静原鸡肌肉组织肌苷酸特异性沉积相关LNC_003828- gga-miR-107-3p-MINPP1的关联分析

doi:10.3864/j.issn.0578-1752.2021.19.017

Abstract

Abstract:

【Objective】The aim of this study was to explore the regulatory role of key regulatory factors in the process of inosine monophosphate deposition in the muscle tissue of Jingyuan chickens, and to use lncRNA-miRNA-mRNA association analysis to identify LNC_003828, gga-miR-107-3p and MINPP1 related to inosine monophosphate specific deposition, so as to provide a theoretical basis for molecular-assisted breeding to improve chicken muscle quality.【Method】 The inosine monophosphate content of the breast and leg muscles of 15 Jingyuan chickens was determined, and three samples of the breast muscles with high inosine monophosphate content and the leg muscles with low inosine monophosphate content were screened to extract total RNA. The cDNA library was constructed after passing the quality test, and PCR amplification test was carried out. Then, the cDNA library quality was evaluated by using Agilent 2100, which was sent the library to the Illumina-Hiseq platform for transcriptome sequencing. Using bioinformatics methods, the differentially expressed MINPP1, gga-miR-107-3p and LNC_003828 in different parts of the muscle tissue of Jingyuan chicken were screened out, and GO annotation and protein interaction network was used to analyze the function of MINPP1. The qRT-PCR method was used to detect the expression of LNC_003828, gga-miR-107-3p and MINPP1 in the breast and leg muscles of Jingyuan chickens, and the correlation between them and the content of inosine monophosphate was analyzed. 【Result】 R², the correlation of gene expression levels between sequenced samples, was greater than 0.9, that is, gene expression between experimental samples could be used for subsequent differential gene analysis. Three differentially expressed genes, including MINPP1, PKM, and ALDH9A1, were detected in the glycolysis/gluconeogenesis pathway involving in the synthesis and metabolism of inosine monophosphate. Interaction analysis found that there were 17 miRNAs (9 up-regulated, 8 down-regulated), 44 mRNAs (16 up-regulated, 28 down-regulated), and 155 lncRNAs (68 up-regulated, 87 down-regulated) in the lncRNA-miRNA-mRNA network diagram, of which the target gene of the core node gga-miR-107-3p interaction was MINPP1, and the target lncRNA was LNC_003828. GO enrichment analysis found that the MINPP1 gene had functions such as phosphatase activity and bisphosphoglycerate phosphatase activity; the MINPP1 gene in the protein interaction network were all interact with PGAM1 and ENO1, which were involved in glycolysis/gluconeogenesis and amino acid biosynthesis pathways BPGM genes. The results of qRT-PCR showed that the relative expression of LNC_003828 and gga-miR-107-3p in breast muscle of Jingyuan chicken was lower than that of leg muscle, but the difference was not significant; the relative expression of MINPP1 in breast muscle was significantly lower than that of leg muscle (P<0.05). The expression of gga-miR-107-3p in the breast and leg muscle tissues of Jingyuan chicken was positively correlated with the expression of LNC_003828 and negatively correlated with the expression of MINPP1. The expression of LNC_003828 and gga-miR-107-3p in breast and leg muscle tissues were positively correlated with inosine monophosphate content, and the difference was not significant; the expression of breast muscle MINPP1 was negatively correlated with inosine monophosphate content and the expression of leg muscle MINPP1. The amount was significantly negatively correlated with the content of inosine monophosphate (P<0.05). In summary, it was speculated that gga-miR-107-3p in the muscle tissue of Jingyuan chicken was used as a core regulator to adsorb LNC_003828, which affected the MINPP1 gene to regulate the specific deposition of muscle inosine monophosphate, thereby improving meat quality. 【Conclusion】 LNC_003828, gga-miR-107-3p, and MINPP1 were selected as candidate regulatory factors affecting the specific deposition of inosine monophosphate.

Key words: jingyuan chicken, inosine monophosphate, gga-miR-107-3p, MINPP1 gene, lncRNA-miRNA-mRNA interaction

YU BaoJun,DENG ZhanZhao,XIN GuoSheng,CAI ZhengYun,GU YaLing,ZHANG Juan. Correlation Analysis of Inosine Monophosphate Specific Deposition Related LNC_003828-gga-miR-107-3P-MINPP1 in Jingyuan Chicken Muscle Tissue[J].Scientia Agricultura Sinica, 2021, 54(19): 4229-4242.

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References 58

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性别 Gender	组织 Tissue	肌苷酸含量 IMP content (g·kg^-1)	肌苷含量 Inosine content (g·kg^-1)
母鸡 Hen	胸肌Chest	1.525±0.294A	0.327±0.067A
	腿肌Leg	1.200±0.213B	0.181±0.063B
	胸肌和腿肌 Chest and leg	1.362±0.157	0.254±0.035

样本名称 Sample name	纯度 Purity (A260/A280)	浓度 Concentration (ng·μL^-1)
胸1 Chest1	2.05	1257.4
胸2 Chest2	1.99	1337.2
胸3 Chest3	2.05	803.7
腿1 Leg1	2.05	736.7
腿2 Leg2	1.99	623.1
腿3 Leg3	1.97	1038.1

引物名称 Primer name	序列 Sequence (5'→3')	退火温度 Tm (℃)
LNC_003828-F LNC_003828-R	CCACATACAACCAGTCTCT CCACCATACATCCACTCT	58
MINPP1-F	GTGGATGAGAGCAGAAGT	58
MINPP1-R	AGAAGTGGCTGAAGTGTT
β-actin-F β-actin-R	ATGGACTCTGGTGATGGTGTTAC TCGGCTGTGGTGGTGAAG	58
gga-miR-107-3p-F gga-miR-107-3p-R U6-F U6-R	CGCGCGAGCTTCTTTACAG CAGTGCAGGGTCCGAGGTAT CTCGCTTCGGCAGCACATATACT ACGCTTCACGAATTTGCGTGTC

步骤 Step	温度 Temperature (℃)	时间 Time	循环数 Number of cycles
预变性Initial denaturation	95	3min	1
变性Denaturation	95	10s	39
退火Annealing	58	30s
溶解曲线分析 Melt curve analysis	60-95	4s	1

成分 Components	终浓度 Final concentration	加样量 Loading volume (μL)
2×SYBR® Green Supermix	1×	5
Forward primer	200 nmol·L^-1	0.5
Reverse primer	200 nmol·L^-1	0.5
cDNA	N/A	1
ddH₂O	N/A	3

Correlation Analysis of Inosine Monophosphate Specific Deposition Related LNC_003828-gga-miR-107-3P-MINPP1 in Jingyuan Chicken Muscle Tissue

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GO号 GO accession	描述 Description	条目类型 Term type	物种 Species
GO:0016791	磷酸酶活性 Phosphatase activity	分子功能 Molecular_function	原鸡 Gallus gallus
GO:0034416	双磷酸甘油酸酯磷酸酶活性 Bisphosphoglycerate Phosphatase activity	分子功能 Molecular_function	原鸡 Gallus gallus
GO:0005783	内质网 Endoplasmic reticulum	细胞组分 Cell component	原鸡 Gallus gallus

部位Part	基因Gene	LNC_003828	gga-miR-107-3p	MINPP1
胸肌Chest	gga-miR-107-3p	0.8788	1.0000	-0.6495
腿肌Leg	gga-miR-107-3p	0.9857	1.0000	-0.3895