用高通量测序技术研究松辽黑猪与长白猪背最长肌 mRNA和lncRNA的差异表达

doi:10.3864/j.issn.0578-1752.2020.04.015

摘要/Abstract

摘要：

【背景】肌肉生长和脂肪沉积是猪生产中非常重要的经济性状,同时也是复杂的数量性状,受到众多因素的影响。长白猪原产于丹麦, 是目前世界上使用最为广泛的瘦肉型猪种之一, 具有生长速度快, 瘦肉率高等特点,但肉质欠佳。松辽黑猪是以民猪、长白猪和杜洛克猪为素材经过三元杂交育成的黑色母系地方培育品种,具有繁殖率高、肉质优良、适应性强、耐粗饲等特性。二者在肉质方面存在较大差异。【目的】采用高通量测序技术对松辽黑猪和长白猪的背最长肌转录组进行分析,筛选影响猪肌肉生长、肉质和脂肪沉积的关键mRNA和长链非编码RNA（long non-coding RNA, lncRNA）,为猪肉品质研究提供新的参考信息。【方法】采集6头松辽黑猪和6头长白猪的背最长肌作为样本,提取其RNA,将每个品种内的个体取等量RNA混池,采用Illumina HiSeq 2500高通量测序技术对混池后的样本RNA进行测序,然后对所获得的Reads进行比对、注释和差异表达等分析,筛选出差异表达mRNA和lncRNA并进行相关生物学功能富集分析。而后在筛选出的差异表达mRNA和lncRNA中各挑选10个基因,包括5个上调基因和5个下调基因,采用荧光定量PCR（Real-time PCR, RT-PCR）方法鉴定所选mRNA和lncRNA的表达水平,并验证测序结果的可靠性。【结果】将得到的原始数据进行质量控制后,每个样本分别得到103 M和150 M reads,其中98%以上reads质量较高,满足进行下一步分析的要求。将reads比对到参考基因组上,共得到26 774个转录本,其中包括8 428个新的转录本。采用edgeR软件包进行差异表达分析,参考标准是错误发现率（false discovery rate, FDR）小于0.05和差异倍数大于2（fold change, FC）。在2个猪种中共得到4 239个显著差异表达的mRNA,其中在松辽黑猪肌肉中2 023个上调,2 216个下调。其中HLCS、BTD、DGKα、LPIN1、FGF1、FGF10、FGFR1和ZNF7等基因参与脂类代谢和肌肉发育相关的调控。差异表达的mRNA的生物学功能富集分析发现,其主要富集在细胞发育、生物代谢调控、肌肉发育、脂类代谢等相关的信号通路和生物代谢途径上。同时,在2个猪种中还检测到的178个差异表达显著的lncRNAs,其中在松辽黑猪肌肉组织中有84个上调,94个下调。联合差异表达lncRNAs的靶基因预测和差异表达基因结果分析,显示有4个差异表达的lncRNA与差异表达mRNA存在潜在的调控关系,分别是TCONS-00041713、TCONS-00041712、ENSSSCT00000034982、ENSSSCT00000034269。同时10个差异表达mRNA和10个差异表达lncRNA的RT-PCR结果显示：其表达水平在两组中同样差异显著,且与转录组测序中表达的趋势相似,表明高通量测序结果具有可靠性。【结论】获得了影响猪肌肉生长、肉质和脂肪沉积的关键mRNA和lncRNA,同时发现了几对差异表达lncRNA和mRNA之间可能存在着潜在的调控关系。这些潜在调控关系的发现,可能对于研究调控肌肉组织代谢活动的分子机制的具有一定意义,并为其提供新的参考信息。

关键词: 松辽黑猪, 长白猪, 背最长肌, mRNA, lncRNAs

Abstract:

【Background】Muscle growth and fat deposition are important economic traits in pig production and are also complex quantitative traits. Landrace, originally from Denmark, is one of the world-famous lean-type, with fast growth and high lean meat rate, but its meat quality is poor. Songliao black pig is a black maternal local cultivar cultivated by ternary cross-breeding with the Min pig, the Landrace pig and the Duroc pig, which has the characteristics of high reproduction rate, good meat quality, strong adaptability, roughage resistant and etc. There is a big difference in meat quality between the Songliao Black Pig and Landrace Pig. 【Objective】 In this study, high-throughput sequencing technique was used to analyze the longissimus dorsi transcriptome of Songliao black pig and Landrace. The key mRNA and long non-coding RNA（lncRNA）, affecting muscle growth, meat quality and fat deposition were selected to provide new reference information for the study of pork quality. 【Method】We collected the longissimus dorsi muscle of 6 Songliao black pigs and 6 Landrace to extract RNA. Individuals in each cultivar were sampled in the same RNA mixing pool and sequenced by Illumina HiSeq 2500 high-throughput sequencing technology. Reads were compared, annotated and differentially expressed. Differentially expressed genes and lncRNAs were screened out and their biological functions were enriched. Moreover then, ten genes, including five up-regulated genes and five down-regulated genes, were selected from the differentially expressed RNA and lncRNA, and the relative expression levels of differentially expressed mRNA and lncRNA were detected by real-time PCR. 【Result】After quality control, each sample received 103 M and 150 M reads, of which more than 98% of the reads were of higher quality, meeting the requirements of further analysis. The reads were aligned to the reference genome and a total of 26 774 transcripts were obtained, of which 8 428 were new transcripts. Using edgeR for differential expression analysis, the reference standards are False Discovery Rate(FDR)＜0.05 and fold change (FC)＞2. A total of 4 239 significant differentially expressed genes were obtained, of which 2 023 were up-regulated and 2 216 were down-regulated in Songliao black pig muscle. Meanwhile, 178 significant differentially expressed lncRNAs were detected, of which 84 were up-regulated and 94 were down-regulated in Songliao black pig muscle tissue. The biological function enrichment analysis of differentially expressed genes revealed that it is mainly enriched in cell components and cell development, biological metabolism regulation, and signaling pathways and biological metabolic pathways involved in muscle development and lipid metabolism. The target gene prediction results of the screened lncRNA showed that there was a potential differential relationship between the four differentially expressed lncRNAs and the differentially expressed genes, namely TCONS-00041713, TCONS-00041712, ENSSSCT00000034982, ENSSSCT00000034269. At the same time, the results of real-time PCR analysis of 10 differentially expressed mRNAs and lncRNAs showed that the results of real-time PCR were similar to the expression of transcriptome sequencing of the longissimus dorsi muscle tissue of Songliao black pig and Landrace, indicating that high throughput sequencing results are reliable.【Conclusion】We obtained the key mRNA and lncRNA that affect the muscle growth, meat quality, and fat deposition of pigs. Meanwhile, it was found that there may be a potential regulatory relationship between several pairs of differentially expressed lncRNA and mRNA.The discovery of these differentially expressed mRNAs and lncRNAs and possible potential regulatory relationships may have a certain significance for studying the molecular mechanisms that regulate metabolic activity in muscle tissue and provide new reference information.

Key words: Songliao black pig, Landrace pig, longissimus dorsi muscles, mRNA, lncRNAs

吴垚群,陈少康,盛熙晖,齐晓龙,王相国,倪和民,郭勇,王楚端,邢凯. 用高通量测序技术研究松辽黑猪与长白猪背最长肌 mRNA和lncRNA的差异表达[J]. 中国农业科学, 2020, 53(4): 836-847.

YaoQun WU,ShaoKang CHEN,XiHui SHENG,XiaoLong QI,XiangGuo WANG,HeMin NI,Yong GUO,ChuDuan WANG,Kai XING. Differential Expression of mRNA and lncRNA in Longissimus Dorsi Muscle of Songliao Black Pig and Landrace Pig Based on High-Throughput Sequencing Technique[J]. Scientia Agricultura Sinica, 2020, 53(4): 836-847.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2020.04.015

https://www.chinaagrisci.com/CN/Y2020/V53/I4/836

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差异表达基因最显著富集的GO条目"

类型 Type	GO条目 GO entries	基因数目 Number of genes	校正后P值 Corrected P value
细胞组成 Cell composition	膜组分 Membrane part	299	1×10^-6
	膜内组分 Intrinsic component of membrane	249	1×10^-6
	细胞器 Organelle	1825	1×10^-6
	细胞内部分 Intracellular part	1890	1×10^-6
	胞内 Intracellular	2003	1×10^-6
	被膜小泡 Membrane-bounded vesicle	212	1×10^-6
	包膜细胞器 Membrane-bounded organelle	1491	1×10^-6
	细胞器膜 Organelle membrane	135	1×10^-6
	胞内细胞器 Intracellular organelle	1624	1×10^-6
	质膜组分 Plasma membrane part	68	1×10^-6
分子功能 Molecular function	阳离子结合 Cation binding	316	1×10^-6
	结合 Binding	1791	1×10^-6
	蛋白质结合 Protein binding	705	1×10^-6
	金属离子结合 Metal ion binding	171	1×10^-6
	转换金属离子结合 Transition metal ion binding	128	1×10^-6
	小分子结合（small molecule binding）	181	1×10^-6
	碳水化合物衍生物结合（carbohydrate derivative binding）	164	1×10^-6
	分子传感活性（molecular transducer activity）	75	1×10^-6
	嘌呤核苷酸结合 Purine nucleoside binding	144	1×10^-6
	核糖核苷结合 Ribonucleotide binding	144	1×10^-6
生物过程 Biological process	调控代谢过程 Regulation of metabolic process	461	1×10^-6
	调控大分子代谢过程 Regulation of macromolecule metabolic process	376	1×10^-6
	生物过程的正调控 Positive regulation of biological process	246	1×10^-6
	调控细胞代谢过程 Regulation of cellular metabolic process	312	1×10^-6
	调控初级代谢过程 Regulation of primary metabolic process	295	1×10^-6
	细胞过程的正调控 Positive regulation of cellular process	206	1×10^-6
	基因表达调控 Regulation of gene expression	275	1×10^-6
	分子功能调控 Regulation of molecular function	183	1×10^-6
	调控刺激反应 Regulation of response to stimulus	196	1×10^-6
	调控蛋白质代谢过程 Regulation of protein metabolic process	158	1×10^-6

表3

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基因Gene	上游引物（5′-3′）Forward primer	下游引物（5′-3′）Reverse primer
HPSE	GCGTTCCTGTCCGTAACCAT	ATGCGGGAGACAAACCTCTG
BTK	TTTCAGCACCATCCCTGAGC	ACGGCGCATTCTTGTTTTGT
SFT2D2	ACTGTCTTCCTCATGGGACC	GTGCAAAACACAACAGCACC
SMAD6	CCCATCTTCGTCAACTCCCC	GCTCGAAGTCGAACACCTTG
FGF10	AGATGTCCGCTGGAGAAAGC	TCCAGGATACTGTACGGGCA
TECRL	CAAAGCATGTGGGCCCTTTT	GTGGTCCAACTGACTTGTTGAC
PRKD2	CTACAACCGCTCACTGGACAT	TGCGTTCTGGATCTGGTCAT
TRAPPC2	ACCAGTTCATAGCACACGCT	GCCGAGACAAACCACTCATTG
HOXD1	CGCACGAATTTCAGCACCAA	TCAGCTGCAATGAGTTGGCT
ATP2B3	GGAGCAGAAGTTCACCGTCA	GGCAGCAGGTCTCCGTATTT
TCONS-OOO52690	GGGAAATCCAGCATCGTGTG	CCACAAGGCACAGTTTTGGTC
TCONS-OOO59635	AGCGTTTATTCCTCCCAGGC	AGGTACTTGTAGGGCCGGTA
TCONS-OOO35174	CACCGTAGGGTCGTTGCA	CATCCTTCCTTCCGTCCAG
TCONS-OOO39610	ACACCTTGGGACCCCAACTA	GGCCAGAATGACGGATGACA
TCONS-OOO59695	CAGGGTTGTCCAGCCAATGT	TGCTGGGCTGTCATCCTCTT
TCONS-OOO16916	ATTACTTTGCCCCGGAGGTCT	TAATTAGGGCTTCCCGGGTCT
TCONS-OOO46917	TGCCCGATACTGTAATGCCA	CATTCCCCGAGCACGGATTC
TCONS-OOO67508	CGTGGACGAAAAACGATCCG	CTTCACCTCGTTCAGGGCTT
TCONS-OOO36675	GGTCTTCCAAATCGGTTGCC	ACAGGGGTTGCTTTGGTGTA
TCONS-OOO52912	GGACCTGGACCCAATCAACT	TGATGATGGCACGGAATGGT
GAPDH	TATGATTCCACCCACGGCAA	CCATTTGATGTTGGCGGGAT

	样本 Sample	Reads数目 No.	Q20(%)	Q30(%)	N(%)	GC含量 GC content (%)
质控前 Before QC	SL	18817856700	96.92%	92.24%	0.01%	49.83%
质控前 Before QC	L	20211575400	96.60%	91.58%	0.01%	51.92%
质控后 After QC	SL	17560551309	98.01%	94.00%	0.01%	49.77%
质控后 After QC	L	18888400280	97.82%	93.52%	0.01%	51.26%