus growth. In FGF5-treated group, differentially expressed LncRNA targets in 10 ^-4g·L ^-1 72 h group The gene was significantly enriched into nine path termes such as Fanconi anemia pathway, Huntington's disease, Metabolic pathway, Aminoacyl-tRNA biosynthesis, among which Metabolic pathway was associated with villus growth; the differentially expressed LncRNA target gene in 10 ^-4g·L ^-1 24 h group had no significant enriched pathway term;10 ^-6g·L ^-1 24 h The differentially expressed LncRNA target genes were only enriched in the Taste transduction pathway. (5) There are two LncRNA corresponding to the target genes TNFα, TNFAIP3 (A20), NFKBIA (IkBα), NFKB2 and IL8 enriched in NF-κB and TNF signaling pathways, respectively (Gene ID): XLOC_005914; XLOC_018763; There are four LncRNA corresponding to the target genes in the Metabolic pathway, namely (Gene ID): XLOC_011424, XLOC_009522, XLOC_009063, XLOC_01115. Real-time PCR results showed that LncRNA XLOC_011424, XLOC_011157, LncRNA XLOC_005914 and XLOC_018763 were consistent with high-throughput sequencing results. 【Conclusion】 LncRNA XLOC_011424, XLOC_011157, LncRNA XLOC_005914 and XLOC_018763 may increase the density and length of cashmere by regulating NF-κB, TNF or Metabolic signaling pathways related to villus growth, and thus improve the yield and quality of cashmere in Liaoning cashmere goat.

Key words: Liaoning cashmere goat, melatonin, FGF5, LncRNA, RNA-seq, signaling pathway

JIN MEI,ZHANG LIJUAN,CAO QIAN,GUO XinYing. The Screening and Identification of LncRNA Related to Villus Growth in Liaoning Cashmere Goats by MT and FGF5[J].Scientia Agricultura Sinica, 2019, 52(4): 738-754.

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Figures/Tables 15

Table 1

Fig. 1

Table 2

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Table 3

M2_72H vs C group differential expression LncRNA target gene GO term classification"

条目类型 Term_type	功能 Features	过表达的P值 Over_represented P value	校正的P值 Corrected P value
生物过程 Biological_process (BP)	细胞代谢过程 Cellular metabolic process	7.15E-06	0.003923
	氮化合物代谢过程 Nitrogen compound metabolic process	8.48E-06	0.0039252
	细胞氮化合物代谢过程 Cellular nitrogen compound metabolic process	1.67E-05	0.0063413
	有机氮化合物代谢过程 Organonitrogen compound metabolic process	8.12E-05	0.019891
	细胞芳香化合物代谢过程 Cellular aromatic compound metabolic process	3.26E-05	0.0097043
	杂环代谢过程 Heterocycle metabolic process	5.36E-05	0.013956
	有机环状化合物代谢过程 Organic cyclic compound metabolic process	3.84E-05	0.01066
	染色体组织 Chromosome organization	9.55E-05	0.022102
	含核碱基的化合物代谢过程 Nucleobase-containing compound metabolic process	0.00011221	0.024598
	染色质修饰 Chromatin modification	0.00025719	0.04581
	细胞成分组织或生物发生 Cellular component organization or biogenesis	0.00026397	0.04581
细胞组分Cellular_component (CC)	核 Nucleus	1.48E-06	0.0031426
	膜结合的细胞器 Membrane-bounded organelle	3.52E-06	0.003923
	细胞内膜结合细胞器 Intracellular membrane-bounded organelle	4.25E-06	0.003923
	染色体 Chromosome	0.00022214	0.042055
分子功能Molecular_function (MF)	核酸酶活性 Nuclease activity	1.51E-06	0.0031426
	腺苷酸核苷酸结合 Adenyl nucleotide binding	5.49E-06	0.003923
	腺苷核糖核苷酸结合 Adenyl ribonucleotide binding	7.54E-06	0.003923
	ATP结合 ATP binding	6.43E-06	0.003923
	蛋白质结合 Protein binding	1.65E-05	0.0063413
	细胞因子活性 Cytokine activity	3.00E-05	0.0096034
	水解酶活性,作用于酯键 Hydrolase activity, acting on ester bonds	0.00012043	0.02508
	催化活性 Catalytic activity	0.00021879	0.042055
	结合物 Binding	1.90E-05	0.0065865

Table 3

Table 4

Fnc_72H vs C group differentially expressed LncRNA target genes GO term classification"

条目类型 Term_type		功能 Features	过表达的P值 Over_represented P value	校正的P值 Corrected P value
生物过程Biological_process （BP）		细胞代谢过程 Cellular metabolic process	1.22E-06	0.002285
		生物合成过程 Biosynthetic process	3.29E-06	0.002285
		有机物生物合成过程 Organic substance biosynthetic process	6.96E-06	0.0034462
		细胞生物合成过程 Cellular biosynthetic process	8.88E-06	0.0034462
		细胞大分子代谢过程 Cellular macromolecule metabolic process	9.10E-06	0.0034462
		基因表达 Gene expression	1.87E-05	0.0051872
		细胞蛋白质代谢过程 Cellular protein metabolic process	2.03E-05	0.0052866
		翻译 Translation	3.43E-05	0.0071461
		高分子生物合成过程 Macromolecule biosynthetic process	7.00E-05	0.013255
		细胞大分子生物合成过程 Cellular macromolecule biosynthetic process	8.41E-05	0.015233
		有机环状化合物代谢过程 Organic cyclic compound metabolic process	9.81E-05	0.017021
		细胞氮化合物代谢过程 Cellular nitrogen compound metabolic process	0.0001207	0.019468
		细胞芳香化合物代谢过程 Cellular aromatic compound metabolic process	0.00012153	0.019468
		细胞芳香化合物代谢过程 Nitrogen compound metabolic process	0.00013872	0.021399
		蛋白质定位 Protein localization	0.00015944	0.023716
		杂环代谢过程 Heterocycle metabolic process	0.00018062	0.025736
		DNA复制,合成RNA引物 DNA replication, synthesis of RNA primer	0.00023573	0.031671
		含核碱基的化合物代谢过程 Nucleobase-containing compound metabolic process	0.00038174	0.045427
胞组分Cellular_component (CC)	细胞内部分 Intracellular part		9.95E-08	0.00041428
	细胞质 Cytoplasm		1.93E-06	0.002285
	细胞内膜结合细胞器 Intracellular membrane-bounded organelle		2.49E-06	0.002285
	膜结合的细胞器 Membrane-bounded organelle		3.16E-06	0.002285
	细胞 Cell		7.88E-06	0.0034462
	细胞部分 Cell part		7.88E-06	0.0034462
	细胞内 Intracellular		1.34E-05	0.0046662
	细胞质部分 Cytoplasmic part		1.65E-05	0.0049423
	核 Nucleus		1.66E-05	0.0049423
	细胞内细胞器 Intracellular organelle		2.56E-05	0.0061674
	细胞器 Organelle		2.69E-05	0.0061674
	大分子复合物 Macromolecular complex		2.81E-05	0.0061674
	核部分 Nuclear part		4.41E-05	0.0087498
	膜封闭的管腔 Membrane-enclosed lumen		0.00018537	0.025736
	细胞器腔 Organelle lumen		0.00025929	0.032725
	细胞内细胞器腔 Intracellular organelle lumen		0.00025929	0.032725
	内膜系统 Endomembrane system		0.00031133	0.038138
	蛋白质复合物 Protein complex		0.00047111	0.049055
	核腔 Nuclear lumen		0.00048565	0.049335
分子功能Molecular_function (MF)	连接酶活性,形成碳 - 氧键 Ligase activity, forming carbon-oxygen bonds		0.00040555	0.045652
	连接酶活性,形成氨酰-tRNA和相关化合物 Ligase activity, forming aminoacyl-tRNA and related compounds		0.00040555	0.045652
	腺苷酸核苷酸结合 Adenyl nucleotide binding		0.00043007	0.047138
	结合物 Binding		0.00046	0.049055

Table 4

Table 5

Table 6

Table 7

Table 8

Table 9

Fig. 7

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基因 Gene	引物 Primer	引物序列（5'→3'） Primer sequence(5'→3')	产物 Products
β-actin	β-actin -F β-actin -R	GATGGCTACTGCTGCGTCG GGCATACAGGTCCTTTCGG	208bp
LncRNA XLOC_005914	LncRNA XLOC_005914 -F LncRNA XLOC_005914-R	CAGACCTTGCCACACCGA CAGTCCCGAACAAAATCAGC	143bp
LncRNA XLOC_018763	LncRNA XLOC_018763 -F LncRNA XLOC_018763-R	CAGCAGAGAAAGGAGAAAATGG GTGATGTAGGTGAGAGGCGAG	299bp
LncRNA XLOC_011424	LncRNA XLOC_011424 -F LncRNA XLOC_011424-R	AAAAGCACATTAGAGGCTGGAG GCAAGCAAATTCATGGCACA	116 bp
LncRNA XLOC_009522	LncRNA XLOC_018763 -F LncRNA XLOC_018763-R	GGGCGTGACATTTTCAGATT TCAGGGGACCTTTGGACTTG	289 bp
LncRNA XLOC_009063	LncRNA XLOC_009063-F LncRNA XLOC_009063-R	GCATCATTTCTTCCGCATTC TGGGTTACCATTTCCTTCTCC	220 bp
LncRNA XLOC_011157	LncRNA XLOC_011157-F LncRNA XLOC_011157-R	CTTTGGAAATGGTTCTGTGCTG GGAAGTAGGAGATGCTGGGTAA	140 bp

样本 Sample	原始序列 Raw reads	过滤后序列 Clean reads	错误率 Error rate (%)	Q20 Q20 (%)	Q30 Q30 (%)	GC含量 GC content(%)
C_1	48045300	46275212	0.03	96.86	93.68	48.39
C_2	48045300	46275212	0.04	94.99	90.67	48.37
M2_72H_1	42277089	40464850	0.03	96.34	92.55	51.77
M2_72H_2	42277089	40464850	0.04	93.42	87.84	51.95
M2_24H_1	50302056	48536611	0.03	96.75	93.50	48.03
M2_24H_2	50302056	48536611	0.03	95.04	90.73	48.03
M1_24H_1	48057476	46336801	0.03	96.80	93.55	48.90
M1_24H_2	48057476	46336801	0.04	94.99	90.66	48.92
F4_24H_1	51394058	49453178	0.03	96.82	93.65	47.67
F4_24H_2	51394058	49453178	0.03	95.31	91.23	47.57
F4_72H_1	49418892	47713121	0.03	96.72	93.27	52.04
F4_72H_2	49418892	47713121	0.04	94.71	90.09	52.26
F6_24H_1	46346088	44739804	0.03	96.88	93.73	48.58
F6_24H_2	46346088	44739804	0.04	94.92	90.55	48.55

通路 Pathway	通路ID Pathway ID	样本编号 Sample number	背景编号 Background number	校正的P值 Corrected P value
细胞周期 Cell cycle	chx04110	62	116	1.30736872661e-07
DNA复制 DNA replication	chx03030	26	34	1.97822480896e-07
范可尼贫血症 Fanconi anemia	chx03460	29	50	0.000199893004074
错配修复 Mismatch repair	chx03430	15	22	0.00320771571904
赖氨酸降解 Lysine degradation	chx00310	25	50	0.0157486842455
癌症中的微小RNA MicroRNAs in cancer	chx05206	50	127	0.0399507008006
阿尔茨海默氏病 Alzheimer's disease	chx05010	61	164	0.0467892160603
类固醇生物合成 Steroid biosynthesis	chx00100	12	20	0.0467892160603
TNF	chx04668	39	97	0.0467892160603
Nod样受体 Nod-like receptor	chx04621	22	47	0.0467892160603
嘧啶代谢 Pyrimidine metabolism	chx00240	37	91	0.0467892160603
RNA转运 RNA transport	chx03013	54	144	0.0467892160603
柠檬酸循环 Citrate cycle	chx00020	16	31	0.0467892160603
同源重组 Omologous recombination	chx03440	14	26	0.0467892160603
NF-kappa B	chx04064	32	77	0.0467892160603

样本名称 Sample name		显著富集的信号通路 Significantly enriched signaling pathway	Q值 Qvalue
F4_24H vs C	cis靶基因 cis target gene	无 No	-
F4_24H vs C	trans靶基因 trans target gene	无 No	-
F4_72H vs C	cis靶基因 cis target gene	无 No	-
F4_72H vs C	trans靶基因 trans target gene	核糖体 Ribosome RNA转运 RNA transport 范可尼贫血途径 Fanconi anemia pathway 亨廷顿氏病 Huntington's disease 代谢途径 Metabolic pathway 氨酰基-tRNA生物合成 Aminoacyl-tRNA biosynthesis 柠檬酸循环（TCA循环） Citrate cycle (TCA cycle) 阿尔茨海默氏病 Alzheimer's disease 泛素介导的蛋白水解 Ubiquitin mediated proteolysis	0.001 0.018 0.018 0.018 0.025 0.029 0.033 0.033 0.037
F6_24H vs C	Cis靶基因 Cis target gene	味觉转导 Taste transduction	0.022
F6_24H vs C	Trans靶基因 Trans target gene	无 No	-

通路 Pathway	通路ID Pathway ID	样本编号 Sample number	背景编号 Background number	校正后的P值 Corrected P-value
核糖体 Ribosome	chx03010	36	129	0.001
RNA转运 RNA transport	chx03013	34	144	0.018
范可尼贫血途径 Fanconi anemia pathway	chx03460	16	50	0.018
亨廷顿氏病 Huntington's disease	chx05016	38	171	0.018
代谢途径 Metabolic pathway	chx01100	178	1126	0.025
氨酰基-tRNA生物合成 Aminoacyl-tRNA biosynthesis	chx00970	14	44	0.029
柠檬酸循环（TCA循环） Citrate cycle (TCA cycle)	chx00020	11	31	0.033
阿尔茨海默氏病 Alzheimer's disease	chx05010	35	164	0.033
泛素介导的蛋白水解 Ubiquitin mediated proteolysis	chx04120	28	124	0.037
味觉转导 Taste transduction	chx04742	2	35	0.022

The Screening and Identification of LncRNA Related to Villus Growth in Liaoning Cashmere Goats by MT and FGF5

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通路 Pathway	基因ID Gene ID	基因名称 Gene name	方式 Style
氨基酸代谢通路 Metabolic pathway	102172231	CTH	UP
	102190784	PSAT1	UP
	102178128	PHGDH	UP
	102184920	NAPRT1	UP
	102187590	GCNT1	UP
	102179919	SHMT2	UP
	102186540	FDPS	UP
	102171848	DHCRT	UP
	102184667	CBS	UP

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