牦牛CCND2基因的克隆及其在不同发情时期卵巢中的表达

doi:10.3864/j.issn.0578-1752.2017.13.018

摘要/Abstract

摘要： 【目的】研究牦牛细胞周期蛋白D2（Cyclin D2, CCND2）基因序列特征及其在牦牛不同发情时期卵巢中的表达。【方法】以牦牛为研究对象，提取卵巢的总RNA进行反转录为第一链cDNA，根据GenBank中黄牛CCND2的mRNA序列（GenBank登录号：NM_001076372.1），使用Primer 5.0 软件设计引物。应用PCR方法对CCND2基因扩增克隆，并测序获得CCND2基因完整的CDS区序列及部分5′端和3′端UTR区。使用Protparam、SOPMA、SWISS-MODEL及SMART分别对牦牛CCND2蛋白的理化性质、二级结构、三级结构及结构域进行预测分析，并进行同源性分析和系统进化树构建，利用半定量PCR方法对CCND2基因在牦牛各组织中的表达进行检测；同时采用实时荧光定量PCR技术分析CCND2基因在牦牛不同发情时期（卵泡期、红体期、黄体期）卵巢中的相对表达量。【结果】克隆获得牦牛CCND2基因序列为909 bp，其中包含可编码289个氨基酸残基的长为870 bp的CDS区。与黄牛相比，牦牛CCND2的CDS区存在5个碱基突变，导致其中一个氨基酸改变。牦牛CCND2基因CDs序列与野牦牛、黄牛、印度水牛、绵羊、野猪、马、家犬、猫、人类的进行比对同源性分别是99.54%、99.31%、99.31%、98.16%、94.81%、90.46%、90.80%、91.49%、88.62%，物种之间同源性较高，与进化树分析显示的亲缘关系远近一致，表明CCND2基因编码区在进化中较为保守。对CCND2蛋白预测分析知牦牛CCND2基因编码蛋白的分子式为C₁₄₄₅H₂₃₁₅N₃₇₇O₄₄₀S₁₈，相对分子质量约为32.59kD，理论等电点为4.75，总平均亲水性为-0.107，不稳定指数为44.56，属亲水不稳定的酸性蛋白，该蛋白无跨膜区和信号肽；二级结构主要包含α-螺旋和无规卷曲，且与三级结构分析结果一致。蛋白含位于第26—152位氨基酸处的Cyclin_N和位于第154—257位氨基酸处的Cyclin_C两个结构域。CCND2 基因在牦牛各组织中均有表达，其中在胃、卵巢和脑中表达相对较高；qRT-PCR结果显示在牦牛不同发情时期卵巢中CCND2 mRNA均有表达，且卵泡期卵巢中的表达水平最高（P＜0.01），在红体期和黄体期卵巢中表达水平无差异（P＞0.05）。【结论】成功获得了牦牛CCND2基因的CDS区全长序列和组织表达谱，对其进行生物信息分析及蛋白功能结构分析为该基因在牦牛繁殖调控中的进一步研究提供理论依据；在牦牛不同发情时期卵巢中CCND2 mRNA表达水平存在差异，可能与卵泡发育过程中颗粒细胞增殖分化、卵泡发生波及卵巢内分泌有关，为进一步研究牦牛卵巢发育机制及改善牦牛繁殖效率提供了基础资料。

关键词: 牦牛, CCND2, 克隆, 发情周期, 卵巢, 表达

Abstract: 【Objective】The aim of the study was to investigate the sequence characteristics and expression level of cyclin D2(CCND2) in yak ovary during different periods of estrus. 【Method】 Total RNA of yak ovary were extracted and reverse transcribed into cDNA. Specific primers were designed according to mRNA sequence of cattle CCND2 gene in the GenBank(GenBank accession No: NM_001076372.1) by software Primer 5.0. The CDs region and part of 5′UTR and 3′UTR in yak CCND2 gene were cloned from yak ovary by polymerase chain reaction(PCR) amplification and sequencing technology. The physicochemical properties, secondary structure, tertiary structure, domain, homology were analyzed and phylogenetic tree of CCND2 was constructed by online software like Protparam, SOPMA, SWISS-MODEL and SMART. Semi-quantitative PCR was used to examine the expression profiles of CCND2 gene in different tissues. Quantitative real-time PCR (qRT-PCR) was used to detect the expression level of CCND2 gene in the ovary during different periods of estrus (ovarian follicular phase, red body phase and luteal phase).【Result】The 909 bp length sequences of CCND2 gene in yak, including 870 bp length CDS region encoding 289 amino acids, were obtained. Alignment with cattle CCND2 gene CDS and amino acid, five bases mutations and one amino acid mutation were found. The CDs homology of CCND2 compared yak to wild yak, cattle, Indian buffalo, sheep, wild boar, horse, dog, cat and human was 99.54%, 99.31%, 99.31%, 98.16%, 94.81%, 90.46%, 90.80%, 91.49% and 88.62%, respectively. There was a high homology among different species, and phyletic evolution was the same as their genetic relationship. The research indicated that the CCND2 gene CDs was conservative in the course of evolution. The formula of protein encoded by CCND2 gene in yak was C₁₄₄₅H₂₃₁₅N₃₇₇O₄₄₀S₁₈, and the molecular weight was 32.59 kD, the theory isoelectric point was 4.75, the grand average of hydroparthicity was -0.107, and the instability index was 44.56. It was an unstable, soluble and acidic protein without transmembrane regions and signal peptide. Random coil and α-helix were mainly in the secondary structure of CCND2, which were the same as the tertiary structure of CCND2 protein. The amino acids sequence of CCND2 contains Cyclin_N domain in the 26-152 amino acids and Cyclin_C domain in the 154-257 amino acids. The CCND2 gene was expressed in various tissues of yak, and relatively higher in stomach, ovary and brain. qRT-PCR results showed that the mRNA expression level of CCND2 in ovary of follicular phase was the highest (P＜0.01), and no difference between the red body phase and luteal phase (P＞0.05).【Conclusion】The complete CDs of CCND2 gene was successfully cloned and the tissues expression profiles were clarified, and its bioinformatics and encoded protein function and structure were investigated. These provide a theoretical basis for further study of CCND2 gene in regulation of yak reproduction. The different expressions of CCND2, in different periods of estrus in yak, might be related to proliferation and differentiation of granulose cells, development process of follicular wave, and ovarian endocrine. These indicated that CCND2 plays an important role during the reproductive cycle, and these results of study will provide basic information for further study on the ovarian development mechanism and improving reproductive competence in yak.

Key words: yak, CCND2, cloning, estrous cycle, ovary, expression

石仙，熊显荣，兰道亮，陈伟明，胡嘉嘉，蔡雯祎，李键. 牦牛CCND2基因的克隆及其在不同发情时期卵巢中的表达[J]. 中国农业科学, 2017, 50(13): 2604-2613.

SHI Xian, XIONG XianRong, LAN DaoLiang, CHEN WeiMing, HU JiaJia, CAI WenYi, LI Jian. Cloning and Expression Analysis of CCND2 Gene in Yak Ovaries During Different Periods of Estrus[J]. Scientia Agricultura Sinica, 2017, 50(13): 2604-2613.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2017.13.018

https://www.chinaagrisci.com/CN/Y2017/V50/I13/2604

参考文献

[1] 兰道亮, 熊显荣, 位艳丽, 徐通, 钟金城, 字向东, 王永, 李键. 基于 RNA-Seq 高通量测序技术的牦牛卵巢转录组研究:进一步完善牦牛基因结构及挖掘与繁殖相关新基因. 中国科学, 2014, 44(3): 307-317.

LAN D L, XIONG X R, WEI Y L, XU T, ZHONG J C, ZI X D, WANG Y, LI J. RNA-Seq analysis of yak ovary: improving yak gene structure information and mining reproduction-related genes. Scientia Sinica, 2014, 44(3): 307-317. (in Chinese)

[2] MALUMBRES M, BARBACID M. Cell cycle, CDKs and cancer: a changing paradigm. Nature Reviews Cancer, 2009, 9(3): 153-166.

[3] ZHANG Q, SAKAMOTO K, WAGNER K U. D-type cyclins are important downstream effectors of cytokine signaling that regulate the proliferation of normal and neoplastic mammary epithelial cells. Molecular and Cellular Endocrinology, 2014, 382(1): 583-559.

[4] COQUERET O. Linking cyclins to transcriptional control. Gene, 2002, 299(1/2): 35-55.

[5] MOTOKURA T, BLOOM T, KIM H G, JÜPPNER H, RUDERMAN J V, KRONENBERG H M, ARNOLD A. A novel cyclin encoded by a bcl1-linked candidate oncogene. Nature, 1991, 350 (6318): 512-515.

[6] SICINSKI P, DONAHER J L, GENG Y, PARKER S B, GARDNER H, PARK M Y, ROBKER R L, RICHARDS J S, MCGINNIS L K, BIGGERS J D, EPPIG J J, BRONSON R T, ELLEDGE S J, WEINBERG R A. Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis. Nature, 1996, 384(6608): 470-474.

[7] PARK Y, MAIZELS E T, FEIGE Z J, ALAM H, PETERS C A, WOODRUF T K, UNTERMAN T G, LEE E J, JAMESON J L, HUNZICKER-DUN M. Induction of cyclin D2 in rat granulosa cells requires FSH-dependent relief from FOX01 repression coupled with positive signals from Smad. Journal Biological Chemistry, 2005, 280(10): 9135-9148.

[8] SHIMIZU T, HIRAI Y, MIYAMOTO A. Expression of cyclins and cyclin-dependent kinase inhibitors in granulosa cells from bovine ovary. Reproduction in Domestic Animals, 2013, 48(5): e65-e69.

[9] LEI X C, CUI K Q , LI Z P, SU J, JIANG J R, ZHANG H H, LIU Q Y, SHI D. BMP-1 participates in the selection and dominance of buffalo follicles by regulating the proliferation and apoptosis of granulosa cells. Theriogenology, 2016, 85(5): 999-1012.

[10] SUMMERS A F, POHLMEIER W E, SARGENT K M, COLE B D, VINTON R J, KURZ S G, MCTEE R M, CUSHMAN R A, CUPP A S, WOOD J R. Altered theca and cumulus oocyte complex gene expression, follicular arrest and reduced fertility in cows with dominant follicle follicular fluid androgen excess. PLoS ONE, 2014, 9(10): e110683.

[11] ZHANG Q, SUN H X, JIANG Y, DING L J, WU S G, FANG T, YAN G J, HU Y L. MicroRNA-181a suppresses mouse granulosa cell proliferation by targeting activin receptor IIA. PLoS ONE, 2013, 8 (3): e59667.

[12] OSHIMO Y, NAKAYAMA H, ITO R, KITADAI Y, YOSHIDA K, CHAYAMA K, YASUI W. Promoter methylation of cyclin D2 gene in gastric carcinoma. Intertional Journal Oncology. 2003, 23(6): 1663-1670.

[13] MORITO N, YOH K, MAEDA A, NAKANO T, FUJITA A, KUSAKABE M, HANADA M, KUDO T, YAMAGATA K, TAKAHASHI S. A novel transgenic mouse model of the human multiple myeloma chromosomal translocationt. Cancer Research. 2011, 71(2): 339-348.

[14] SONG C L, REN J H, RAN L K, LI Y G, LI X S, CHEN X, LI W Y, HUANG A L, CHEN J. Cyclin D2 plays a regulatory role in HBV replication. Virology, 2014, 8(462/463): 149-157.

[15] CHAGANTI R S, HOULDSWORTH J. Genetics and biology of adult human male germ cell tumors. Cancer Research, 2000, 60(6): 1475-1482.

[16] ANTTONEN M, PIHLAJOKI M, ANDERSSON N, GEORGES A, LHOTO D, VATTULAINEN S, FARKKILA A, UNKILA-KALLIO L, VEITIA R A, HEIKINHEIMO M. FOXL2, GATA4 and SMAD3 co-operatively modulate gene expression, cell viability and apoptosis in ovarian granulosa cell tumor cells. PLoS ONE, 2014, 9(1): e85545.

[17] SHAFIEE M N, MALIK D A, YUNOS R I, ATIMO W, OMAR M H, GHANI N A, HATTA A Z, SEEDHOUSE C, Chapman C, MOKHTAR N M. The effect of Metformin on endometrial tumor-regulatory genes and systemic metabolic parameters in polycystic ovarian syndrome--a proof-of-concept study. Gynecological Endocrinology, 2015, 31(4): 286-290.

[18] SAHARE M, OTOMO A, KOMATSU K, MINAMI N, YAMADA M, IMAI H. The role of signaling pathways on proliferation and self-renewal of cultured bovine primitive germ cells. Reproduction Medical Biology, 2015,14(1): 17-25.

[19] LEE J, KANATSU-SHINOHARA M, MORIMOTO H, KAZUKI Y, TAKASHIMA S. Genetic reconstruction of mouse spermatogonial stem cell self-renewal in vitro by Ras-cyclin D2 activation. Cell Stem Cell, 2009, 5(1): 76-86.

[20] ROBKER R L, RIEHARDS J S. Hormone-induced proliferation and differentiation of granulose cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27Kipl. Molecular Endocrinology, 1998, 12(7): 924-940.

[21] WIANNY F, REAL F X, MUMMERY C L, VAN-ROOIJEN M, LAHTI J, SAMARUT J, SAVATIER P. G1-phase regulators, cyclin D1,cyclin D2, and cyclin D3:up-regulation at gastrulation and dynamic expression during neurulation. Developmental Dynamics, 1998, 212(1): 49-62.

[22] ADAMS G P, JAISWAL R, SINGH J, MALHI P. Progress in understanding ovarian follicular dynamics in cattle. Theriogenology, 2008, 69(1): 72-80.

[23] 郑红飞, 潘阳阳, 刘鹏刚, 彭秀梅, 崔燕, 余四九. 牦牛Tp53基因特征分析及其在不同发情时期卵巢中的表达. 华北农学报, 2015, 30(5) : 93-99.

ZHENG H F, PAN Y Y, LIU P G, PENG X M, CUI Y, YU S J. Characteristics of yak tumor protein p53 Gene(Tp53) and its expression in different periods of estrus ovaries. Acta Agriculturae Boreali-Sinica, 2015, 30(5): 93-99. (in Chinese )

[24] MASTUDA-MINEHATA F, INOUE N, GOTO Y, MANABE N. The regulation of ovarian granulosa cell death by pro- and anti-apoptotic molecules. The Journal of Reproduction and Development, 2006, 52(6): 695-705.

[25] MCRAE R S, JOHNSTON H M, MIHM M, O'SHAUGHNESSY P J. Changes in mouse granulosa cell gene expression during early luteinization. Endocrinology, 2005, 146(1): 309-317.

[26] HAMEL M, DUFORT I, ROBERT C, GRAVEL C, LEVEILLE M C, LEADER A, SIRARD M A. Identification of differentially expressed markers in human follicular cells associated with competent oocytes. Human Reproduction, 2008, 23(5): 1118-1127.

[27] SHIINA H, MATSUMOTO T, SATO T, IGARASI K, MIYAMOTO J, TAKEMASA S, SAKARI M, TAKADA I, NAKAMURA T, METZGER D, CHAMBON P, KANNO J, YOSHIKAWA H, KATO S. Premature ovarian failure in androgen receptor-deficient mice. Proceedings of the National Academy of Sciences of the U nited States of America, 2006, 103(1): 224-229.

[28] NEGANOVA I, AI-QASSAB H, HEFFRON H, SELMAN C, CHOUDHURY A I, LINGARD S J, DIAKONOV I, PATTERSON M, GHATEI M, BLOOM S R, FRANKS S, HUHTANIEMII, HARDY K, WITHERS D J. Role of central nervous system and ovarian insulin receptor substrate 2 signaling in female reproductive function in the mouse. Biology Reproduction, 2007, 76(6): 1045-1053.

[29] GILCHRIST R B, LANE M, THOMPSON J G. Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Human Reproduction Update, 2008, 14(2):159-177.

[30] RJCHARDS J S, RUSSELL D L, OCHSNER S, ESPEY L L. Ovulation: new dimensions and new regulators of the inflammatory- like response. Annual Review Physiology, 2002, 64: 69-92.

[31] KAYANI A R, GLISTER C, KNIGHT P G. Evidence for an inhibitory role of bone morphogenetic protein(s) in the follicular-luteal transition in cattle. Reproduction, 2009, 137(1): 67-78.

[32] KWINTKIEWICE J, GIUDICE L C. The interplay of insulin-like growth factors, gonadotropins, and endocrine disruptors in ovarian follicular development and function. Seminars Reproductive Medicine,2009, 27(1): 43-51.

[33] YOSHIOKA S, ABE H, SAKUMOTO R, OKUDA K. Proliferation of luteal Steroidogenic Cells in Cattle. PLoS ONE, 2013, 8(12): e84186.

[34] GINTHER O J, BERGFELT D R, KULICK L J, KOT K. Selection of the dominant follicle in cattle: establishment of follicle deviation in less than 8 hours through depression of FSH concentrations. Theriogenology, 1999, 52(6): 1079-1093.

[35] 应诗家, 彭中友, 李燕，蔡柳萍, 施振旦. 绵羊黄体期卵巢类固醇激素调节基因的表达研究. 畜牧兽医学报, 2013, 44(11): 1775-1780.

YING S J, PENG Z Y, LI Y, CAN L P, SHI Z D. Study on ovarian sterol-regulatory genes expression in sheep during luteal phase. Acta Veterinaria et Zootechnica Sinica, 2013, 44(11): 1775-1780. (in Chinese)

[36] 王锋. 动物繁殖学. 北京: 中国农业大学出版社, 2012.

WANG F. Animal Reproduction. Press, 2012. (in Chinese) Beijing: China Agricultural University

[37] HILLIER S G, WHITELAW P F, SMYTH C D. Follicular oestrogen synthesis: the 'two-cell, two-gonadotrophin' model revisited. Molecular Cellular Endocrinology, 1994, 100(1/2): 51-54.

[38] HERMANDEZ-GONZALEZ I, GONZALEZ-ROBAYNA I, SHIMADA M, WAYNE C M, OCHSNER S A, WHITE L, RICHARDS J S. Gene expression profiles of cumulus cell oocyte complexes during ovulation reveal cumulus cells express neuronal and immune-related genes: does this expand their role in the ovulation process? Molecular Endocrino, 2006, 20(6): 1300-1321.logy

[39] 李泰云, 韩小康, 丁彦红, 王树迎, 王丽, 石中强. 沂蒙黑山羊发情周期不同阶段卵巢中FSHR和LHR基因表达规律的研究. 中国兽医科学, 2011, 41(09): 954-959.

LI T Y, HAN X K, DING Y H, WANG S Y, WANG L, SHI Z Q. Expression of FSHR gene and LHR gene in the ovary of Yimeng Black goat in different stages of estrous cycle. Chinese Veterinary Science, 2011, 41(09): 954-959. (in Chinese)

[40] 葛利江, 徐常林, 石瑞青, 谭景和, 罗明久. 德州驴发情周期中卵泡动力学和激素变化规律的研究. 畜牧兽医学报, 2011, 42(9): 1256-1263.

GE L J, XU C L, SHI R Q, TAN J H, LUO M J. Ovarian follicular and hormonal dynamics in dezhou jenny during estrous cycle monitored by real-time ultrasonography. Acta Veterinaria et Zootechnica Sinica, 2011, 42(9): 1256-1263. (in Chinese)