Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (4): 836-847.doi: 10.3864/j.issn.0578-1752.2020.04.015

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Differential Expression of mRNA and lncRNA in Longissimus Dorsi Muscle of Songliao Black Pig and Landrace Pig Based on High-Throughput Sequencing Technique

YaoQun WU1,ShaoKang CHEN3,XiHui SHENG1,XiaoLong QI1,XiangGuo WANG1,HeMin NI1,Yong GUO1,ChuDuan WANG2(),Kai XING1()   

  1. 1 College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206
    2 College of Animal Science and Technology, China Agricultural University, Beijing 100193
    3 Beijing Animal Husbandry Station, Beijing 100101
  • Received:2018-12-10 Accepted:2019-12-05 Online:2020-02-16 Published:2020-03-09
  • Contact: ChuDuan WANG,Kai XING E-mail:cdwang@cau.edu.cn;xk181986@163.com

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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

Table 1

Primer sequences"

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

Table 2

Quality of sample sequencing data before and after QC"

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%
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%
L 18888400280 97.82% 93.52% 0.01% 51.26%

Fig. 1

Transcript distribution abundance distribution function map The abscissa is the log10 (FPKM) value. The higher the value, the higher the transcript expression; the ordinate is the density of the transcript, which is the number of transcripts corresponding to the horizontal axis expression/detection of the total number of expressed transcripts; The peak of the distribution curve represents the region where the expression of the entire sample transcript is most concentrated"

Fig. 2

Results of differential expression analysis of genes. In the graph A, the abscissa indicates the logarithm of the difference between the samples, the ordinate indicates the -log10 (FDR) value of the two samples, and the red indicates the up-regulation of the sample expression and the green expression of the Landrace pig relative to the Songliao black pig. Down, there is no difference in the black dot mark. In the B diagram, the abscissa indicates the pairwise comparison of the samples, and the ordinate indicates the number of genes differentially expressed"

Table 3

GO entries with the most significant enrichment of differentially expressed genes"

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

Fig. 3

Functional enrichment analysis of KEGG pathways of differentially expressed genes The size and color of the circle represent the enrichment pathway and the amount of differentially expressed genes with significant enrichment, respectively"

Fig. 4

Differential expression analysis results of lncRNA A figure shows the identified new LNCRNA, blue shows the LNCRNA expressed in Songliao black pig, red shows the LNCRNA expressed in landrace pig, and the middle intersection shows the LNCRNA expressed in both Songliao black pig and landrace pig; B figure shows lncRNA of difference expression between Songliao black pig and landrace pig, horizontal coordinate shows logarithm of difference multiple between samples, vertical coordinate shows-log10(FDR) value of two samples; The red color showed that the expression of landrace pig was up-regulated and the green color showed down-regulated compared with Songliao black pig"

Fig. 5

Potential regulatory relationships between lncRNA and genes Different colors indicate the level of gene expression. Yellow indicates that gene expression is high in Songliao Black pig, and blue indicates that gene expression is high in Landrace pigs. Arrows indicate potential regulatory relationships between lncRNA and genes. One-way arrows represent upstream and downstream relationships of genes, arrows point to downstream genes, and two-way arrows represent complementary pairing relationships between genes"

Fig. 6

RT-PCR verification of differentially expressed genes The differential expression of genes in the longissimus dorsi transcriptome of Songliao black pig and Landrace was verified by RT-PCR"

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