牦牛跨膜附睾蛋白1基因的克隆及其在卵丘卵母细胞复合体中的表达分析

doi:10.16431/j.cnki.1671-7236.2021.04.015

摘要/Abstract

摘要： 试验旨在分析牦牛跨膜附睾蛋白1（transmembrane epididymal protein 1，TEDDM1）基因的分子特征及其在卵泡发育中的时序表达特性。研究分别收集发情期牦牛大卵泡（φ≥8 mm）、中卵泡（φ=5~7 mm）和小卵泡（φ≤3 mm）中的卵丘-卵母细胞复合体（COCs）；提取总RNA并反转录，对TEDDM1基因CDS区全序列进行克隆测序，用生物信息学软件分析其编码蛋白分子特征；以牛GAPDH基因为内参，采用实时荧光定量PCR技术分析TEDDM1基因在卵泡发育不同时期的表达水平。结果表明，牦牛TEDDM1基因CDS区全长903 bp，共编码300个氨基酸，与野牦牛、牛、野牛、绵羊和水牛氨基酸序列有极高的相似性，在进化中较为保守；牦牛TEDDM1蛋白为非分泌型不稳定疏水蛋白，共存在7个螺旋结构，整条肽链7次横跨胞膜，属于典型的G蛋白偶联受体；其潜在的磷酸化位点共21个，其中酪氨酸、苏氨酸和丝氨酸磷酸化位点分别为1、3和17个，仅存在1个N-糖基化位点，无O-糖基化位点；存在未知功能结构域蛋白家族（domains of unknow function protein families，DUFs）716结构域，且该结构域涵盖多个跨膜区域；实时荧光定量PCR检测结果发现，中卵泡中TEDDM1基因表达水平显著高于大卵泡和小卵泡（P<0.05），而大卵泡与小卵泡的表达水平差异不显著（P>0.05）。本研究分析了牦牛TEDDM1基因序列及其在牦牛COCs中的表达特性，为进一步揭示该基因在牦牛卵泡发育过程中的调控作用奠定了理论基础。

关键词: 牦牛; 卵泡; 跨膜附睾蛋白1(TEDDM1)基因; 克隆; 实时荧光定量PCR

Abstract: The aim of this study was to analyze the molecular characteristics and the expression level of transmembrane epididymal protein 1 (TEDDM1) gene during follicle development of the yak.In this study,cumulus-oocyte complexes (COCs) were collected from small follicles (φ≤3 mm),medium follicles (φ=5-7 mm) and large follicles (φ≥8 mm) of yak during estrus,and total RNA was extracted and reverse transcription was conducted.The complete sequence of the CDS region of the TEDDM1 gene was sequenced.Bioinformatics soft-wares were used to analyze the molecular characteristics of CDS region sequence structure.Using GAPDH as reference gene,Real-time quantitative PCR was performed to reveal expression level of TEDDM1 in small,medium and large follicles.The results showed that the CDS region of yak TEDDM1 gene was 903 bp,encoding 300 amino acid,which was highly similar to Bos mutus,Bos taurus,Bison,Ovis and Bubalus bubalis,and was conserved in evolution.Yak TEDDM1 protein was a non-secretory unstable hydrophobic protein with seven helix structures,and the whole peptide chain crosses the cell membrane seven times,which was a typical G protein-coupled receptor.Twenty-one potential phosphorylation sites were distributed in the protein,including 1 tyrosine,3 threonine and 17 serine phosphorylation sites,only one N-glycosylation site and no O-glycosylation prediction site.Functional domain analysis showed that unknown functional domain protein family 716 domain existed in TEDDM1 protein,covering multiple transmembrane regions.Result of Real-time quantitative PCR analysis showed that the expression level of TEDDM1 in medium follicles was significantly higher than that in large follicles and small follicles (P<0.05),but there was no significant difference between large follicles and small follicles (P>0.05).The study provided the sequence of yak TEDDM1 gene and its expression characteristics in yak COCs,laid a theoretical foundation for further revealing the regulatory role of the yak TEDDM1 gene in follicular development.

Key words: yak; follicles; transmembrane epididymal protein 1(TEDDM1) gene; cloning; Real-time quantitative PCR

中图分类号:

S823

李恒, 字向东. 牦牛跨膜附睾蛋白1基因的克隆及其在卵丘卵母细胞复合体中的表达分析[J]. 中国畜牧兽医, 2021, 48(4): 1284-1293.

LI Heng, ZI Xiangdong. Cloning and Expression in Cumulus-oocyte Complexes of Yak TEDDM1 Gene[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(4): 1284-1293.

参考文献

[1] ZI X D,ZHONG G H,WEN Y L,et al.Growth performance,carcass composition and meat quality of Jiulong-yak (Bos grunniens)[J].Asian-Australasian Journal of Animal Science,2004,17:410-441.
[2] QIU Q,ZHANG G,MA T,et al.The yak genome and adaptation to life at high altitude[J].Nature Genetics,2012,44(8):946-949.
[3] 罗晓林.中国牦牛[M].成都:四川科学技术出版社,2019. LUO X L.China Yak[M].Chengdu:Sichuan Science and Technology House,2019.(in Chinese)
[4] ZI X D.Reproduction in female yaks (Bos grunniens) and opportunities for improvement[J].Theriogenology,2003,59(5-6):1303-1312.
[5] ZI X D,YIN R H,CHEN S W,et al.Developmental competence of embryos derived from reciprocal in vitro fertilization between yak (Bos grunniens) and cattle (Bos taurus)[J].Journal of Reproduction and Development,2009,55:480-483.
[6] YANG R F,XIONG X R,ZIX D.Effect of cysteine,insulin-like growth factor-1 and epidermis growth factor during in vitro oocyte maturation and in vitro culture of yak-cattle crossbred embryos[J].Journal of Applied Animal Research,2019,47(1):463-466.
[7] 王琴,熊燕,字向东.体外成熟液中添加PAF对牦牛卵母细胞发育能力及基因表达的影响[J].畜牧兽医学报,2020,51(3):514-523. WANG Q,XIONG Y,ZI X D.Effects of PAF supplementation during in vitro maturation medium on yak oocyte development competence and gene expression[J].Acta Veterinaria et Zootechnica Sinica,2020,51(3):514-523.(in Chinese)
[8] YAMAZAKI K,ADACHI T,SATO K,et al.Identification and characterization of novel and unknown mouse epididymis-specific genes by complementary DNA microarray technology[J].Biology of Reproduction,2006,75(3):462-468.
[9] ZIMIN A V,DELCHER A L,FLOREA L,et al.A whole-genome assembly of the domestic cow,Bos taurus[J].Genome Biology,2009,10(4):R42.
[10] LIU Y,LUO J,DOU J,et al.The sequence and de novo assembly of the wild yak genome[J].Science Letter,2020,7(1):66.
[11] 李鹏飞.牛卵泡可卡因-苯丙胺调节转录肽(CART)受体的筛选[D].太谷:山西农业大学,2014. LI P F.Screening of cocaine-and amphetamine-regulate transcript peptide (CART) receptor of cattle follicle[D].Taigu:Shanxi Agricultural University,2014.(in Chinese)
[12] SMITH G W,SEN A,FOLGER J K,et al.Putative role of cocaine- and amphetamine-regulated transcript (CARTPT) in dominant follicle selection in cattle[J].Society of Reproduction and Fertility Supplement,2010,67:105-117.
[13] LV L H,JIMENEZ-KRASSEL F,SEN A,et al.Evidence supporting a role for cocaine-and amphetamine-regulated transcript (CARTPT) in control of granulosa cell estradiol production associated with dominant follicle selection in cattle[J].Biology of Reproduction,2009,81(3):580-586.
[14] 朱芷葳,郝庆玲,侯淑宁,等.牛卵泡TEDDM1表达特点及其功能分析[J].畜牧兽医学报,2019,50(6):1189-1197. ZHU Z W,HAO Q L,HOU S N,et al.Expression characteristic and function analysis of TEDDM1 in bovine follicle[J].Acta Veterinaria et Zootechnica Sinica, 2019,50(6):1189-1197.(in Chinese)
[15] SINNER M K,SCHMIDT M,SAVENKOVA M I,et al.Regulation of granulosa and theca cell transcriptomes during ovarian antral follicle development[J].Molecular Reproduction and Develop-ment,2008,75(9):1457-1472.
[16] PFAFFL M W.A new mathematical model for relative quantification in Real-time RT-PCR[J].Nucleic Acids Research,2001,29(9):e45.
[17] 郭松长,刘建全,祁得林,等.牦牛的分类学地位及起源研究:mtDNA D-loop序列的分析[J].兽类学报,2006,4:325-330. GUO S C,LIU J Q,QI D L,et al.Taxonomic placement and origin of yaks:Implications from analyses of mtDNA D-loop fragment sequences[J].Acta Theriologica Sinica,2006,4:325-330.(in Chinese)
[18] GUPTA R,BRUNAK S.Prediction of glycosylation across the human proteome and the correlation to protein function[J].Pacific Symposium Biocomputing,2002,7:310-322.
[19] PUNTA M,COGGILL P C,EBERHARDT R Y,et al.The Pfam protein families database[J].Nucleic Acids Research,2012,40:D290-301.
[20] HOU C,TIAN W,KLEIST T,et al.DUF221 proteins are a family of osmosensitive calcium-permeable cation channels conserved across eukaryotes[J].Cell Research,2014,24(5):632-635.
[21] PAVY N,BOYLE B,NELSON C,et al.Identification of conserved core xylem gene sets:Conifer cDNA microarray development,transcript profiling and computational analyses[J].New Phytologist,2008,180(4):766-786.
[22] YOSHIDA A,SUZAKI T,TANAKA W,et al.The homeotic gene long sterile lemma (G1) specifies sterile lemma identity in the rice spikelet[J].Proceedings of National Academy Sciences of the United States of America,2009,106(47):20103-20108.
[23] SUN D,WANG L,YANG L,et al.Ectopic expression of a proteinase inhibitor I4(MtPiI4) gene from medicago truncatula confers plant resistance to Pseudomonas syringae pv.tomato DC3000[J].Plant Molecular Biology Reporter,2015,33(6):1686-1696.
[24] GERBER P A,HEVEZI P,BUHREN B A,et al.Systematic identification and characterization of novel human skin-associated genes encoding membrane and secreted proteins[J].PLoS One.2013,8(6):e63949.
[25] JUSTESEN J,HARTMANN R,KJELDGAARD N O.Gene structure and function of the 2'-5'-oli goad-enylate synthetase family[J].Celluar and Molecular Life Sciences, 2000,57(11):1593-1612.
[26] 杨世华.几种生殖激素对体外培养的牦牛卵泡颗粒细胞凋亡的影响[D].兰州:甘肃农业大学,2002. YANG S H.Effects of several reproductive hormones on apoptosis of ovarian granulose cells of yaks in vitro[D].Lanzhou:Gansu Agricultural University,2002.(in Chinese)
[27] BASINI G,BAIONI L,BUSSOLATI S,et al.Prolactin is a potential physiological modulator of swine ovarian follicle function[J].Regulatory Peptides,2014,189:22-30.
[28] LAVOIEH A.Transcriptional control of genes mediating ovarian follicular growth,differentiation,and steroidogenesis in pigs[J].Molecular Reproduc-tion and Development,2017,84(9):788-801.
[29] WOO M M,TAI C J,KANG S K,et al.Direct action of melatonin in human granulosa-luteal cells[J].The Journal of Clinical Endocrinology and Metabolism,2001,86(10):4789-4797.
[30] BASINI G,BUSSOLATI S,CICCIMARRA R,et al.Melatonin potentially acts directly on swine ovary by modulating granulosa cell function and angiogenesis[J].Reproduction Fertility and Development,2017,29(12):2305-2312.
[31] SEN A,BETTEGOWDA A,JIMENEZ-KRASSEL F,et al.Cocaine- and amphetamine-regulated transcript regulation of follicle-stimulating hormone signal transduction in bovine granulosa cells[J].Endocrinology,2007,148(9):4400-4410.
[32] SEN A,LV L,BELLO N,et al.Cocaine- and amphetamine-regulated transcript accelerates termina-tion of follicle-stimulating hormone-induced extracellularly regulated kinase 1/2 and Akt activation by regulating the expression and degradation of specific mitogen-activated protein kinase phosphatases in bovine granulosa cells[J].Molecular Endocrinology,2008,22(12):2655-2676.
[33] 阿尔祖古丽·阿依丁.大豆异黄酮对牦牛卵巢颗粒细胞增殖与凋亡的影响[D].兰州:西北民族大学,2020. AERZUGULI AYIDING.Effects of soy isoflavones on the proliferation ang apoptosis of yak ovary granulosa cells[D].Lanzhou:Northwest Minzu University,2020.(in Chinese)
[34] EPPIG J J.Reproduction:Oocytes call,granulosa cells connect[J].Current Biology,2018,28(8):R354-R356.
[35] 熊显荣,王艳,李键,等.SIRT1对牦牛卵母细胞体外成熟与老化的影响[J].畜牧兽医学报,2019,50(12):2440-2448. XIONG X R,WANG Y,LI J,et al.Effects of SIRT1 on the in vitro maturation and aging of yak oocytes[J].Acta Veterinaria et Zootechnica Sinica,2019,50(12):2440- 2448.(in Chinese)
[36] AUDET M,BOUVIER M.Restructuring G-protein- coupled receptor activation[J].Cell,2012,151(1):14-23.
[37] 郭振伟.褪黑素对水牛卵母细胞体外成熟的影响及其机制的探究[D].南宁:广西大学,2016. GUO Z W.A preliminary study on the effect of melatonin on the in vitro maturation of buffalo oocytes and its mechanism[D].Nanning:Guangxi University,2016.(in Chinese)
[38] LIN T,LEE J E,KANG J W,et al.Melatonin supplementation during prolonged in vitro maturation improves the quality and development of poor-quality porcine oocytes via anti-oxidative and anti-apoptotic effects[J].Molecular Reproduction and Development,2018,85(8-9):665-681.
[39] YANG L,WANG Q,CUI M,et al.Effect of melatonin on the in vitro maturation of porcine oocytes,development of parthenogenetically activated embryos,and expression of genes related to the oocyte developmental capability[J].Animals (Basel),2020,10(2):209.
[40] TIAN X,WANG F,HE C,et al.Beneficial effects of melatonin on bovine oocytes maturation:A mechanistic approach[J].Journal of Pineal Research,2014,57(3):239-247.