Cloning, Expression Analysis and Functional Identification of SOCS3 Gene in Anhui White Goat (Capra hircus)
CUI Heng-Yuan1, MA Cun-Xia1, CUI Shuang-Shuang1, GUO Yu-Zhu1, LIU Hong-Yu1,2, ZHANG Yun-Hai1,2, SONG Ning1,2,*
1 College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China; 2 Anhui Province Key Laboratory of Local Livestock and Poultry Genetic Resource Conservation and Bio-breeding, Hefei 230036, China
Abstract:Suppressor of cytokine signaling 3 (SOCS3) is a protein that responds to signal transduction and regulates milk protein synthesis. αS1-casein is one of the most important allergens in ruminant milk. However, the effect of SOCS3 on αS1-casein synthesis in goats (Capra hircus) is still unclear. To elucidate the gene sequence, structure and expression of SOCS3, and the effect of SOCS3 on αS1-casein expression in goat mammary gland, in this study, Anhui white goat mammary tissue was used as experimental material, SOCS3 gene coding region sequence was cloned by PCR. Bioinformatics analysis was conducted by online software, the expression of SOCS3 in different lactation periods was detected, interference and overexpression of SOCS3 were performed for functional analysis. The results showed that the complete coding region of SOCS3 gene in goats was 690 bp, encoding 229 amino acids. The molecular weight of SOCS3 protein was 25.09 kD, the theoretical isoelectric point was 8.97. There were 34 phosphorylation sites, no signal peptide and transmembrane helix structure, and SOCS3 was an unstable protein carrying positive charge. SOCS3 protein interacted with SOCS2, Janus kinase (JAK), tyrosine kinase 2 (TYK2), insulin receptor substrate 2 (IRS2), leptin receptor (LEPR) and interleukin 6 (IL6) and other proteins. SOCS3 gene of goat was most closely related to sheep, followed by cattle (Bos taurus). The expression of SOCS3 gene was the highest in the early and peak lactation periods. In goat mammary epithelial cells, interference with SOCS3 gene significantly up-regulated αS1-casein expression, and overexpression of SOCS3 gene significantly down-regulated αS1-casein expression. These results indicated that SOCS3 had a negative regulatory effect on αS1-casein synthesis in goats. This study provides a basis for studying the regulatory mechanism of SOCS3 gene in goat αS1-casein synthesis.
崔恒源, 马存霞, 崔双双, 郭玉柱, 刘洪瑜, 张运海, 宋宁. 安徽白山羊SOCS3基因克隆、表达分析及其功能鉴定[J]. 农业生物技术学报, 2024, 32(9): 2071-2080.
CUI Heng-Yuan, MA Cun-Xia, CUI Shuang-Shuang, GUO Yu-Zhu, LIU Hong-Yu, ZHANG Yun-Hai, SONG Ning. Cloning, Expression Analysis and Functional Identification of SOCS3 Gene in Anhui White Goat (Capra hircus). 农业生物技术学报, 2024, 32(9): 2071-2080.
[1] Caubet J C, Lin J, Ahrens B, et al.2017. Natural tolerance development in cow's milk allergic children: IgE and IgG4 epitope binding[J]. Allergy, 72(11): 1677-1685. [2] Chandrakar P, Parmar N, Descoteaux A, et al.2020. Differential induction of socs isoforms by leishmania donovani impairs macrophage-t cell cross-talk and host defense[J]. Journal of Immunology, 204(3): 596-610. [3] Clark S, Garcia M B M.2017. A 100-year review: Advances in goat milk research[J]. Journal of Dairy Science, 100(12): 10026-10044. [4] Faida P, Attiogbe M K I, Majeed U, et al.2023. Lung cancer treatment potential and limits associated with the stat family of transcription factors[J]. Cellular Signalling, 109: 110797. [5] Fan Y, Arbab A A I, Zhang H, et al.2021. Lactation associated genes revealed in holstein dairy cows by weighted gene co-expression network analysis (wgcna)[J]. Animals, 11(2): 314. [6] Feng S, Wang J, Xu X, et al.2018. The expression of socs and nf-kappa b p65 in hypopharyngeal carcinoma[J]. Iranian Journal of Public Health, 47(12): 1874-1882. [7] Geng Z, Shan X, Lian S, et al.2021. Lps-induced socs3 antagonizes the jak2-stat5 pathway and inhibits beta-casein synthesis in bovine mammary epithelial cells[J]. Life Sciences, 278: 119547. [8] Gillespie A L, Green B D.2016. The bioactive effects of casein proteins on enteroendocrine cell health, proliferation and incretin hormone secretion[J]. Food Chemistry, 211: 148-159. [9] Huang Y, Zhao F, Luo C, et al.2013. Socs3-mediated blockade reveals major contribution of jak2/stat5 signaling pathway to lactation and proliferation of dairy cow mammary epithelial cells in vitro[J]. Molecules, 18(10): 12987-13002. [10] Jiao B, Zhang X, Wang S, et al.2019. Microrna-221 regulates proliferation of bovine mammary gland epithelial cells by targeting the stat5a and irs1 genes[J]. Journal of Dairy Science, 102(1): 426-435. [11] Khan M Z, Khan A, Xiao J, et al.2020. Role of the jak-stat pathway in bovine mastitis and milk production[J]. Animals, 10(11): 2107. [12] Kobayashi K.2023. Culture models to investigate mechanisms of milk production and blood-milk barrier in mammary epithelial cells: A review and a protocol[J]. Journal of Mammary Gland Biology and Neoplasia, 28(1): 8. [13] Kusza S, Cziszter L T, Ilie D E, et al.2018. Kompetitive allele specific pcr (kasp (tm)) genotyping of 48 polymorphisms at different caprine loci in french alpine and saanen goat breeds and their association with milk composition[J]. PeerJ, 6: e4416. [14] Lin G, Qi H, Guo X, et al.2023. Arid1b blocks methionine-stimulated mtor activation to inhibit milk fat and protein synthesis in and proliferation of mouse mammary epithelial cells[J]. Journal of Nutritional Biochemistry, 114: 109274. [15] Liu R, Zhu H, Zhao J, et al.2022. Lycium barbarum polysaccharide inhibits E. coli-induced inflammation and oxidative stress in mammary epithelial cells of dairy cows via socs3 activation and mapk suppression[J]. Agriculture, 12(5): 598. [16] Masuzaki R, Kanda T, Sasaki R, et al.2022. Suppressors of cytokine signaling and hepatocellular carcinoma[J]. Cancers, 14(10): 2549. [17] Mavangira V, Sordillo L M.2018. Role of lipid mediators in the regulation of oxidative stress and inflammatory responses in dairy cattle[J]. Research in Veterinary Science, 116: 4-14. [18] Pelham S J, Caldirola M S, Avery D T, et al.2022. Stat5b restrains human b-cell differentiation to maintain humoral immune homeostasis[J]. Journal of Allergy and Clinical Immunology, 150(4): 931-946. [19] Putman A, Brown J, Gandy J, et al.2018. Changes in biomarkers of nutrient metabolism, inflammation, and oxidative stress in dairy cows during the transition into the early dry period[J]. Journal of Dairy Science, 101(10): 9350-9359. [20] Schulmeister U, Hochwallner H, Swoboda I, et al.2009. Cloning, expression, and mapping of allergenic determinants of alpha s1-casein, a major cow's milk allergen[J]. Journal of Immunology, 182(11): 7019-7029. [21] Shin H Y, Hennighausen L, Yoo K H.2019. Stat5-driven enhancers tightly control temporal expression of mammary-specific genes[J]. Journal of Mammary Gland Biology and Neoplasia, 24(1): 61-71. [22] Song N, Luo J, Huang L, et al.2021. Mir-204-5p and mir-211 synergistically downregulate the alpha(s1)-casein content and contribute to the lower allergy of goat milk[J]. Journal of Agricultural and Food Chemistry, 69(18):5353-5362. [23] Song N, Luo J, Huang L, et al.2023. Mir-380-3p promotes beta-casein expression by targeting alphas1-casein in goat mammary epithelial cells[J]. Animal Bioscience, 36(10): 1488-1498. [24] Xu Y, Lv S.2016. The effect of jak2 knockout on inhibition of liver tumor growth by inducing apoptosis, autophagy and anti-proliferation via stats and pi3k/akt signaling pathways[J]. Biomedicine & Pharmacotherapy, 84: 1202-1212. [25] Yang J, Kuang H, Xiong X, et al.2024. Alteration of the allergenicity of cow's milk proteins using different food processing modifications[J]. Critical Reviews in Food Science and Nutrition, 64(14): 4622-4642. [26] Zhang X, Wang Y, Yuan J, et al.2018. Macrophage/microglial ezh2 facilitates autoimmune inflammation through inhibition of socs3[J]. Journal of Experimental Medicine, 215(5): 1365-1382. [27] Zong J, Shen J, Liu X, et al.2023. Lithium chloride promotes milk protein and fat synthesis in bovine mammary epithelial cells via hif-1 alpha and beta-catenin signaling pathways[J]. Biological Trace Element Research, 201(1): 180-195.