皱皮香猪泛素化连接酶2基因结构变异的差异表达研究

doi:10.11843/j.issn.0366-6964.2025.01.013

摘要/Abstract

摘要：

旨在解析CUL2基因的结构变异是否影响基因表达及其与香猪皱皮严重程度之间的联系。本研究取200头6月龄皱皮香猪和普通香猪的皮肤和血样，采用PCR方法检测基因组中CUL2-I3-sv300位点的多态性分布，利用UCSC等在线软件分析该结构变异包含的功能元件，预测CUL2基因与其它基因间的互作网络，并应用RT-qPCR和Western blot技术检测CUL2以及皮肤胶原蛋白生成相关基因的表达量变化。结果表明，猪群中结构变异CUL2-I3-sv300表现出丰富的多态性，普通香猪中检测到缺失突变基因型MM、杂合型WM和野生型WW，而皱皮香猪中未检测到WW基因型；且皱皮香猪的M等位基因频率显著增加(P < 0.05)。生物信息学分析提示，结构变异CUL2-I3-sv300中含有一个短散在元件(short interspersed nuclear element, SINE)、3个内含子剪切抑制子(intronic splicing silencers, ISS)和4个RNA结合蛋白位点(RNA binding protein sites, RBPs)等功能元件；预测CUL2可直接调控HIF-1α，进一步调控VEGFA、MMP-1、MMP-9，最终影响皮肤胶原蛋白的生成。经皮肤样品检测，MM型皱皮CUL2的mRNA表达量明显高于其它基因型；且MM型皱皮CUL2的蛋白表达量高于MM型普通皮肤的相应值。同时，MM型皱皮的MMP-1、MMP-9表达量高于普通皮肤，HIF-1α、VEGFA、COL1A1、COL3A1、COL6A3基因表达量却低于普通皮肤。推测缺失型结构变异因丢失了SINE等功能元件，可促进皱皮CUL2等的表达，抑制胶原蛋白生成，进而加重香猪体侧皮肤褶皱的严重程度。

关键词: 皱皮香猪, CUL2基因, 胶原蛋白, 皮肤褶皱, 生物信息学分析

Abstract:

The present study aimed to explore whether the structural variation in CUL2 affected the gene expression and wrinkle severity on skin of Xiang pigs. The 200 skins and blood samples were collected from Xiang pigs with wrinkled skin and common Xiang pigs in 6 months old. The polymorphic distribution of the CUL2-I3-sv300 site was examined using PCR method. Based on the prediction of potential functional elements within the structural variation region, and the interaction between CUL2 gene and other related genes by softwares online such as UCSC, the expression levels of CUL2 and those collagen formation-related genes were determined using RT-qPCR and Western blot methods. The results showed that the structural variation CUL2-I3-sv300 exhibited abundant polymorphism within the pig populations. Genotypes of the mutant (MM), the heterozygous (WM) and the wild (WW) were detected from skins of the common Xiang pigs, while type WW was not detected from the Xiang pig with wrinkled skin. Furthermore, the allele M was significantly more abundant in Xiang pigs with wrinkled skin compared with that in the common Xiang pigs (P < 0.05). Based on the bioinformatics analysis, several elements were screened from the structural variation region including a short interspersed nuclear element (SINE), three intronic splicing silencers (ISS) and four RNA binding protein sites (RBPs). And CUL2 might regulate HIF-1α, indirectly change VEGFA, MMP-1, and MMP-9, and eventually affect the formation of skin collagen. After detection by using both of the wrinkled skin and common skin, the mRNA and protein levels of CUL2 expression were higher in the wrinkled skin with MM genotypes than that with other genotypes in wrinkled and common skins. Moreover, the expression of CUL2, MMP-1 and MMP-9 were up-regulated, while mRNA levels of HIF-1α, VEGFA, COL1A1, COL3A1 and COL6A3 genes were down-regulated in Xiang pig with wrinkled skin compared with samples from the common Xiang pig.It is suggested that the loss of those functional elements in the structural variation region might promote the expression of CUL2 while inhibit collagen formation, thereby aggravating the severity of skin wrinkles on Xiang pig body.

Key words: Xiang pigs with wrinkled skin, CUL2 gene, collagen, wrinkled skin, bioinformatics analysis

中图分类号:

S828.2

周显珊, 黄世会, 牛熙, 冉雪琴, 王嘉福. 皱皮香猪泛素化连接酶2基因结构变异的差异表达研究[J]. 畜牧兽医学报, 2025, 56(1): 136-146.

ZHOU Xianshan, HUANG Shihui, NIU Xi, RAN Xueqin, WANG Jiafu. Differential Expression Study of Structural Variation in the Ubiquitin Ligase 2 Gene of Xiang Pigs with Wrinkled Skin[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 136-146.

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引物名称 Primer	引物序列(5′→3′) Primers sequence	预期长度/bp Fragments size
PCR-CUL2	F: CTTTTACAACCCTGAACTCGATGA R: TGGCGCTGCTACAGAAGACAC	589
q-PCR-CUL2	F: ACAGACGAGAAGTGCTGTCG R: AAACCGAGCTCTCGATTGGC	136
q-PCR- HIF-1α	F: TGAAAGCCTTGGATGGTTTTGT R: GCTCCGCTGTGTATTTTGCT	221
q-PCR-MMP-1	F: CAAGGTCTCCGAGGGTCAAG R: TGGGACAGCTGAACATCACC	300
q-PCR-MMP-9	F: TTCTGCCCTAGCGAGAGACT R: TTGTCCTGGTCGTAGTTGGC	164
q-PCR-VEGFA	F: GTGCCCACTGAGGAGTTCAA R: GCAACGCGAGTCTGTGTTTT	234
q-PCR- COL1A1	F: AGCCCTGGTGAAAATGGAGC R: CACCCTTAGCACCAACAGCA	190
q-PCR- COL3A1	F: TGGAACATGCCAGTGGGAAT R: TAGTCTCACAGCCTTGCGTG	182
q-PCR- COL6A3	F: GATCACGGAGAACAGTGCCA R: GACCGTGAGGTTCTGCTTCA	124
GAPDH	F: ACGACCACTTCGTCAAGCTC R: CAGGAGATGCTCGGTGTGTT	208

类别 Type	软件 Software	网址 Website
短散在元件Short interspersed nuclear element	UCSC	https://genome.ucsc.edu/
内含子剪切抑制子Intronic splicing silencer	SpliceAid	http://www.introni.it/splicing.html
RNA结合蛋白结合位点RBP binding site	RBP suite	http://www.csbio.sjtu.edu.cn/bioinf/RBPsuite/
RNA类型RNA type	Web Circ RNA	https://rth.dk/resources/webcircrna/submit
蛋白互作网络Protein interaction network	STRING	https://string-db.org/

猪群 Population	样本数 Number of sample	样本数Number of sample			等位基因频率Allele frequency
猪群 Population	样本数 Number of sample	MM	WM	WW	W	M
皱皮香猪ZXP	50	47	3	0	0.03^a	0.97^b
普通香猪XP	150	97	42	11	0.213^c	0.787^d

元件Element	位置/bp^* Location^*
短散在元件Short interspersed nuclear element(SINE)	42~300
内含子剪切抑制子Intronic splicing silencer(ISS)	81~85、236~241、281~286

RNA结合蛋白RBPs	评分Score	RBPs结合的碱基序列Sequence of RBP binding
FMRP	0.689	CGACUAG
IGF2BP2	0.638	AACCAUGAGGU
LIN28B	0.511	UGGUGUA
LIN28A	0.665	UGGUGUA