五指山猪与长白猪骨骼肌miRNA转录组比较分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-0137

摘要/Abstract

摘要：

为研究microRNA（miRNA）在五指山猪和长白猪肌肉发育过程中的差异及其对骨骼肌发育转录后调控的影响,以8月龄的五指山猪和长白猪背最长肌为材料,通过Solexa测序技术结合生物信息学筛选与骨骼肌发育相关的差异miRNAs。结果从2个文库中共鉴定获得311种己知的猪miRNAs,五指山猪和长白猪分别鉴定出300和293个已知miRNAs,同时分别发现了96和79新的miRNAs。筛选到的17个差异的miRNAs中有15个miRNAs在五指山猪背肌中表达量显著下调,2个miRNAs表达量显著上调。这些差异miRNAs靶基因预测共靶向574个基因,富集到30个差异显著生物学通路（P<0.05）,其中富集到胰岛素信号通路的miRNAs有5个（ssc-miR-362,ssc-miR-455-3p,ssc-miR-497,ssc-miR-499-5p和ssc-miR-874）。本研究从miRNA层面揭示了8月龄五指山猪和长白猪骨骼肌生长发育的差异,为进一步研究骨骼肌生长发育的分子机理提供基础数据。

关键词: 五指山猪, 骨骼肌, miRNAs, Solexa测序

Abstract:

In order to investigate the differences of microRNAs（miRNAs）in growth and development of skeletal muscle between Wuzhishan and Landrace pigs and to explore the effects of differential miRNAs on post-transcriptional regulation of skeletal muscle development,we used the longissimus dorsi muscle of 8-month-old Wuzhishan pig and of Landrace pig to screen their differential miRNAs related to the development of skeletal muscle via Solexa sequencing combined with bioinformatics analysis. A total of 311 known porcine miRNAs were identified from 2 libraries,300 and 293 known miRNAs were identified in Wuzhishan and Landrace pigs respectively,and new identified miRNAs were 96 and 79 respectively. Fifteen of 17 screened differential miRNAs significantly down-regulated in the Landrace pig,while 2 miRNAs significantly up-regulated. A total of 574 target genes were predicted by the 17 differential miRNAs that enriched to 30 significantly different biological pathways（P<0.05）,of which 5 miRNAs enriched to the insulin signaling pathway（ssc-miR-362,ssc-miR-455-3p,ssc-miR-497,ssc-miR-499-5p,and ssc-miR-874）. This study reveals the differences in the growth and development of skeletal muscle between 8-month-old Wuzhishan pig and Landrace pig at the level of miRNA and provides basic data for further research on the molecular mechanism of skeletal muscle growth.

Key words: Wuzhishan pig, skeletal muscle, microRNAs, Solexa sequencing

孙瑞萍, 王峰, 晁哲, 刘海隆, 邢漫萍, 刘圈炜, 郑心力, 黄丽丽, 魏立民. 五指山猪与长白猪骨骼肌miRNA转录组比较分析[J]. 生物技术通报, 2020, 36(10): 40-48.

SUN Rui-ping, WANG Feng, CHAO Zhe, LIU Hai-long, XING Man-ping, LIU Quan-wei, ZHENG Xin-li, HUANG Li-li, WEI Li-min. Comparative Analysis on miRNA Transcriptome of Skeletal Muscle Between Wuzhishan Pig and Landrace[J]. Biotechnology Bulletin, 2020, 36(10): 40-48.

图/表 10

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基因名	引物序列（5'-3'）	Tm/℃
ssc-miR-874	RT：GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACgctgta	57
	F：ACACTCCAGCTGGGCTGCCCTGGCCCGAGGG
ssc-miR-208b	RT：GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACacaaac	60
	F：ACACTCCAGCTGGGATAAGACGAACAAAAG
ssc-miR-369	RT：GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGAatcttt	58
	F：ACACTCCAGCTGGGAATAATACATGGTTG
ssc-miR-499-5p	RT：GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACaaacat	57
	F：ACACTCCAGCTGGGTTAAGACTTGCAGTG
ssc-miR-455-5p	RT：GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACcgatgt	56
	F：ACACTCCAGCTGGGTATGTGCCTTTGGACT
ssc-miR-497	RT：GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACacaaac	56
	F：ACACTCCAGCTGGGCAGCAGCACACTGTG
ssc-miR-144	RT：GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACgtacat	58
	F：ACACTCCAGCTGGGTACAGTATAGATG
ssc-miR-451	RT：GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACaactca	56
	F：ACACTCCAGCTGGGAAACCGTTACCATTAC
ssc-miR-6782-3p	RT：GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACcagggc	58
	F：ACACTCCAGCTGGGTGACCTCTGGTCTCCC
U6	F：CTCGCTTCGGCAGCACA	58
	R：AACGCTTCACGAATTTGCGT
茎环上引物	TAGTGCGTGTCGTGGAGT

基因名	引物序列（5'-3'）	Tm/℃
ssc-miR-874	RT：GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACgctgta	57
	F：ACACTCCAGCTGGGCTGCCCTGGCCCGAGGG
ssc-miR-208b	RT：GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACacaaac	60
	F：ACACTCCAGCTGGGATAAGACGAACAAAAG
ssc-miR-369	RT：GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGAatcttt	58
	F：ACACTCCAGCTGGGAATAATACATGGTTG
ssc-miR-499-5p	RT：GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACaaacat	57
	F：ACACTCCAGCTGGGTTAAGACTTGCAGTG
ssc-miR-455-5p	RT：GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACcgatgt	56
	F：ACACTCCAGCTGGGTATGTGCCTTTGGACT
ssc-miR-497	RT：GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACacaaac	56
	F：ACACTCCAGCTGGGCAGCAGCACACTGTG
ssc-miR-144	RT：GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACgtacat	58
	F：ACACTCCAGCTGGGTACAGTATAGATG
ssc-miR-451	RT：GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACaactca	56
	F：ACACTCCAGCTGGGAAACCGTTACCATTAC
ssc-miR-6782-3p	RT：GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACcagggc	58
	F：ACACTCCAGCTGGGTGACCTCTGGTCTCCC
U6	F：CTCGCTTCGGCAGCACA	58
	R：AACGCTTCACGAATTTGCGT
茎环上引物	TAGTGCGTGTCGTGGAGT

类型	长白猪	五指山猪
Total Raw Reads	18 644 880	23 249 997
Total Clean Reads	17 644 044	22 003 289
Total Clean Reads Ratio/%	90.99	91.54
Total Uniquely mapped reads	415 134	716 221
Matched Unique_Count/%	49.37	55.29
Q30/%	97.37	97.30

类型	长白猪	五指山猪
Total Raw Reads	18 644 880	23 249 997
Total Clean Reads	17 644 044	22 003 289
Total Clean Reads Ratio/%	90.99	91.54
Total Uniquely mapped reads	415 134	716 221
Matched Unique_Count/%	49.37	55.29
Q30/%	97.37	97.30

项目	长白猪	五指山猪
#of_miRNA-5p	78	76
#of_miRNA-3p	73	75
Total known miRNAs	300	293
Known miRNAs precursors	271	266
#total_sRNAs_match_hairpin	10 158 324	13 250 243
#unique_sRNAs_match_hairpin	21 220	23 700
novel_miRNA total_sRNAs_match_hairpin	129 067	86 179
Novel miRNAs	79	96