猪miR-378种子序列的多态性对其功能以及胴体性状的影响

doi:10.13560/j.cnki.biotech.bull.1985.2020-1165

摘要/Abstract

摘要：

本研究旨在探索miR-378种子序列多态性对其结构、功能和猪胴体性状的影响。利用荣昌猪和亚洲野猪的单核苷酸多态性(SNP)进行群体遗传选择分析,预测miR-378种子序列的多态性对其结构的改变,统计不同猪种中等位基因频率;采用microRNA类似物验证种子序列多态性对miR-378在脂肪细胞中功能以及相关基因表达的影响;运用猪屠宰测定数据与miR-378基因型进行关联分析。结果发现,荣昌猪miR-378位点具有极强的受选择信号,miR-378-3p种子序列第5位出现了A>G的突变,在不同猪种中该位点等位基因频率不同,其中人工选择越强的猪种A等位基因频率越高;该突变会抑制miR-378在脂肪细胞中促进脂质生成的功能,同时会消除miR-378对脂肪分化相关基因(PGC-1α和PGC-1β)以及脂肪分解代谢相关基因(HSL、ATGL和CGI-58)的调控作用;而且该位点的基因型与猪胴体性状显著相关,GG型的背膘厚显著高于AG型和AA型,AA型眼肌面积显著高于GG型。本研究发现的miR-378种子序列这个多态性位点可作为猪生长、胴体性状辅助选择的分子标记应用于猪的育种实践中。

关键词: miR-378, 种子序列, 多态性, 脂质生成, 背膘厚, 眼肌面积

Abstract:

The aim of this study is to explore the effects of miR-378 on its structure,function,and porcine carcass traits for the polymorphism of its seed sequence. The single nucleotide polymorphism(SNP)of Rongchang pig and Asian wild boar was used to analyze population genetic selection,to predict the changes of its structure by the polymorphism of miR-378 seed sequence,and to count the allele frequencies of miR-378 in different pig breeds. microRNA mimics were used to verify the function of miR-378 and related gene expressions in adipocytes for the polymorphism of its seed sequence. The association between the slaughter data of pig and the allele frequency of miR-378 were analyzed. The results showed that there was a strong selection signal at the miR-378 locus of Rongchang pig,and A> G mutation was found in the 5th position of miR-378-3p seed sequence. The allele frequency of this locus differed in different pig breeds,and the frequency of A allele in the pig breeds with the stronger artificial selection was higher. This mutation inhibited the function of miR-378 promoting lipid production in adipocytes,and eliminated the regulation of miR-378 on the adipocyte differentiation-related genes(PGC-1α and PGC-1 β)and the adipolysis-related genes(HSL,ATGL and CGI-58). Moreover,the genotype of this locus was significantly correlated with porcine carcass traits. Compared with the GG genotype,the thickness of backfat in AA and AG genotypes was significantly lower,and the eye-muscle area of AA genotype was significantly higher. The polymorphism of miR-378 seed sequence identified in this study could be used as a molecular marker for assisted selection of pig growth and carcass traits in pig breeding practice.

Key words: miR-378, seed sequence, polymorphism, lipogenesis, backfat thickness, eye-muscle area

张廷焕, 张利娟, 陈四清, 郭宗义. 猪miR-378种子序列的多态性对其功能以及胴体性状的影响[J]. 生物技术通报, 2021, 37(6): 154-162.

ZHANG Ting-huan, ZHANG Li-juan, CHEN Si-qing, GUO Zong-yi. Effects of the Polymorphism of the Seed Sequence in Porcine miR-378 on Its Function and Carcass Traits[J]. Biotechnology Bulletin, 2021, 37(6): 154-162.

图/表 10

表1 RT-PCR引物

Table 1 RT-PCR primers

引物名称 Name	引物序列 Sequence	长度Length/bp
ap2-F	GGCGTGACTTCCACAAGAGTTTA	23
ap2-R	GCCTCTTCCTTTGGCTCATG	20
Ppar-γ-F	GTGAAGGATGCAAGGGTT	18
Ppar-γ-R	CCTGATGGCATTGTGAGA	18
C/EBP-α-R	TGGACAAGAACAGCAACGAG	20
C/EBP-α-F	TCACTGGTCAACTCCAGCAC	20
PGC-1α-F	AGCGCCGTGTGATTTACGTT	20
PGC-1α-R	CCGCAGATTTACGGTGCATT	20
PGC-1β-F	CAGACGTGAGAGCAGAGGGC	20
PGC-1β-R	CGAATGTATACCACACGGCCT	21
Resistin-F	CAACTCCCTGTTTCCAAATGC	21
Resistin-R	GTCCAGCAATTTAAGCCAATGTT	23
HSL-F	AACCAACCCTAGGCCAACTG	20
HSL-R	GCTGTGTGCACCAAACTACG	20
ATGL-F	AGGCCAATGTCTGCAGCACAT	21
ATGL-R	CAAGTTGTCTGAAATGCCGCC	21
CGI-58-F	CCCTCAGGTTGGACAAAATGA	21
CGI-58-R	AGGAAAACCCCATGGCTCTAC	21
GAPDH-F	ACCACAGTCCATGCCATCAC	20
GAPDH-R	TCCACCACCCTGTTGCTGTA	20
U6-F	CGCTTCGGCAGCACATATA	19
U6-R	TTCACGAATTTGCGTGTCAT	20
miR-378W	ACTGGACTTGGAGTCAGAAGGC	22
miR-378M	ACTGGGCTTGGAGTCAGAAGGC	22

表2 miR-378扩增引物

Table 2 Amplification primer of miR-378

引物名称 Name	引物序列 Sequence	长度 Length/bp
miR-378-F	TAACCCCTAGGTGGGTCTGAG	21
miR-378-R	TCAGATGAGCAGGACAGTTCAG	22

图1 miR-378位点在荣昌猪和亚洲野猪中的群体遗传选择分析黄色标注的地方表示受选择的区域;红色五角星表示miR-378位点

Fig.1 Population genetic selection analysis of miR-378 locus in Rongchang pig and Asian wild boar The areas marked in yellow indicate the selected regions and the red five-pointed star indicates miR-378 locus

图2 miR-378野生型(miR-378W)和突变型(miR-378M)二级结构以及自由能分析红色字体表示突变位点

Fig.2 Secondary structure and free energy analysis of miR-378 wild type(miR-378W)and mutated type(miR-378 M) The red font indicates the mutation site

图3 miR-378种子序列第5位碱基(A/G)在不同猪种中的等位基因频率黄色代表 A 等位基因,红色代表 G 等位基因

Fig.3 Allele frequency of the fifth base(A/G)of miR-378 seed sequence in different pig breeds Yellow represents A allele and red represents G allele

图4 miR-378W和miR-378M对脂肪细胞脂质生成的影响 A:miRNA类似物的转染效率;B:油红 O 染色脂肪细胞,标尺:50 µm;C:定量分析脂质含量。**代表差异显著(P <0.01),下同

Fig.4 Effects of miR-378W and miR-378 M on the lipid production of adipocytes A:Transfection efficiency of miRNA mimics. B:Oil red O staining adipocytes,scale bar:50 μm. C:Quantitative analysis of the lipid content. **refers to significant difference(P < 0.01),the same below

图5 miR-378W和miR-378M对脂质相关基因表达的影响 A:脂肪生成相关基因表达情况;B:脂肪分解相关基因表达情况。*代表差异显著(P <0.05),下同

Fig.5 Effects of miR-378W and miR-378 M on the expres-sion of lipid related genes A:The expression of genes related to adipogenesis. B:The expression of genes related to lipolysis. * refers to significant difference(P < 0.05),the same below

图6 miR-378在10个物种间的碱基序列比对红色字母代表miR-378的种子序列

Fig.6 Base sequence alignment of miR-378 among 10 species The red letters represent the seed sequence of miR-378

图7 荣昌猪杂交群体中miR-378种子序列多态性测序峰图

Fig.7 Polymorphism of miR-378 seed sequence in Rong-chang pig cross population

表3 miR-378种子序列3种基因型在群体中的个体数及与屠宰性状之间的关联分析

Table 3 Number of individuals for three genotypes of miR-378 seed sequence in the population and their association with slaughter traits

	GG(24)	AG(51)	AA(32)	加性效应Additive effect	显性效应Dominant effect
最后肋骨处背膘厚 Backfat thickness at the last rib/cm	2.77^a±0.20	2.66^b±0.13	2.63^b±0.16	0.07	-0.04
眼肌面积 Eye-muscle area/cm²	22.16^b±1.08	23.25^ab±0.63	23.80^a±0.62	1.22	0.445
胴体瘦肉率 Lean percentage/%	48.12±1.06	48.28±0.64	48.32±0.55	0.32	0.07

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