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

doi:10.7668/hbnxb.20195468

Abstract

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

CLC Number:

S826

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.

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Figures/Tables 7

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

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

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

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

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.

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

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.

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