多浪羊PROKR2基因克隆、生物信息学及组织差异表达分析

doi:10.16431/j.cnki.1671-7236.2024.04.002

摘要/Abstract

摘要： 【目的】克隆多浪羊促动力素受体2(prokineticin receptor 2,PROKR2)基因,并检测初情期启动过程中PROKR2基因在多浪羊不同组织中的表达水平,为探究PROKR2基因在绵羊初情期启动过程中的作用提供依据。【方法】以初情期后多浪羊下丘脑cDNA为模板,PCR扩增PROKR2基因并克隆测序。利用DNAMAN软件对测序结果进行拼接,采用MegAlign软件进行物种间相似性比对并构建系统进化树,并利用生物信息学软件预测多浪羊PROKR2蛋白理化性质和结构功能。使用实时荧光定量PCR技术检测PROKR2基因在多浪羊下丘脑、垂体、卵巢、输卵管及子宫中初情期前、初情期及初情期后的表达水平。【结果】克隆获得PROKR2基因序列大小为2 641 bp,包括5'-UTR 143 bp、3'-UTR 1 343 bp和CDS区1 155 bp,编码384个氨基酸,与GenBank中绵羊预测mRNA序列(登录号:XM_004014342.5)相似性为99.83%。系统进化树表明,多浪羊PROKR2基因的遗传距离与山羊最近,与鸡最远。生物信息学分析表明,PROKR2蛋白为疏水稳定碱性蛋白,有7个跨膜结构,属于跨膜蛋白,存在31个磷酸化位点,主要在质膜上发挥作用。多浪羊PROKR2蛋白与GnRH1、PROK1、ANOS1等蛋白存在相互作用关系。实时荧光定量PCR结果显示,在多浪羊下丘脑各时期中PROKR2基因表达量均显著高于其他组织(P＜0.05),且在初情期后的表达量最高;垂体中PROKR2基因表达量无显著变化(P＞0.05);子宫中PROKR2基因在初情期前的表达量显著高于初情期和初情期后(P＜0.05);卵巢和输卵管中PROKR2基因在初情期的表达量显著高于初情期前和初情期后(P＜0.05)。【结论】试验成功克隆了多浪羊PROKR2基因CDS区序列,PROKR2蛋白属于疏水稳定碱性蛋白,含有7个跨膜结构,主要在下丘脑中表达,且在卵巢和输卵管中的总趋势为先升后降。研究结果可为进一步探究PROKR2基因在绵羊初情期启动过程中的调控作用提供参考。

关键词: 多浪羊; PROKR2基因; 克隆; 初情期; 表达

Abstract: 【Objective】 This study was aimed to clone prokineticin receptor 2 (PROKR2) gene,and detect the expression of PROKR2 gene in different tissues during the initiation of puberty in Dolang sheep,so as to provide a basis for exploring the role of PROKR2 gene in the initiation of puberty in sheep.【Method】 Using the hypothalamic cDNA in Dolang sheep after first puberty as a template,PROKR2 gene was amplified by PCR, cloned and sequenced.DNAMAN software was used to splice the sequencing results,MegAlign software was used to compare the similarity among different species and construct a phylogenetic tree,and the physicochemical property and structural function of PROKR2 protein in Dolang sheep were predicted by bioinformatics software.Real-time quantitative PCR was used to detect the expression of PROKR2 gene in hypothalamus,hypophysis,ovary,oviduct and uterus of Dolang sheep in prepuberty,puberty and postpuberty.【Result】 The cloned sequence size of PROKR2 gene was 2 641 bp,including 5'-UTR 143 bp,3'-UTR 1 343 bp and CDS region 1 155 bp,encoding 384 amino acids,and the similarity was 99.83% with the predicted mRNA sequence in sheep on GenBank (accession No.:XM_004014342.5).The phylogenetic tree showed that the genetic distance of the PROKR2 gene in Dolang sheep was the closest to Capra hircus,and the farthest to Gallus gallus.Bioinformatics analysis showed that PROKR2 protein was a hydrophobic and stable basic protein with 7 transmembrane structures,which belonged to the transmembrane protein,and there were 31 phosphorylation sites,which mainly played a role in the plasma membrane.There was an interaction relationship between proteins such as GnRH1,PROK1,ANOS1 and PROKR2 protein in Dolang sheep.The results of Real-time quantitative PCR showed that the expression of PROKR2 gene in each stage of hypothalamus was significantly higher than that of other tissues (P＜0.05),and the expression of PROKR2 gene was the highest in the postpuberty stage,while there was no significant change in hypophysis (P＞0.05).The expression of PROKR2 gene in uterus in prepuberty was significantly higher than that in puberty and postpuberty (P＜0.05).The expression of PROKR2 gene in ovary and oviduct tissues were significantly higher than those in the prepuberty and postpuberty stages (P＜0.05).【Conclusion】 In this study,the CDS region sequence of PROKR2 gene was successfully cloned,and PROKR2 protein was a hydrophobic stable basic protein containing 7 transmembrane structures,which was mainly expressed in hypothalamus,and the general trend in the ovary and oviduct were first rising and then decreasing,which provided a reference for further exploring its regulation in the initiation of early puberty in sheep.

Key words: Dolang sheep; PROKR2 gene; cloning; puberty; expression

中图分类号:

S826
Q78

黄巧艳, 李伟, 王鑫昆, 邢凤. 多浪羊PROKR2基因克隆、生物信息学及组织差异表达分析[J]. 中国畜牧兽医, 2024, 51(4): 1339-1348.

HUANG Qiaoyan, LI Wei, WANG Xinkun, XING Feng. Cloning,Bioinformatics and Tissue Differential Expression Analysis of PROKR2 Gene in Dolang Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1339-1348.

参考文献

[1] 邢凤,廖秋萍.哺乳动物初情期启动相关基因研究进展[J].家畜生态学报,2017,38(7):1-4 XING F,LIAO Q P.Advances on related genes at the onset of puberty for mammals[J].Journal of Domestic Animal Ecology,2017,38(7):1-4.(in Chinese)
[2] YANG L K,HOU Z S,TAO Y X.Biased signaling in naturally occurring mutations of G protein-coupled receptors associated with diverse human diseases[J].Biochimica Biophysica Acta-Molecular Basis of Disease,2021,1867(1):165973.
[3] SUZUKI E,MIYADO M,KUROKI Y,et al.Genetic variants of G-protein coupled receptors associated with pubertal disorders[J].Reproductive Medicine and Biology, 2023,22(1):e12515.
[4] NOEL S D,KAISER U B.G protein-coupled receptors involved in GnRH regulation:Molecular insights from human disease[J].Molecular and Cellular Endocrinology,2011,346(1-2):91-101.
[5] SOGA T,MATSUMOTO S I,ODA T,et al.Molecular cloning and characterization of prokineticin receptors[J].Biochimica et Biophysica Acta,2002,1579(2-3):173-179.
[6] BASSI I,LUZZANI F,MARELLI F,et al.Knocking-down of the prokineticin receptor 2 affects reveals its complex role in the regulation of the hypothalamus-pituitary-gonadal axis in the zebrafish model[J].Scientific Reports,2020,10(1):7632.
[7] MASUDA Y,TAKATSU Y,TERAO Y,et al.Isolation and identification of EG-VEGF/prokineticins as cognate ligands for two orphan G-protein-coupled receptors[J].Biochemical and Biophysical Research Communications,2002,293(1):396-402.
[8] NEGRI L,LATTANZI R,GIANNINI E,et al.Bv8/prokineticin proteins and their receptors[J].Life Sciences,2007,81(14):1103-1116.
[9] CHENG M Y,LESLIE F M,ZHOU Q Y.Expression of prokineticins and their receptors in the adult mouse brain[J].Journal of Comparative Neurology,2006,498(6):796-809.
[10] KOYAMA Y,KIYO-OKA M,OSAKADA M,et al.Expression of prokineticin receptors in mouse cultured astrocytes and involvement in cell proliferation[J].Brain Research,2006,1112(1):65-69.
[11] ZHANG C,TRUONG K K,ZHOU Q Y.Efferent projections of prokineticin 2 expressing neurons in the mouse suprachiasmatic nucleus[J]. Public Library of Science One,2009,4(9):e7151.
[12] MATSUMOTO S,YAMAZAKI C,MASUMOTO K H,et al.Abnormal development of the olfactory bulb and reproductive system in mice lacking prokineticin receptor PKR2[J]. Proceedings of the National Academy of Sciences of the United States of America,2006,103(11):4140-4145.
[13] NEGRI L,LATTANZI R,GIANNINI E,et al.Melchiorri P.Bv8/prokineticins and their receptors A new pronociceptive system[J].International Review of Neurobiology,2009,85:145-157.
[14] MA L,LI Z,XI S,et al.Tubal ectopic pregnancy occurrence is associated with high expressions of prokineticin receptors and aberrant secretion of inflammatory cytokines[J].American Journal of Translational Research,2020,12(9):5741-5751.
[15] FUKAMI M,SUZUKI E,IZUMI Y,et al.Paradoxical gain-of-function mutant of the G-protein-coupled receptor PROKR2 promotes early puberty[J]. Journal of Cellular and Molecular Medicine,2017,21(10):2623-2626.
[16] CAO Y L,ZHANG Z F,WANG J,et al.Association between polymorphisms of prokineticin receptor (PKR1 rs4627609 and PKR2 rs6053283) and recurrent pregnancy loss[J].Journal of Zhejiang University.Science B,2016,17(3):218-224.
[17] 周晓涛,陈丹娜,刘化蝶,等.人PROKR2蛋白274位缬氨酸突变质粒的构建及表达[J].生物技术通讯,2017,28(2):90-95. ZHOU X T,CHEN D N,LIU H D,et al.Construction and expression of the pKR5-mGlu-Flag-hPROKR2(V274D/R/T/A)[J].Letters in Biotechnology,2017,28(2):90-95.(in Chinese)
[18] WHITLOCK K E,POSTLETHWAIT J,EWER J.Neuroendocrinology of reproduction:Is gonadotropin-releasing hormone (GnRH) dispensable?[J]. Frontiers in Neuroendocrinology,2019,53:100738.
[19] GORYSZEWSKA E,KACZYNSKI P,BALBONI G,et al.Prokineticin 1-prokineticin receptor 1 signaling promotes angiogenesis in the porcine endometrium during pregnancy[J].Biology of Reproduction,2020,103(3):654-668.
[20] KAUR K K,ALLAHBADIA G,SINGH M.An update on the role of prokineticins in human reproduction-potential therapeutic implications[J].Open Journal of Genetics,2013,3:201-215.
[21] HU Y,BOULOUX P M.X-linked GnRH deficiency:Role of KAL-1 mutations in GnRH deficiency[J].Molecular and Cellular Endocrinology,2011,346(1-2):13-20.
[22] ZHAO X,LI H,CHEN X,et al.Long non-coding RNA MSTRG.5970.28 regulates proliferation and apoptosis of goose follicle granulosa cells via the miR-133a-3p/ANOS1 pathway[J].Poultry Science,2023,102(3):102451.
[23] ZHONG Y,DI R,YANG Y,et al.Transcriptome analysis of neuroendocrine regulation of ovine hypothalamus-pituitary-ovary axis during ovine anestrus and the breeding season[J].Genes,2021,12(12):1861.