海兰褐蛋鸡ZP3基因结构及其在卵泡中表达规律分析

doi:10.11843/j.issn.0366-6964.2021.012.005

摘要/Abstract

摘要： 旨在探讨ZP3基因在海兰褐蛋鸡中的表达特性，为后期研究ZP3基因在蛋鸡卵泡发育中的作用提供理论支撑。本研究以产蛋期50周龄（50 W）海兰褐蛋鸡为试验对象，利用RACE技术克隆ZP3基因全长，并对编码区进行生物信息学分析。选择健康、体重相近的海兰褐蛋鸡6只，分别取心、肝、肺、肾、卵巢、胸肌、腿肌和不同等级卵泡构建表达谱。卵巢颗粒细胞经不同浓度（PBS、5 ng·mL^-1、10 ng·mL^-1、20 ng·mL^-1）促卵泡素（follicle-stimulating hormone，FSH）处理后，提取总RNA，采用实时荧光定量检测ZP3基因在各个样品的表达水平。结果表明，鸡ZP3基因全长1 415 bp，其中编码区1 341 bp，共编码446个氨基酸，位于10号染色体上，包含9个外显子；其亲缘关系与猪最远；跨膜结构预测表示，其含有一个跨膜结构域和一个信号肽区域；对其亲疏水性进行分析，预测该蛋白为弱的疏水性蛋白，蛋白质结构主要由无规则卷曲构成。组织表达谱显示，ZP3基因在鸡的卵巢中相对表达量最高。各等级卵泡ZP3表达分析显示，随着卵泡发育，ZP3基因在成熟卵泡（F1）颗粒细胞层表达量最高；在使用FSH处理等级颗粒细胞后，发现ZP3表达量显著上调（P<0.05），暗示ZP3基因在颗粒细胞中受到FSH激素调节。本试验对ZP3基因的结构进行了分析并预测该基因存在跨膜结构和信号肽区域。基因表达谱分析结果显示，ZP3基因在卵泡发育过程中表达量逐渐升高且主要在颗粒细胞中表达，可能受到FSH的调节作用，因此预测，ZP3基因可能与海兰褐蛋鸡繁殖功能相关。

关键词: 海兰褐蛋鸡, ZP3基因, 基因克隆, 生物信息学, 组织表达谱

Abstract: The study aimed to explore the expression characteristics of ZP3 gene in Hailan laying hens, which could lay the theoretical foundations for elucidating the function of ZP3 gene in follicle development in chicken. Chicken ZP3 gene was cloned with rapid amplification of cDNA ends (RACE) using the follicle tissue from 50-week-old (50 W) Hailan laying hens. The sequence of CDS region of ZP3 was analyzed by bioinformatics tools. The expression profile of ZP3 gene was constructed using heart, liver, lung, kidney, ovary, chest muscle, leg muscle tissues and follicles of different grades from 6 healthy Hailan brown laying hens with similar body weight. Total RNA was obtained from the ovarian granulosa cells with the treatment of follicle-stimulating hormone (FSH) in different concentrations (PBS, 5 ng·mL^-1,10 ng·mL^-1,20 ng·mL^-1). The expression levels of ZP3 gene in different tissues were detected by real-time PCR. The total length of chicken ZP3 gene was 1 415 bp containing 9 exons, in which the coding region was 1 341 bp, encoding 446 amino acids, located at chromosome 10. Its genetic relationship was the farthest from swine. The prediction of transmembrane structure indicated that it contained a transmembrane domain and a signal peptide; By analyzing its hydrophobicity, it was predicted that the protein was a weak hydrophobic protein, and the protein structure was mainly composed of random curl. According to the results of gene expression profile, the expression of ZP3 gene was the highest in chicken ovary. Besides, the expression of ZP3 gene was the highest in granulosa cells of mature follicles (F1) relative to follicles of other degrades. After treating granulosa cells of different grades with FSH, the expression level of ZP3 gene was significantly increased (P<0.05), suggesting that ZP3 gene was regulated by FSH in granulosa cells. This stuay analyzed the structure of ZP3 gene and predicted that it contained the transmembrane structure and signal peptide region. Expression profiles revealed that the expression level of ZP3 gene increased gradually during follicular develop ment and was mainly expressed in granulosa cells, which may be regulated by FSH. Therefore, it was predicted that ZP3 gene might be involved in reproductive function of Hailan brown laying hens.

Key words: Hailan brown laying hens, ZP3 gene, gene cloning, bioinformatics, tissue expression profile

中图分类号:

S831.2

朱帅鹏, 王东雪, 刘聪, 孙君卫, 杜振伟, 孙桂荣, 李文婷. 海兰褐蛋鸡ZP3基因结构及其在卵泡中表达规律分析[J]. 畜牧兽医学报, 2021, 52(12): 3366-3374.

ZHU Shuaipeng, WANG Dongxue, LIU Cong, SUN Junwei, DU Zhenwei, SUN Guirong, LI Wenting. ZP3 Gene Structure of Hailan Brown Laying Hens and Its Expression Regularity in Follicles[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(12): 3366-3374.

参考文献 31

[1]	WASSARMAN P M, LIU C Y, LITSCHER E S.Constructing the mammalian egg zona pellucida:some new pieces of an old puzzle[J].J Cell Sci, 1996, 109(Pt 8):2001-2004.
[2]	LIU K Q, LI K M, BAI D D, et al.Dosage effects of ZP2 and ZP3 heterozygous mutations cause human infertility[J].Hum Genet, 2017, 136(8):975-985.
[3]	MACKENZIE S M, MCLAUGHLIN E A, PERKINS H D, et al.Immunocontraceptive effects on female rabbits infected with recombinant myxoma virus expressing rabbit ZP2 or ZP3[J].Biol Reprod, 2006, 74(3):511-521.
[4]	KIEFER S M, SALING P.Proteolytic processing of human zona pellucida proteins[J].Biol Reprod, 2002, 66(2):407-414.
[5]	REPIN V S, AKIMOVA I M.A study of the protein composition of the zona pellucida of mammalian oocytes and zygotes by a method of micro-electrophoresis in polyacrylamide gel[J].Biokhimiia, 1976, 41(1):50-57.(in Russian)
[6]	BLEIL J D, WASSARMAN P M.Structure and function of the zona pellucida:identification and characterization of the proteins of the mouse oocyte's zona pellucida[J].Dev Biol, 1980, 76(1):185-202.
[7]	WASSARMAN P M, MORTILLO S.Structure of the mouse egg extracellular coat, the zona pellucida[J].Int Rev Cytol, 1991, 130:85-110.
[8]	徐华伟, 字向东, 黄磊, 等.牦牛ZP3基因的克隆及蛋白质结构的预测[J].生物技术通报, 2010(10):156-161, 167.XU H W, ZI X D, HUANG L, et al.Cloning of ZP3 gene and prediction of ZP3 protein structure in yak[J].Biotechnology Bulletin, 2010(10):156-161, 167.(in Chinese)
[9]	WASSARMAN P M, JOVINE L, LITSCHER E S.A profile of fertilization in mammals[J].Nat Cell Biol, 2001, 3(2):E59-E64.
[10]	BRONSON R A, MCLAREN A.Transfer to the mouse oviduct of eggs with and without the zona pellucida[J].J Reprod Fertil, 1970, 22(1):129-137.
[11]	LITSCHER E S, QI H Y, WASSARMAN P M.Mouse zona pellucida glycoproteins mZP2 and mZP3 undergo carboxy-terminal proteolytic processing in growing oocytes[J].Biochemistry, 1999, 38(38):12280-12287.
[12]	RANKIN T, TALBOT P, LEE E, et al.Abnormal zonae pellucidae in mice lacking ZP1 result in early embryonic loss[J]. Development, 1999, 126(17):3847-3855.
[13]	RANKIN T, FAMILARI M, LEE E, et al.Mice homozygous for an insertional mutation in the ZP3 gene lack a zona pellucida and are infertile[J].Development, 1996, 122(9):2903-2910.
[14]	陈泰来.ZP3基因突变引起空卵泡综合征导致女性不孕[D].济南:山东大学, 2017.CHEN T L.A recurrent missense mutation in ZP3 causes empty follicle syndrome and female infertility[D].Ji'nan:Shandong University, 2017.(in Chinese)
[15]	ZHANG D Z, ZHU L X, LIU Z X, et al.A novel mutation in ZP3 causes empty follicle syndrome and abnormal zona pellucida formation[J].J Assist Reprod Genet, 2021, 38(1):251-259.
[16]	BIANCHI E, WRIGHT G J.Sperm meets egg:the genetics of mammalian fertilization[J].Annu Rev Genet, 2016, 50:93-111.
[17]	WASSARMAN P M, JOVINE L, LITSCHER E S.Mouse zona pellucida genes and glycoproteins[J].Cytogenet Genome Res, 2004, 105(2-4):228-234.
[18]	HARDY C M, TEN HAVE J F, PEKIN J, et al.Contraceptive responses of mice immunized with purified recombinant mouse zona pellucida subunit 3(mZP3) proteins[J].Reproduction, 2003, 126(1):49-59.
[19]	孙君卫, 马向飞, 朱帅鹏, 等.蛋鸡INHBA的克隆分析及其对卵泡发育调控的研究[J].中国家禽, 2019, 41(22):9-15.SUN J W, MA X F, ZHU S P, et al.Cloning and analysis of INHBA in layers and its regulation for follicular development[J].China Poultry, 2019, 41(22):9-15.(in Chinese)
[20]	GUPTA S K.Role of zona pellucida glycoproteins during fertilization in humans[J].J Reprod Immunol, 2015, 108:90-97.
[21]	BLEIL J D, WASSARMAN P M.Galactose at the nonreducing terminus of O-linked oligosaccharides of mouse egg zona pellucida glycoprotein ZP3 is essential for the glycoprotein's sperm receptor activity[J].Proc Natl Acad Sci U S A, 1988, 85(18):6778-6782.
[22]	张昊, 陈芳, 梁振华, 等.家禽卵泡闭锁研究进展[J].湖北畜牧兽医, 2014, 35(10):61-65.ZHANG H, CHEN F, LIANG Z H, et al.Research progress of poultry follicular atresia[J].Hubei Journal of Animal and Veterinary Sciences, 2014, 35(10):61-65.(in Chinese)
[23]	林金杏.局部性促生长因子对鸡卵泡发育的调控及其机理的研究[D].杭州:浙江大学, 2011.LIN J X.Regulation of local growth-promoting factors on follicular development in the laying chickens[D].Hangzhou:Zhejiang University, 2011.(in Chinese)
[24]	金艳梅.颗粒细胞对卵泡发育的影响[J].中国畜牧兽医, 2010, 37(8):69-72.JIN Y M.Effect of granulosa cells on follicle development[J].China Animal Husbandry & Veterinary Medicine, 2010, 37(8):69-72.(in Chinese)
[25]	艾浩.颗粒细胞对机体卵泡发育产生的影响研究分析[C].中国中药杂志2015/专集:基层医疗机构从业人员科技论文写作培训会议论文集.北京:中国中药杂志社, 2016:1.AI H.Research and analysis on the influence of granulosa cells on follicular development[C]//Proceedings of 2015/Special Collection:Proceedings of the Training Conference on Writing Scientific Papers for Employees in Primary Medical Institutions. Beijing:China Journal of Traditional Chinese Medicine, 2016:1.(in Chinese)
[26]	EDWARDS L J, KIND K L, ARMSTRONG D T, et al.Effects of recombinant human follicle-stimulating hormone on embryo development in mice[J].Am J Physiol Endocrinol Metab, 2005, 288(5):E845-E851.
[27]	ABEL M H, WOOTTON A N, WILKINS W, et al.The effect of a null mutation in the follicle-stimulating hormone receptor gene on mouse reproduction[J].Endocrinology, 2000, 141(5):1795-1803.
[28]	DEVERAUX Q L, TAKAHASHI R, SALVESEN G S, et al.X-linked IAP is a direct inhibitor of cell-death proteases[J].Nature, 1997, 388(6639):300-304.
[29]	HAMILTON K J, HEWITT S C, ARAO Y, et al.Estrogen hormone biology[J].Curr Top Dev Biol, 2017, 125:109-146.
[30]	WANG Y F, ASSELIN E, TSANG B K.Involvement of transforming growth factor alpha in the regulation of rat ovarian X-linked inhibitor of apoptosis protein expression and follicular growth by follicle-stimulating hormone[J].Biol Reprod, 2002, 66(6):1672-1680.
[31]	罗茜, 陈伟, 郑春田, 等.家禽卵泡颗粒细胞体外培养方法研究进展[J].动物营养学报, 2016, 28(9):2681-2686.LUO Q, CHEN W, ZHENG C T, et al.The overview of granulosa cells culture in vitro of fowl follicle[J].Chinese Journal of Animal Nutrition, 2016, 28(9):2681-2686.(in Chinese)