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

摘要/Abstract

摘要：

【目的】探究静原鸡肌肉组织肌苷酸沉积过程中关键调控因子的调节作用,利用lncRNA-miRNA-mRNA关联分析鉴定与肌苷酸特异性沉积相关的LNC_003828、gga-miR-107-3p和MINPP1,其作为肉质研究的候选基因,为分子辅助育种提高肌肉品质提供理论基础。【方法】测定15只静原鸡胸肌和腿肌的肌苷酸含量,筛选高肌苷酸含量的胸肌和低肌苷酸含量的腿肌各3个样本提取总RNA,质量检测合格后构建cDNA文库、PCR扩增,利用Agilent 2100对文库质量进行评价,库检合格后送Illumina-Hiseq平台进行转录组测序。利用生物信息学方法筛选出静原鸡肌肉组织不同部位差异表达的MINPP1、gga-miR-107-3p和LNC_003828,进行GO注释和蛋白互作网络分析MINPP1的功能。采用qRT-PCR方法检测LNC_003828、gga-miR-107-3p和MINPP1在静原鸡胸肌和腿肌组织中的表达情况,并分析其与肌苷酸含量的相关性。【结果】测序样品间基因表达水平相关性R²>0.9,即试验样本之间基因表达可用于后续的差异基因分析。参与肌苷酸合成和代谢的糖酵解/糖异生途径中检测出3个差异表达基因MINPP1、PKM和ALDH9A1。互作分析发现lncRNA-miRNA-mRNA网络图中共有17个miRNA（9个上调,8个下调）、44个mRNA（16个上调,28个下调）和155个lncRNA（68个上调、87个下调）,核心节点gga-miR-107-3p互作的靶基因有MINPP1、靶lncRNA有LNC_003828。GO富集分析发现MINPP1基因具有磷酸酶活性、双磷酸甘油酸酯磷酸酶活性等功能;蛋白互作网络中MINPP1基因与参与糖酵解/糖异生和氨基酸生物合成通路中的PGAM1、ENO1、BPGM基因均有互作关系。qRT-PCR结果表明,静原鸡胸肌LNC_003828和gga-miR-107-3p的相对表达量低于腿肌,但差异不显著;胸肌MINPP1的相对表达量显著低于腿肌（P<0.05）。静原鸡胸肌和腿肌组织中gga-miR-107-3p的表达量与LNC_003828表达量均呈正相关,与MINPP1的表达量均呈负相关。胸肌和腿肌组织中LNC_003828、gga-miR-107-3p的表达量与肌苷酸含量均呈正相关,且差异均不显著;胸肌MINPP1表达量与肌苷酸含量呈负相关,腿肌MINPP1表达量与肌苷酸含量呈显著负相关（P<0.05）。综上所述,推测静原鸡肌肉组织中gga-miR-107-3p作为核心调节因子吸附LNC_003828,影响MINPP1基因调控肌肉肌苷酸特异性沉积,从而改善肉质。【结论】筛选出LNC_003828、gga-miR-107-3p和MINPP1为影响肌苷酸特异性沉积的候选调控因子。

关键词: 静原鸡, 肌苷酸, gga-miR-107-3p, MINPP1基因, lncRNA-miRNA-mRNA互作

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

禹保军,邓占钊,辛国省,蔡正云,顾亚玲,张娟. 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[J]. 中国农业科学, 2021, 54(19): 4229-4242.

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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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