基于山羊40K液相芯片鉴定的RHOU基因调控山羊产羔数的机制探究

doi:10.11843/j.issn.0366-6964.2026.01.022

摘要/Abstract

摘要：

旨在筛选与山羊产羔数关联的候选基因，探究RHOU基因调控山羊产羔数的分子机制。本研究利用GenoBaits^®山羊40K液相芯片对337只正在选育的波尔山羊与海门山羊的杂交群体进行SNP 基因分型，结合头胎产羔表型进行全基因组关联分析；PCR方法克隆山羊RHOU基因编码区，进行序列和结构特征分析；分离山羊卵巢大、小卵泡和卵巢颗粒细胞，采用RT-qPCR、CCK-8、ELISA等方法检测基因表达水平、细胞增殖及雌激素水平。结果鉴定到山羊第28号染色体的44 302 456处即RHOU基因上游177 365 bp处的C/T突变与头胎产羔数显著关联；克隆得到山羊RHOU基因编码区全长768 bp，编码255个氨基酸残基，与其他哺乳动物高度同源；RHOU基因在山羊各组织中广泛表达，在卵巢组织中极显著高表达（P<0.01），且在小卵泡中的表达水平极显著高于大卵泡（P<0.01）；过表达RHOU基因显著降低细胞增殖能力（P<0.05）和细胞增殖标志基因PCNA（P<0.05）、MKI67（P<0.01）的mRNA表达水平，显著下调山羊卵巢颗粒细胞中CYP19A1基因表达水平和培养液中E2浓度（P<0.05）。本研究基于山羊40K液相芯片鉴定到RHOU基因与山羊产羔数显著关联，研究发现其通过负调控山羊卵巢颗粒细胞增殖，降低颗粒细胞CYP19A1的表达，抑制E2的合成从而调控山羊的产羔性能。

关键词: 山羊40K液相芯片, RHOU, 细胞增殖, 雌激素合成

Abstract:

The study aimed to screen candidate genes associated with litter size in goats and investigate the molecular mechanism by which the RHOU gene regulates goat litter size. The GenoBaits^® Goat 40K liquid chip was used to perform SNP genotyping on a hybrid population of 337 Boer goats and Haimen goats under selection， and a genome-wide association study was conducted in combination with the first-parity litter size phenotype. The coding region of the goat RHOU gene was cloned by the PCR method， and its sequence and structural characteristics were analyzed. The large and small follicles and ovarian granulosa cells of goats were isolated， and methods such as RT-qPCR， CCK-8， and ELISA were used to detect gene expression levels， cell proliferation， and estrogen levels. A C/T mutation on chromosome 28 （position 44 302 456， 177 365 bp upstream of RHOU） was significantly associated with first-parity litter size. The cloned RHOU coding region spanned 768 bp， encoding 255 amino acids with high homology to other mammals. The RHOU gene was widely expressed in various tissues of goats， and its expression was extremely significantly higher in ovarian tissue （P<0.01）， and the expression level in small follicles was extremely significantly higher than that in large follicles （P<0.01）. Overexpression of the RHOU gene significantly reduced cell proliferation ability （P<0.05） and the mRNA expression levels of cell proliferation marker genes PCNA （P<0.05） and MKI67 （P<0.01）， and significantly down-regulated the expression level of the CYP19A1 gene and the E2 concentration in the culture medium of goat ovarian granulosa cells （P<0.05）. This study identified that the RHOU gene was significantly associated with goat litter size based on the goat 40K liquid chip. The study found that it regulates goat litter performance by negatively regulating the proliferation of goat ovarian granulosa cells， reducing the expression of CYP19A1 in granulosa cells， and inhibiting the synthesis of E2.

Key words: goat 40K liquid chip, RHOU, cell proliferation, estrogen synthesis

中图分类号:

S827.2

谢蓓伊庭, 沈阳阳, 安振江, 孟春花, 刘丰慧, 张俊, 张建丽, 曹少先, 李隐侠, 钱勇. 基于山羊40K液相芯片鉴定的RHOU基因调控山羊产羔数的机制探究[J]. 畜牧兽医学报, 2026, 57(1): 258-269.

XIE Beiyiting, SHEN Yangyang, AN Zhenjiang, MENG Chunhua, LIU Fenghui, ZHANG Jun, ZHANG Jianli, CAO Shaoxian, LI Yinxia, QIAN Yong. Mechanism Investigation of RHOU Gene Regulation on Goat Litter Size Based on the Goat 40K Liquid Chip[J]. Acta Veterinaria et Zootechnica Sinica, 2026, 57(1): 258-269.

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引物 Primer	基因 Gene	引物序列（5′→3′） Primers sequence	退火温度/℃ Tm	产物长度/bp Products length	用途 Usage
P1	RHOU	F： ATAAAGCTTACCTACCCGACCGCCTTTGT R： ATACTCGAGACTTTCACCCTGGACACC	63	1 190	CDS扩增及表达载体构建
P2	RHOU	F： GTCAGTGAGAAATGGGTGC R： TGTAGGAGGTGGCTTTGAT	60	187	RT-qPCR
P3	CYP19A1	F： ATCCCGATTTGGCAGTAA R： TGTAAAGAAAGGTCGAACAG	60	109	RT-qPCR
P4	MKI67	F： GAGGTGGCTCAGGTTCGT R： TCGGATCTGGGTTTCATA	60	114	RT-qPCR
P5	PCNA	F： AAGTGCTGGAGGCGCTTAA R： TTGGACATGCTGGTGAGG	60	190	RT-qPCR
P6	GAPDH	F： TCGGAGTGAACGGATTCG R： ATCTCGCTCCTGGAAGATG	60	227	RT-qPCR

物种 Species	登录号 Accession No.
物种 Species	核苷酸登录号 Nucleotide No.	氨基酸登录号 Amino acid No.
人 Homo sapiens	NM_021205.6	NP_067028.1
牛 Bos taurus	NM_001098147.1	NP_001091616.1
猪 Sus scrofa	XM_021072737.1	XP_020928396.1
鼠 Mus musculus	NM_133955.5	NP_598716.1