甘加型藏羊<i>TACR3</i>基因克隆及在发情周期和乏情期输卵管、子宫中的表达

doi:10.7668/hbnxb.20195468

摘要/Abstract

摘要：

为探究速激肽3受体基因(TACR3)功能及其编码产物神经激肽B受体(NK3R)在甘加型藏羊输卵管和子宫中的表达与分布,以发情周期和乏情期甘加型藏羊下丘脑、输卵管和子宫组织为样本,通过巢式PCR反应克隆TACR3基因编码区序列(CDS),利用STRING蛋白质互作数据库、基因本体论(GO)和京都基因与基因组百科全书(KEGG)预测分析TACR3互作蛋白的生物学功能;通过实时荧光定量PCR(qRT-PCR)、蛋白免疫印迹(WB)和免疫组织化学(IHC)方法分析TACR3基因及其编码蛋白NK3R在甘加型藏羊发情周期和乏情期输卵管和子宫的分布及表达规律。结果显示,甘加型藏羊TACR3基因开放阅读框全长1 95 bp,编码464个氨基酸,未发现信号肽结构,存在7次跨膜结构;TACR3可能参与神经肽信号通路、GnRH信号通路和钙信号通路等生物学过程。TACR3编码的NK3R蛋白主要表达在输卵管的黏膜上皮、纤毛细胞、分泌细胞和子宫的子宫腺、基质细胞中。输卵管中TACR3 mRNA在发情后期表达量最高,NK3R蛋白在发情期表达量最高;子宫中TACR3 mRNA和蛋白均在发情后期表达量最高。结果表明,TACR3基因在动物进化中较为保守,不同发情时期的输卵管和子宫组织均有TACR3 mRNA及蛋白表达,其可能在甘加型藏羊生殖过程中发挥重要作用。

关键词: 甘加型藏羊, 速激肽3受体基因(TACR3), 神经激肽B受体(NK3R), 基因克隆, 发情周期

Abstract:

To investigate the function of tachykinin 3 receptor gene(TACR3)and the expression and distribution of its encoded product neurokinin B receptor(NK3R)in the oviduct and uterus of Ganjia Tibetan sheep,hypothalamus,oviduct and uterus tissues during estrous cycle and anestrus of Ganjia Tibetan sheep were taken as samples.The TACR3 gene coding sequence(CDS)was cloned by nested PCR.The biological function of TACR3 interacting proteins was predicted using STRING protein interaction database,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).Real-time Quantitative PCR(qRT-PCR),Western Blot and immunohistochemistry(IHC)were used to analyze the distribution and expression of TACR3 gene and NK3R in the oviduct and uterus during estrous cycle and anestrus of Ganjia Tibetan sheep.The results showed that the total length of the open reading frame of TACR3 gene in Ganjia Tibetan sheep was 1 395 bp,encoding 464 amino acids,and no signal peptide structure was found,with seven transmembrane structures.TACR3 may participate in biological processes such as neuropeptide signaling pathway,GnRH signaling pathway and calcium signaling pathway.The NK3R protein encoded by TACR3 was mainly expressed in the mucosal epithelium,ciliary cells,secretory cells of oviduct,uterine glands and stromal cells of uterus.In oviduct,the expression level of TACR3 mRNA was the highest in the metestrus,and the expression level of NK3R protein was the highest in the estrus.The expression levels of TACR3 mRNA and protein in uterus were the highest in the metestrus.The results showed that TACR3 was conserved in animal evolution,and TACR3 mRNA and protein were expressed in oviduct and uterus tissues at different estrus periods,which may play an important role in the reproductive process of Ganjia Tibetan sheep.

Key words: Ganjia Tibetan sheep, Tachykinin receptor 3 gene(TACR3), Neurokinin B receptor(NK3R), Gene cloning, Estrous cycle

中图分类号:

S826

赵国勇, 高何璇, 刘晓洁, 何玉琴, 陶乐凯, 蔡永强, 张光敏. 甘加型藏羊TACR3基因克隆及在发情周期和乏情期输卵管、子宫中的表达[J]. 华北农学报, 2025, 40(3): 182-189. doi: 10.7668/hbnxb.20195468.

ZHAO Guoyong, GAO Hexuan, LIU Xiaojie, HE Yuqin, TAO Lekai, CAI Yongqiang, ZHANG Guangmin. Cloning of TACR3 Gene and Its Expression in Oviduct and Uterus During Estrous Cycle and Anestrus of Ganjia Tibetan Sheep[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 182-189. doi: 10.7668/hbnxb.20195468.

http://www.hbnxb.net/CN/Y2025/V40/I3/182

图/表 7

表1 引物序列信息

Tab.1 Sequence information of primers

基因 Gene	引物序列(5'—3') Primer sequences(5'—3')	产物大小/bp Product length	退火温度/℃ Annealing temperature
TACR3	F:AACTGGACGGACAGAGGAATGG	142	60.0
	R:GGGAGAAGGAAGCGGAAAGATTG
β-actin	F:ATATTGCTGCGCTCGTGGTT	173	60.0
	R:CTTGCTCTGAGCCTCATCCC
TACR3	1F:AGCGACAGGGAGGCAGAGAAGAACT	807	68.1
	1F':ATGGCCTCGCTCCCCGTAGC		68.1
	1R':CAACGATGGTGTATGTGATGCCCATG		69.9
	1R:AGGTGTCCCCTGGGATCTCTCCTCC		70.8
TACR3	2F:AAACCAAAGTCATGCCAGGCCGTAC	734	69.4
	2F':AGAAGGCCCCAAGCAACATTTCACT		68.4
	2R':TGGTGACACCATGATGGCCTCACTC		69.8
	2R:GAAACACAATTGCCTAAAAATTGCCCTTC		69.5

图1 TACR3基因扩增结果 M.DNA Marker;1—2.TACR3基因2条分段引物扩增产物。

Fig.1 Results of TACR3 gene amplification M.DNA Marker;1—2.The amplification products of two sectionalized primers of TACR3 gene.

图2 TACR3三级结构(A)和TACR3基因系统进化树(B)

Fig.2 TACR3 tertiary structure(A)and phylogenetic tree of TACR3 genes(B)

图3 TACR3蛋白PPI预测(A)、GO(B)与KEGG(C)分析

Fig.3 The PPI prediction(A),GO(B),and KEGG analysis(C)of TACR3 protein

图4 甘加型藏羊不同时期输卵管和子宫TACR3 mRNA的表达不同小写字母代表同一组织不同时期差异显著(P<0.05)。图5同。

Fig.4 Expression of TACR3 mRNA in oviduct and uterus tissues in different periods of Ganjia Tibetan sheep Different lowercase letters indicate significant differences among different periods in the same tissues(P<0.05).The same as Fig.5.

图5 甘加型藏羊不同时期输卵管和子宫组织NK3R蛋白的表达

Fig.5 Expression of NK3R protein in oviduct and uterus tissues in different periods of Ganjia Tibetan sheep

图6 甘加型藏羊不同时期输卵管和子宫组织NK3R蛋白的分布 A1—F1.输卵管;A2—F2.子宫。A1—2.发情前期;B1—2.发情期;C1—2.发情后期;D1—2.间情期;E1—2.乏情期;F1—2.阴性对照。L.管腔;C.纤毛;LP.固有层;EM.黏膜上皮;UG.子宫腺;SC.基质细胞。

Fig.6 Distribution of NK3R protein in oviduct and uterus tissues in different periods of Ganjia Tibetan sheep A1—F1.Oviduct;A2—F2.Uterus.A1—2.Proestrus;B1—2.Estrus;C1—2.Metestrus;D1—2.Diestrus;E1—2.Anestrus;F1—2.Negative control. L.Lumen;C.Cilia;LP.Lamina propria;EM.Epithelium mucosae;UG.Uterine glands;SC.Stromal cells.

参考文献 28

[1]	Zhang W W, Wang Y Q, Chu Y X. Tacr3/NK3R:beyond their roles in reproduction[J]. ACS Chemical Neuroscience, 2020, 11(19):2935-2943.doi:10.1021/acschemneuro.0c00421.
[2]	Garcia J P, Guerriero K A, Keen K L, Kenealy B P, Seminara S B, Terasawa E. Kisspeptin and neurokinin B signaling network underlies the pubertal increase in GnRH release in female Rhesus monkeys[J]. Endocrinology, 2017, 158(10):3269-3280.doi:10.1210/en.2017-00500. pmid: 28977601
[3]	Cejudo Roman A, Pinto F M, Dorta I, Almeida T A, Hernández M, Illanes M, Tena-Sempere M, Candenas L. Analysis of the expression of neurokinin B,kisspeptin,and their cognate receptors NK3R and KISS1R in the human female genital tract[J]. Fertility and Sterility, 2012, 97(5):1213-1219.doi:10.1016/j.fertnstert.2012.02.021.
[4]	Zhang Z X, Wen H S, Li Y, Li Q, Li W J, Zhou Y Y, Wang L Y, Liu Y, Lyu L K, Qi X. TAC3 gene products regulate brain and digestive system gene expression in the spotted sea bass(Lateolabrax maculatus)[J]. Frontiers in Endocrinology, 2019, 10:556.doi:10.3389/fendo.2019.00556.
[5]	王世冉, 田占庄. 神经激肽B及其生殖内分泌作用[J]. 生理科学进展, 2012, 43(2):107-110.doi:10.3969/j.issn.0559-7765.2012.02.005.
	Wang S R, Tian Z Z. Neurokinkin B and it's function on reproductive endocrine[J]. Progress in Physiological Sciences, 2012, 43(2):107-110.
[6]	Sun W J, Yang F, Zhang H H, Yuan Q N, Ling S L, Wang Y X, Lyu P, Li Z L, Luo Y F, Liu D S, Yin W C, Shi P, Xu H E, Tian C L. Structural insights into neurokinin 3 receptor activation by endogenous and analogue peptide agonists[J]. Cell Discovery, 2023, 9(1):66.doi:10.1038/s41421-023-00564-w. pmid: 37391393
[7]	王婷. Tac3/Tacr3在黑线仓鼠繁殖过程中的调控作用[D]. 曲阜: 曲阜师范大学, 2016.doi:10.7666/d.D01063407.
	Wang T. Regulation of Tac3/Tacr3 in the reproductive process of striped hamster[D]. Qufu: Qufu Normal University, 2016.
[8]	杨勇. 甘南州藏羊产业发展现状、存在的问题及建议[J]. 畜牧兽医杂志, 2017, 36(6):47-48.doi:10.3969/j.issn.1004-6704.2017.06.018.
	Yang Y. Current situation,existing problems and suggestions of Tibetan sheep industry in Gannan prefecture[J]. Journal of Animal Science and Veterinary Medicine, 2017, 36(6):47-48.
[9]	Topaloglu A K, Reimann F, Guclu M, Yalin A S, Kotan L D, Porter K M, Serin A, Mungan N O, Cook J R, Ozbek M N, Imamoglu S, Akalin N S, Yuksel B, O'Rahilly S, Semple R K. TAC3 and TACR3 mutations in familial hypogonadotropic hypogonadism reveal a key role for Neurokinin B in the central control of reproduction[J]. Nature Genetics, 2009, 41(3):354-358.doi:10.1038/ng.306. pmid: 19079066
[10]	Cañete H, Dorta I, Hernández M, Cejudo Roman A, Candenas L, Pinto F M, Valladares F, Báez D, Montes de Oca F, Bello A R, Almeida T A. Differentially regulated expression of neurokinin B(NKB)/NK3 receptor system in uterine leiomyomata[J]. Human Reproduction, 2013, 28(7):1799-1808.doi:10.1093/humrep/det128.
[11]	Hu W P, Wang X Y, Sun X D, Di R, Liu Q Y, Pan Z Y, Guo X F, Cao X H, Wang J Y, Zhang Y J, Jin M, Yang Y Z, Chu M X. Cloning, polymorphism and tissue expression of TAC3 gene in sheep[J]. Pakistan Journal of Zoology, 2020, 52(4):1541-1546.doi:10.17582/journal.pjz/20170303090335.
[12]	赵国勇, 高何璇, 刘晓洁, 何玉琴, 陶乐凯, 蔡永强, 张光敏. 甘加型藏羊发情周期和乏情期血浆NKB动态变化及HPO轴NK3R的表达[J]. 核农学报, 2024, 38(9):1699-1706.doi:10.11869/j.issn.1000-8551.2024.09.1699.
	Zhao G Y, Gao H X, Liu X J, He Y Q, Tao L K, Cai Y Q, Zhang G M. Dynamic changes of NKB in plasma and expression of NK3R in HPO axis during estrous cycle and anestrus of Ganjia Tibetan sheep[J]. Journal of Nuclear Agricultural Sciences, 2024, 38(9):1699-1706. doi: 10.11869/j.issn.1000-8551.2024.09.1699
[13]	Jayasena C N, Comninos A N, Stefanopoulou E, Buckley A, Narayanaswamy S, Izzi-Engbeaya C, Abbara A, Ratnasabapathy R, Mogford J, Ng N, Sarang Z, Ghatei M A, Bloom S R, Hunter M S, Dhillo W S. Neurokinin B administration induces hot flushes in women[J]. Scientific Reports, 2015, 5:8466.doi:10.1038/srep08466. pmid: 25683060
[14]	Pintado C O, Pinto F M, Pennefather J N, Hidalgo A, Baamonde A, Sanchez T, Candenas M L. A role for tachykinins in female mouse and rat reproductive function[J]. Biology of Reproduction, 2003, 69(3):940-946.doi:10.1095/biolreprod.103.017111. pmid: 12773411
[15]	韩志磊, 史军红, 何玉琴, 火静萍, 王文芳, 刘英. 甘加型藏羊发情周期阴道细胞变化规律[J]. 中国兽医学报, 2016, 36(7):1241-1246.doi:10.16303/j.cnki.1005-4545.2016.07.31.
	Han Z L, Shi J H, He Y Q, Huo J P, Wang W F, Liu Y. Observation on the changes of vaginal cells of Ganjia Tibetan sheep during the estrous cycle[J]. Chinese Journal of Veterinary Science, 2016, 36(7):1241-1246.
[16]	赵兴绪. 羊的繁殖调控[M]. 北京: 中国农业出版社, 2008.
	Zhao X X. Reproductive regulation of sheep[M]. Beijing: China Agriculture Press, 2008.
[17]	杜晓华, 米晓钰, 胡桂玲, 王海芳, 刘霞. 双峰驼NGB基因克隆及序列分析[J]. 中国草食动物科学, 2020, 40(4):1-7.doi:10.3969/j.issn.2095-3887.2020.04.001.
	Du X H, Mi X Y, Hu G L, Wang H F, Liu X. Cloning and sequence analysis of NGB gene in Bactrian camel[J]. China Herbivore Science, 2020, 40(4):1-7.
[18]	石军, 陈文丽, 白旭, 李建富, 袁宝, 赵兴绪, 张全伟. 牦牛STAG3基因克隆及其在不同年龄段牦牛睾丸中的表达定位[J]. 农业生物技术学报, 2024, 32(2):344-354.doi:10.3969/j.issn.1674-7968.2024.02.009.
	Shi J, Chen W L, Bai X, Li J F, Yuan B, Zhao X X, Zhang Q W. Clone of STAG3 gene and its expression and localization in yak(Bos grunniens)testis with different ages[J]. Journal of Agricultural Biotechnology, 2024, 32(2):344-354.
[19]	Naomi E R C. Menopause and the human hypothalamus:evidence for the role of kisspeptin/neurokinin B neurons in the regulation of estrogen negative feedback[J]. Peptides, 2009, 30(1):111-122.doi:10.1016/j.peptides.2008.05.016. pmid: 18614256
[20]	李月勤, 王君君, 张利卫, 彭永帅, 王军, 张曼, 罗琴, 宋予震. NKB及其受体NK3R基因在发情周期不同阶段小鼠下丘脑-垂体-卵巢中的表达研究[J]. 黑龙江畜牧兽医, 2023(10):132-136.doi:10.13881/j.cnki.hljxmsy.2022.08.0142.
	Li Y Q, Wang J J, Zhang L W, Peng Y S, Wang J, Zhang M, Luo Q, Song Y Z. Expression of NKB and its receptor NK3R gene in hypothalamic-pituitary-ovary in mice with different stages of estrus cycle[J]. Heilongjiang Animal Science and Veterinary Medicine, 2023(10):132-136.
[21]	Decourt C, Beltramo M. New insights on the neuroendocrine control of puberty and seasonal breeding in female sheep[J]. Animal Reproduction, 2018, 15(1):856-867.doi:10.21451/1984-3143-AR2018-0047.
[22]	Li X H, Liang C Y, Yan Y. Novel insight into the role of the Kiss1/GPR54 system in energy metabolism in major metabolic organs[J]. Cells, 2022, 11(19):3148.doi:10.3390/cells11193148.
[23]	Zhu M T, Zhang H M, Yang H, Zhao Z S, Blair H T, Liang H H, Wu P, Yu Q. Targeting GNAQ in hypothalamic nerve cells to regulate seasonal estrus in sheep[J]. Theriogenology, 2022, 181:79-88.doi:10.1016/j.theriogenology.2022.01.005. pmid: 35065460
[24]	Thorson J F, Prezotto L D, Adams H, Petersen S L, Clapper J A, Wright E C, Oliver W T, Freking B A, Foote A P, Berry E D, Nonneman D J, Lents C A. Energy balance affects pulsatile secretion of luteinizing hormone from the adenohypophesis and expression of neurokinin B in the hypothalamus of ovariectomized gilts[J]. Biology of Reproduction, 2018, 99(2):433-445.doi:10.1093/biolre/ioy069. pmid: 30101293
[25]	杨珊珊, 何翃闳, 刘敏清, 赵凌, 王靖雷, 余四九, 蔡得琪, 潘阳阳. E-cadherin在牦牛不同繁殖周期输卵管不同部位中的表达与定位[J]. 核农学报, 2022, 36(1):57-65.doi:10.11869/j.issn.100-8551.2022.01.0057.
	Yang S S, He H H, Liu M Q, Zhao L, Wang J L, Yu S J, Cai D Q, Pan Y Y. Expression and localization of E-cadherin in different parts of the oviduct at different reproduction cycles of yak[J]. Journal of Nuclear Agricultural Sciences, 2022, 36(1):57-65. doi: 10.11869/j.issn.100-8551.2022.01.0057
[26]	Candenas M L, Magraner J, Armesto C P, Anselmi E, Nieto P M, Martín J D, Advenier C, Pinto F M. Changes in the expression of tachykinin receptors in the rat uterus during the course of pregnancy[J]. Biology of Reproduction, 2001, 65(2):538-543.doi:10.1095/biolreprod65.2.538. pmid: 11466223
[27]	Casañ E M, Raga F, Bonilla-Musoles F, Polan M L. Human oviductal gonadotropin-releasing hormone:possible implications in fertilization,early embryonic development,and implantation[J]. The Journal of Clinical Endocrinology and Metabolism, 2000, 85(4):1377-1381.doi:10.1210/jcem.85.4.6503.
[28]	Pinto F M, Bello A R, Gallardo-Castro M, Valladares F, Almeida T A, Tena-Sempere M, Candenas L. Analysis of the expression of tachykinins and tachykinin receptors in the rat uterus during early pregnancy[J]. Biology of Reproduction, 2015, 93(2):51.doi:10.1095/biolreprod.115.130617. pmid: 26157068

选择文件类型/文献管理软件名称

选择包含的内容

甘加型藏羊TACR3基因克隆及在发情周期和乏情期输卵管、子宫中的表达

Cloning of TACR3 Gene and Its Expression in Oviduct and Uterus During Estrous Cycle and Anestrus of Ganjia Tibetan Sheep

RichHTML

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 28

相关文章 15

编辑推荐

Metrics

本文评价

[1]	李忠莹, 郭震楠, 朱洪林, 李佳琳, 施会彬, 韩浩园, 李君, 权凯. *奶山羊SIRT7基因序列特征、组织表达及其对乳脂合成的影响*[J]. 华北农学报, 2025, 40(3): 199-206.
[2]	侯培珂, 程宇坤, 王继庆, 孙玲, 王建鹏, 耿洪伟. *小麦TaSOD-B1基因克隆及KASP标记开发与验证*[J]. 华北农学报, 2025, 40(3): 1-8.
[3]	马峻, 连凯琪, 闪金研, 刘玉玲, 李晓龙, 赵黎明, 李学军, 彭仁海. 淇河鲫bmp6基因的克隆及表达特性分析[J]. 华北农学报, 2025, 40(2): 218-229.
[4]	司比努尔·牙生江, 者玉琦, 钟金城, 武志娟, 柴志欣. *九龙牦牛ELOVL3基因克隆、真核表达载体构建及组织表达谱分析*[J]. 华北农学报, 2025, 40(2): 201-209.
[5]	黄友举, 于泳波, 逄翠晶, 孙轼絮, 路晨, 于延冲. *大豆GmWRKY44生物信息学分析及功能验证*[J]. 华北农学报, 2025, 40(1): 29-35.
[6]	李俊仁, 陈秀珍, 吴带娣. *广藿香Trihelix家族PatASIL2基因克隆、亚细胞定位及表达分析*[J]. 华北农学报, 2025, 40(1): 104-112.
[7]	林宏峻, 吕珂, 潘萍萍, 董莉莉, 徐志浩, 王忠华. *金线莲C4H基因克隆与表达及在黄酮合成途径中的作用*[J]. 华北农学报, 2024, 39(S1): 31-38.
[8]	李俊仁, 吴带娣, 赵公政, 陈秀珍. *广藿香Trihelix转录因子家族基因PatGTL1的克隆、亚细胞定位及表达分析*[J]. 华北农学报, 2024, 39(6): 84-93.
[9]	赵安琪, 尹跃, 何军, 安巍, 秦小雅, 胡体旭. *枸杞LbaHY5基因克隆、亚细胞定位及表达分析*[J]. 华北农学报, 2024, 39(6): 76-83.
[10]	余道宁, 王佟, 洛桑顿珠, 平措占堆, 张强, 卓玛次仁, 尼玛加措, 张德荣, 梁春年. *牦牛PID1基因克隆、生物信息学及组织表达分析*[J]. 华北农学报, 2024, 39(6): 216-223.
[11]	龚意辉, 张灿美, 周桂花, 陈玫羽, 谭倩, 皮水勤, 曾永贤. *锦绣黄桃漆酶基因 PpLAC7* 的克隆、亚细胞定位及表达分析**[J]. 华北农学报, 2024, 39(5): 72-79.
[12]	高婉婷, 刘志达, 孙学桃, 李志平, 吕文霞, 李爱珍, 赵君, 张之为. *马铃薯StRac5、StRac7和StRac13基因的克隆及其表达模式分析*[J]. 华北农学报, 2024, 39(5): 33-41.
[13]	甘露, 谢美娟, 卢振华, 李明, 丁博, 邱丽娜, 谢晓东, 王俊斌. *小麦钙依赖蛋白激酶基因TaCDPK17的克隆及其抗逆功能初步分析*[J]. 华北农学报, 2024, 39(5): 1-8.
[14]	张元臣, 苏圣盈, 张家祺, 薛爽, 王景顺. *东方黏虫溶菌酶基因MseLYS的克隆与表达分析*[J]. 华北农学报, 2024, 39(4): 206-214.
[15]	王广龙, 许吴俊, 陈洋洋, 胡珍珠, 孙敏, 熊爱生. *大蒜类钙调蛋白基因AsCML15和AsCML42的分离及其在渗透胁迫中的功能分析*[J]. 华北农学报, 2024, 39(4): 80-86.

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

甘加型藏羊TACR3基因克隆及在发情周期和乏情期输卵管、子宫中的表达

Cloning of TACR3 Gene and Its Expression in Oviduct and Uterus During Estrous Cycle and Anestrus of Ganjia Tibetan Sheep

RichHTML

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 28

相关文章 15

编辑推荐

Metrics

本文评价