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

Abstract

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

CLC Number:

S827.2

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.

Figures/Tables 13

Table 1

Table2

Fig.1

Fig.2

Table 3

Fig.3

Fig.4

Fig.5

Fig.6

Fig.7

Fig.8

Fig.9

Fig.10

References 43

[1]	ZHU J H，ZHANG H Y，BAI Y Y，et al.Relationship between SNPs of POU1F1 gene and litter size and growth traits in Shaanbei White Cashmere goats［J］.Animals （Basel），2019，9（3）：114.
[2]	刘泽林，杨娟，许铭洙，等.山羊ZBP1基因多态性及与产羔性能的关联分析［J］.中国畜牧兽医，2023，50（1）：161-173.
	LIU Z L，YANG J，XU M Z，et al.Association analysis between polymorphisms of ZBP1 gene andambing performance in goats［J］.China Animal Husbandry & Veterinary Medicine，2023，50（1）：161-173.（in Chinese）
[3]	兰蓉，朱兰，姚新荣，等.山羊产羔数全基因组关联分析［J］.畜牧兽医学报，2015，46（4）：549-554.
	LAN R，ZHU L，YAO X R，et al.Genome-wide association study of lambing number in goat［J］.Acta Veterinaria et Zootechnica Sinica，2015，46（4）：549-554.（in Chinese）
[4]	ZHANG T Y，WANG J X，BAI Y Y，et al.A functional SNP of the core promoter region within goat CDC25A gene affects litter size［J］.Front Vet Sci，2025，11：1471123.
[5]	JEET V，MAGOTRA A，BANGAR Y C，et al.Evaluation of candidate point mutation of kisspeptin 1 gene associated with litter size in Indian goat breeds and its effect on transcription factor binding sites［J］.Domest Anim Endocrinol，2022，78：106676.
[6]	MAHMOUDI P，RASHIDI A，NAZARI-GHADIKOLAEI A，et al.Genome-wide association study reveals novel candidate genes for litter size in Markhoz goats［J］.Front Vet Sci，2022，9：1045589.
[7]	王有栋，曹志平，李玉茂，等.SNP芯片技术原理及其在鸡遗传育种中的应用［J］.畜牧兽医学报，2025，56（9）：4165-4175.
	WANG Y D，CAO Z P，LI Y M，et al.The principle of SNP chip technology and its application in chicken genetic breeding［J］.Acta Veterinaria et Zootechnica Sinica，2025，56（9）：4165-4175.（in Chinese）
[8]	张嘉良，黄畅，杨永林，等.基于50K液相芯片的中国绵羊群体遗传结构与羊毛性状选择信号分析［J］.畜牧兽医学报，2025，56（7）：3164-3176.
	ZHANG J L，HUANG C，YANG Y L，et al.Genetic structure and wool trait selection signatures analysis of Chinese sheep populations based on 50K liquid SNP chip［J］.Acta Veterinaria et Zootechnica Sinica，2025，56（7）：3164-3176.（in Chinese）
[9]	李聪，刘书琴，高峰，等.家驴40K液相芯片开发及其初步应用［J］.畜牧兽医学报，2024，55（12）：5538-5548.
	LI C，LIU S Q，GAO F，et al.Development and preliminary application of domestic donkey 40K liquid chip［J］.Acta Veterinaria et Zootechnica Sinica，2024，55（12）：5538-5548.（in Chinese）
[10]	FAURE S，FORT P.Atypical RhoV and RhoU GTPases control development of the neural crest［J］.Small GTPases，2011，2（6）：310-313.
[11]	CANOVAS NUNES S，MANZONI M，PIZZI M，et al.The small GTPase RhoU lays downstream of JAK/STAT signaling and mediates cell migration in multiple myeloma［J］.Blood Cancer J，2018，8（2）：20.
[12]	CHUANG Y Y，VALSTER A，CONIGLIO S J，et al.The atypical Rho family GTPase Wrch-1 regulates focal adhesion formation and cell migration［J］.J Cell Sci，2007，120（Pt 11）：1927-1934.
[13]	BRAZIER H，KRANC W，CHERMULA B，et al.The Rho GTPase Wrch1 regulates osteoclast precursor adhesion and migration［J］.Int Biochem Cell Biol，2009，41（6）：1391-1401.
[14]	BRADY D C，ALAN J K，MADIGAN J P，et al.The transforming Rho family GTPase Wrch-1 disrupts epithelial cell tight junctions and epithelial morphogenesis［J］.Mol Cell Biol，2009，29（4）：1035-1049.
[15]	FORT P，GUÉMAR L，VIGNAL E，et al.Activity of the RhoU/Wrch1 GTPase is critical for cranial neural crest cell migration［J］.Dev Biol，2011，350（2）：451-463.
[16]	TAO W，PENNICA D，XU L，et al.Wrch-1，a novel member of the Rho gene family that is regulated by Wnt-1［J］.Genes Dev，2001，15（14）：1796-1807.
[17]	GUEMAR L，SANTA BARBARA P，VIGNAL E，et al.The small GTPase RhoV is an essential regulator of neural crest induction in Xenopus［J］.Dev Biol，2007，310（1）：113-128.
[18]	DICKOVER M，HEGARTY J M，LY K，et al.The atypical Rho GTPase，RhoU，regulates cell-adhesion molecules during cardiac morphogenesis［J］.Dev Biol，2014，389（2）：182-191.
[19]	WOO S，STRASSER L.Atypical RhoUV GTPases in development and disease［J］.Biochem Soc Trans，2024，52（1）：89-97.
[20]	NOTARNICOLA C，LE G L，FORT P，et al.Dynamic expression patterns of RhoV/Chp and RhoU/Wrch during chicken embryonic development［J］.Dev Dyn，2008，237（4）：1165-1171.
[21]	NANDI S，GUPTA P S P，MONDAL S.Ammonia concentrations in different size classes of ovarian follicles of sheep （Ovis aries）：possible mechanisms of accumulation and its effect on oocyte and granulosa cell growth in vitro［J］.Theriogenology，2016，85（4）：678-687.
[22]	史露露，胡明月，赖伟宁，等.基于50K SNP芯片的夏洛来羊遗传结构及选择信号分析［J］.黑龙江畜牧兽医，2023，（6）：49-53，59，139-140.
	SHI L L，HU M Y，LAI W N，et al.Genetic structure and selection signal analysis of charolais sheepbased on 50K SNP chip［J］.Heilongjiang Animal Science and Veterinary Medicine，2023，（6）：49-53，59，139-140.（in Chinese）
[23]	徐颂为，黄忍，杨璐，等.山羊不同组织实时荧光定量PCR内参基因的筛选［J］.黑龙江畜牧兽医，2021，（24）：31-35，40，145.
	XU S W，HUANG R，YANG L，et al.Screening of reference genes for real-time fluorescence quantification PCR in different goat tissues［J］.Heilongjiang Animal Science and Veterinary Medicine，2021，（24）：31-35，40，145.（in Chinese）
[24]	SHUTES A，BERZAT A C，CHENETTE E J，et al.Biochemical analyses of the Wrch atypical Rho family GTPases［J］.Methods Enzymol，2006，406：11-26.
[25]	BRAZERT M，KRANC W，CHERMULA B，et al.Human ovarian granulosa cells isolated during an IVF procedure exhibit differential expression of genes regulating cell division and mitotic spindle formation［J］.J Clin Med，2019，8（12）：2026.
[26]	BOUREUX A，VIGNAL E，FAURE S，et al.Evolution of the Rho family of ras-like GTPases in eukaryotes［J］.Mol Biol Evol，2007，24（1）：203-216.
[27]	CSÉPÁNYI-KÖMI R，SÁFÁR D，GRÓSZ V，et al.In silico tissue-distribution of human Rho family GTPase activating proteins［J］.Small GTPases，2013，4（2）：90-101.
[28]	MALOSIO M L，GILARDELLI D，PARIS S，et al.Differential expression of distinct members of Rho family GTP-binding proteins during neuronal development：identification of Rac1B，a new neural-specific member of the family［J］.J Neurosci，1997，17（17）：6717-6728.
[29]	YAN H，ZHANG J，WEN J，et al.CDC42 controls the activation of primordial follicles by regulating PI3K signaling in mouse oocytes［J］.BMC Biol，2018，16（1）：73.
[30]	PAN Z N，ZHANG H L，ZHANG K H，et al.Insufficient MIRO1 contributes to declined oocyte quality during reproductive aging［J］.Sci China Life Sci，2025，68（3）：764-776.
[31]	TERA DOLEBO A，MELESSE A，PORCU C，et al.Increased number of large non-atretic follicles and co-dominance effects account for high litter sizes in Bonga sheep［J］.Anim Sci J，2020，91（1）：e13384.
[32]	XU Q，WANG C，WANG L，et al.Correlation analysis of serum reproductive hormones and metabolites during multiple ovulation in sheep［J］.BMC Vet Res，2022，18（1）：290.
[33]	BOTTINO J P，PÉREZ-CLARIGET R，RODRIGUEZ M G K，et al.Multiple matings modify the estrous length，the moment of ovulation，and the estradiol and LH patterns in ewes［J］.Anim Reprod Sci，2021，18（3）：e20210045.
[34]	ZHANG，Y，ZHANG S，LU J H，et al.Genes encoding aromatases in teleosts：evolution and expression regulation［J］.Gen Comp Endocrinol，2014，205：151-158.
[35]	LUO，X，ZHANG D，ZHENG M J，et al.Casein kinase 1alpha mediates estradiol secretion via CYP19A1 expression in mouse ovarian granulosa cells［J］.BMC Biol，2024，22（1）：176.
[36]	LI Q Q，DU X，PAN X Z，et al.The transcription factor SMAD4 and miR-10b contribute to E2 release and cell apoptosis in ovarian granulosa cells by targeting CYP19A1［J］.Mol Cell Endocrinol，2018，476：84-95.
[37]	VLACHOS S，HARDEN N.Genetic evidence for antagonism between Pak protein kinase and Rho1 small GTPase signaling in regulation of the actin cytoskeleton during Drosophila oogenesis［J］.Genetics，2011，187（2）：501-512.
[38]	高丕尧，龙军先，甘嘉亮，等.CCK-8法与Alamar blue法对RKO细胞株增殖活力测试条件的对比分析［J］.华西药学杂志，2015，30（1）：38-41.
	GAO P Y，LONG J X，GAN J L，et al.Comparative analysis of CCK-8 and alamar blue on RKO celproliferation activity test conditions［J］.West China Journal of Pharmaceutical Sciences，2015，30（1）：38-41.（in Chinese）
[39]	DONG X，LU S，TIAN Y，et al.Bavachinin protects the liver in NAFLD by promoting regeneration via targeting PCNA［J］.J Adv Res，2024，55：131-144.
[40]	FROUIN I，MAGA G，DENEGRI M，et al.Human proliferating cell nuclear antigen，poly（ADP-ribose） polymerase-1，and p21waf1/cip1.A dynamic exchange of partners［J］.J Biol Chem，2003，278（41）：39265-39268.
[41]	XIONG D D，ZENG C M，JIANG L，et al.Ki-67/MKI67 as a predictive biomarker for clinical outcome in gastric cancer patients：an updated meta-analysis and systematic review involving 53 studies and 7078 patients［J］.J Cancer，2019，10（22）：5339-5354.
[42]	UXA S，CASTILLO-BINDER P，KOHLER R，et al.Ki-67 gene expression［J］.Cell Death Different，2021，28（12）：3357-3370.
[43]	SLAYMI C，VIGNAL E，CRÈS G，et al.The atypical RhoU/Wrch1 Rho GTPase controls cell proliferation and apoptosis in the gut epithelium［J］.Biol Cell，2019，111（5）：121-141.

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

Mechanism Investigation of RHOU Gene Regulation on Goat Litter Size Based on the Goat 40K Liquid Chip

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 13

References 43

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	XIA Chunqiu, MIAO Shu, LI Zhiqing, LIU Lei, WAN Fachun, SHEN Weijun. Valine Regulates Bovine Myoblast Proliferation through the AMPK/mTOR Signaling Pathway [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4491-4506.
[2]	HU Jinling, ZHONG Qiqi, HUANG Cheng, LEI Minggang. AKR1B1 Regulates Proliferation and Differentiation of Porcine Skeletal Muscle Satellite Cells via the AMPK/mTOR/S6 Signaling Pathway [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 3722-3733.
[3]	ZHANG Zhengyu, YANG Peihong, GUO Hong, LI Xin, ZHANG Linlin, GUO Yiwen, HU Debao, DING Xiangbin. Effects of Sirt1 Deacetylase on Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 603-610.
[4]	ZHAO Xiaoyi, ZHU Longlong, LIU Hui, ZHANG Dongyan, CAI Long, WANG Yalei, WANG Jing, ZHAO Junxing, CHEN Meixia. Effects of L-Malic Acid on the Proliferation and Transcriptional Profile of Porcine Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5563-5574.
[5]	HUANG Xinhe, LI Haonan, ZHOU Xiao, XU Jiajing, ZHANG Yuanshu, HAN Zhengkang. Effects and Mechanism on the Synthesis of Milk Components and Cell Proliferation in Mouse Mammary Epithelial Cells by Phytoestrogen Daidzein [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 417-429.
[6]	古丽米热·阿布都热依木, Xinru ZHANG, Yangsheng WU, Ying CHEN, Liqin WANG, Xinming XU, Juncheng HUANG, Jiapeng LIN. Effects of FKBP5 on Function of Sheep Follicular Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 3947-3956.
[7]	Ziyan WANG, Yahui WANG, Tianyi WU, Chen GAO, Zhenwei DU, Fei GE, Xiaobei ZHANG, Wenxuan ZHAO, Lupei ZHANG, Huijiang GAO, Huansheng DONG, Junya LI. INTS11 Promotes the Proliferation of Bovine Myoblasts by Mediating the Transcription of CDK2 and CYCLIND1 [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 2927-2939.
[8]	LI Qiuyun, TIAN Xinyuan, LIAO Wensheng, ZHANG Huanrong, REN Yupeng, YANG Falong, ZHU Jiangjiang, XIANG Hua. Effects of SOCS2 on Proliferation, Cycle and Apoptosis of Turbinate Bone Cells in Goats [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2226-2240.
[9]	ZHANG Wanfeng, ZHAO Tianzhi, LI Jiao, YOU Ziwei, YANG Yang, CAI Chunbo, GAO Pengfei, CAO Guoqing, GUO Xiaohong, LI Bugao. Study on NR2F2 Gene Regulating Proliferation and Apoptosis of Porcine PK15 Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3242-3251.
[10]	WANG Wanjie, CHEN Nanzhu, ZOU Huiying, ZHOU Xinyi, HAO Haisheng, PANG Yunwei, ZHU Huabin, ZHAO Xueming, YU Dawei, DU Weihua. Effects of Histone Methyltransferase ASH1L Overexpression on Proliferation and Apoptosis of Bovine Cumulus Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3358-3368.
[11]	ZHANG Peng, WANG Mingxiu, JING Kemin, PENG Wei, TIAN Yuan, LI Yuqian, FU Changqi, SHU Shi, ZHONG Jincheng, CAI Xin. Abnormal Expression of FGFs/FGFRs and Their Mediated Signaling Pathway Genes Affect the Proliferative Activity of Undifferentiated Spermatogonia in Cattleyak [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2886-2897.
[12]	XU Tiantian, ZHANG Tongtong, WANG Meng, WANG Xin. Transcription Factor Foxq1 Affects the Proliferation of Hair Follicle Stem Cells in Cashmere Goats via WNT/β-catenin Signaling Pathway [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2653-2661.
[13]	REN Xiaoli, FAN Yuying, HUANGFU Heping, LIU Yun, SHI Dongmei. Effect of GSK126 on Epithelial-mesenchymal Transition of Canine Mammary Tumor Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1721-1729.
[14]	YANG Guang, XU Jing, LI Xin, HU Debao, GUO Yiwen, DING Xiangbin, GUO Hong, ZHANG Linlin. Effect of Interfering lncbMD on Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1015-1025.
[15]	SUN Jinkui, XU Houqiang, SHI Pengfei, RUAN Yong. Construction of MEF2A Gene Interference Vector and Effect of Its Transfection on Myoblasts in Guanling Cattle [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 584-595.