牦牛和犏牛睾丸组织DDX4基因mRNA表达水平 与启动子区甲基化

doi:10.3864/j.issn.0578-1752.2013.03.020

摘要/Abstract

摘要： 【目的】了解牦牛和犏牛睾丸组织中DDX4基因mRNA表达水平和启动子区甲基化状态。【方法】采用real-time PCR技术检测牦牛和犏牛睾丸组织DDX4基因mRNA表达水平，采用克隆测序技术获得牦牛和犏牛DDX4基因启动子区序列，采用亚硫酸氢钠测序法检测牦牛和犏牛睾丸组织中DDX4基因启动子区甲基化状态。【结果】牦牛睾丸组织中DDX4基因mRNA表达水平极显著高于犏牛（P＜0.01）；牦牛和犏牛DDX4基因启动子区1 370 bp，含有核心启动子区（251 bp）和CpG岛（918 bp）。犏牛睾丸组织中DDX4基因启动子区甲基化水平（86.5%）极显著高于牦牛（67.0%）（P＜0.01）。【结论】牦牛睾丸组织DDX4基因表达水平极显著高于犏牛，获得了牦牛和犏牛DDX4基因启动子区序列，且犏牛睾丸组织中DDX4基因启动子区甲基化水平极显著高于牦牛（P＜0.01）。

关键词: 牦牛 , 犏牛 , DDX4基因 , 表达水平 , 甲基化

Abstract: 【Objective】The study was aimed to investigate the expression levels and promoter methylation status of DDX4 gene in the testes of yak and cattle-yak. 【Method】Real-time PCR was employed to examine the expression levels in the testes of yak and cattle-yak. Clone sequencing was applied to acquire the promoter sequences of DDX4 gene of yak. The promoter methylation status of DDX4 gene in the testes of yak and cattle-yak was analyzed by using sodium bisulfite sequencing.【Result】The mRNA expression level of DDX4 gene in the testis of yak was significantly higher than that of cattle-yak (P＜0.01). The length of the promoter sequences of the DDX4 gene was 1 370 bp, which included a core promoter (251 bp) and a CpG island (918 bp). The promoter methylation level of DDX4 gene in the testes of cattle-yaks (86.5%) was significantly higher than that in yak (67.0%) (P＜0.01). 【Conclusion】Results of the study showed that the testicular expression level of DDX4 might be involved in cattle-yak male sterility, and the promoter hypermethylation was correlated to the lower expression of DDX4 in the testes of cattle-yak.

Key words: yak , cattle-yak , DDX4 gene , expression level , methylation

周阳, 骆骅, 李伯江, 贾超, 谢庄, 赵兴波, 钟金城, 李齐发. 牦牛和犏牛睾丸组织DDX4基因mRNA表达水平与启动子区甲基化[J]. 中国农业科学, 2013, 46(3): 630-638.

ZHOU Yang, LUO Hua, LI Bo-Jiang, JIA Chao, XIE Zhuang, ZHAO Xing-Bo, ZHONG Jin-Cheng, LI Qi-Fa. mRNA Expression Level and Promoter Methylation of DDX4 Gene in Testes of Yak and Cattle-Yak[J]. Scientia Agricultura Sinica, 2013, 46(3): 630-638.

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.0578-1752.2013.03.020

https://www.chinaagrisci.com/CN/Y2013/V46/I3/630

参考文献

[1]Lee H Y, Chou J Y, Cheong L, Chang N H, Yang S Y, Leu J Y. Incompatibility of nuclear and mitochondrial genomes causes hybrid sterility between two yeast species. Cell, 2008, 135(6): 1065-1073.

[2]Douglas K C, Halbert N D, Kolenda C, Childers C, Hunter D L, Derr J N. Complete mitochondrial DNA sequence analysis of Bison bison and bison-cattle hybrids: function and phylogeny. Mitochondrion, 2011, 11(1): 166-175.

[3]Zhang Q B, Li J H, Li Q F, Li X F, Liu Z S, Song D W, Xie Z. Cloning and characterization of the gene encoding the bovine BOULE protein. Molecular Genetics and Genomics, 2009, 281(1): 67-75.

[4]Liu Z S, Li Q F, Pan Z X, Qu X G, Zhang C X, Xie Z. Comparative analysis on mRNA expression level and methylation status of DAZL gene between cattle-yaks and their parents. Animal Reproduction Science, 2011, 126(3/4): 258-264.

[5]潘增祥, 刘振山, 李隐侠, 于莎莉, 李明桂, 谢庄, 李齐发. 犏牛及其亲本睾丸组织中印记基因SNRPN DMR甲基化与mRNA表达差异研究. 中国农业科学, 2010, 43(22): 4709-4716.

Pan Z X, Liu Z S, Li Y X, Yu S L, Li M G, Li Q F , Xie Z. Difference of SNRPN methylation status and its mRNA expression in testes between cattle-yaks and their parents. Scientia Agricultura Sinica, 2010, 43(22): 4709-4716. (in Chinese)

[6]Schupbach T, Wieschaus E. Germline autonomy of maternal-effect mutations altering the embryonic body pattern of Drosophila. Developmental Biology, 1986, 113(2): 443-448.

[7]Hickford D E, Frankenberg S, Pask A J, Shaw G, Renfree M B. DDX4 (VASA) is conserved in germ cell development in marsupials and monotremes. Biology of Reproduction, 2011, 85(4): 733-743.

[8]Reynolds N, Collier B, Maratou K, Bingham V, Speed R M, Taggart M, Semple C A, Gray N K, Cooke H J. Dazl binds in vivo to specific transcripts and can regulate the pre-meiotic translation of Mvh in germ cells. Human Molecular Genetics, 2005, 14(24): 3899-3909.

[9]Gustafson E A, Yajima M, Juliano C E, Wessel G M. Post-translational regulation by gustavus contributes to selective Vasa protein accumulation in multipotent cells during embryogenesis. Developmental Biology, 2011, 349(2): 440-450.

[10]Soultanas P, Dillingham M S, Velankar S S, Wigley D B. DNA binding mediates conformational changes and metal ion coordination in the active site of PcrA helicase. Journal of Molecular Biology, 1999, 290(1): 137-148.

[11]Fujiwara Y, Komiya T, Kawabata H, Sato M, Fujimoto H, Furusawa M, Noce T. Isolation of a DEAD-family protein gene that encodes a murine homolog of Drosophila vasa and its specific expression in germ cell lineage. Proceedings of the National Academy of Sciences of the United States of America, 1994, 91(25): 12258-12262.

[12]Anderson R A, Fulton N, Cowan G, Coutts S, Saunders P T. Conserved and divergent patterns of expression of DAZL, VASA and OCT4 in the germ cells of the human fetal ovary and testis. BMC Developmental Biology, 2007, 7: 136-135.

[13]Toyooka Y, Tsunekawa N, Takahashi Y, Matsui Y, Satoh M, Noce T. Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development. Mechanisms of Development, 2000, 93(1/2): 139-149.

[14]Singleton M R, Dillingham M S, Wigley D B. Structure and mechanism of helicases and nucleic acid translocases. Annual Review of Biochemistry, 2007, 76: 23-50.

[15]Tanaka S S. The mouse homolog of Drosophila Vasa is required for the development of male germ cells. Genes and Development, 2000, 14(7): 841-853.

[16]李贤, 李齐发, 赵兴波, 徐洪涛, 顾垚, 朱翔, 谢庄, 刘红林. 牦牛和犏牛Dmc1基因序列分析及睾丸组织转录水平研究. 中国农业科学, 2010, 43(15): 3221-3229.

Li X, Li Q F, Zhao X B, Xu H T, Gu Y, Zhu X, Xie Z, Liu H L. Sequence analysis and study on the expression level of Dmc1 mRNA in yak and cattle-yak testis. Scientia Agricultura Sinica, 2010, 43(15): 3221-3229. (in Chinese)

[17]Wang S, Pan Z, Zhang Q, Xie Z, Liu H, Li Q. Differential mRNA expression and promoter methylation status of SYCP3 gene in testes of yaks and cattle-yaks. Reproduction in Domestic Animals, 2012, 47(3): 455-462.

[18]Liu Z S, Li Q F, Zhang Q B, Qu X G, Dong L Y, Yangzon Q B, Xie Z, Liu H L. Analysis of IGF2 mRNA expression and its methylation status between cattle yaks and their parents. Progress in Nature Science, 2009, 19: 1063-1069.

[19]李明桂, 徐洪涛, 李隐侠, 于莎莉, 赵兴波, 潘增祥, 朱翔, 谢庄, 李齐发. b-Boule基因5′调控序列的克隆与睾丸组织DMR甲基化分析. 中国农业科学, 2011, 44(18): 3859-3867.

Li M G, Xu H T, Li Y X, Yu S L, Zhao X B, Pan Z X, Zhu X, Xie Z, Li Q F. Cloning and testicular DMR methylation analysis of b-boule gene 5′-flanking regulation regions. Scientia Agricultura Sinica, 2011, 44(18): 3859-3867. (in Chinese)

[20]Castrillon D H, Quade B J, Wang T Y, Quigley C, Crum C P. The human VASA gene is specifically expressed in the germ cell lineage. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(17): 9585-9590.

[21]Onohara Y, Fujiwara T, Yasukochi T, Himeno M, Yokota S. Localization of mouse vasa homolog protein in chromatoid body and related nuage structures of mammalian spermatogenic cells during spermatogenesis. Histochemistry and Cell Biology, 2010, 133(6): 627-639.

[22]Medrano J V, Ramathal C, Nguyen H N, Simon C, Reijo P R A. Divergent RNA-binding proteins, DAZL and VASA, induce meiotic progression in human germ cells derived in vitro. Stem Cells, 2012, 30(3): 441-451.

[23]Cordin O, Banroques J, Tanner N K, Linder P. The DEAD-box protein family of RNA helicases. Gene, 2006, 367: 17-37.

[24]Weston A, Sommerville J. Xp54 and related (DDX6-like) RNA helicases: roles in messenger RNP assembly, translation regulation and RNA degradation. Nucleic Acids Research, 2006, 34(10): 3082-3094.

[25]Megosh H B, Cox D N, Campbell C, Lin H. The role of PIWI and the miRNA machinery in Drosophila germline determination. Current Biology, 2006, 16(19): 1884-1894.

[26]Gustafson E A, Wessel G M. Vasa genes: emerging roles in the germ line and in multipotent cells. Bioessays, 2010, 32(7): 626-637.

[27]Kuramochi-Miyagawa S, Watanabe T, Gotoh K, Takamatsu K, Chuma S, Kojima-Kita K, Shiromoto Y, Asada N, Toyoda A, Fujiyama A, Totoki Y, Shibata T, Kimura T, Nakatsuji N, Noce T, Sasaki H, Nakano T. MVH in piRNA processing and gene silencing of retrotransposons. Genes and Development, 2010, 24(9): 887-892.

[28]Ando M, Yamaguchi K, Chiba K, Miyake H, Fujisawa M. Expression of VASA mRNA in testis as a significant predictor of sperm recovery by microdissection testicular sperm extraction in patient with non-obstructive azoospermia. Journal of Andrology, 2012, 33(4): 711-716.

[29]Guo X, Gui Y T, Tang A F, Lu L H, Gao X, Cai Z M. Differential expression of VASA gene in ejaculated spermatozoa from normozoospermic men and patients with oligozoospermia. Asian Journal of Andrology, 2007, 9(3): 339-344.

[30]Medrano J V, Marqués-Marí A I, Aguilar C E, Riboldi M, Garrido N, Martínez-Romero A, O’Connor E, Gil-Salom M, Simón C. Comparative analysis of the germ cell markers c-KIT, SSEA-1 and VASA in testicular biopsies from secretory and obstructive azoospermias. Molecular Human Reproduction, 2010, 16(11): 811-817.

[31]Weber M, Hellmann I, Stadler M B, Ramos L, Pääbo S, Rebhan M, Schübeler D. Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nature Genetics, 2007, 39(4): 457-466.

[32]Bauer A P, Leikam D, Krinner S, Notka F, Ludwig C, Längst G, Wagner R. The impact of intragenic CpG content on gene expression. Nucleic Acids Research, 2010, 38(12): 3891-3908.

[33]Kitamura E, Igarashi J, Morohashi A, Hida N, Oinuma T, Nemoto N, Song F, Ghosh S, Held W A, Yoshida-Noro C, Nagase H. Analysis of tissue-specific differentially methylated regions (TDMs) in humans. Genomics, 2007, 89(3): 326-337.

[34]Sugimoto K, Koh E, Sin H S, Maeda Y, Narimoto K, Izumi K, Kobori Y, Kitamura E, Nagase H, Yoshida A, Namiki M. Tissue-specific differentially methylated regions of the human VASA gene are potentially associated with maturation arrest phenotype in the testis. Journal of Human Genetics, 2009, 54(8): 450-456.

[35]Lin Z Y, Imamura M, Sano C, Nakajima R, Suzuki T, Yamadera R, Takehara Y, Okano H J, Sasaki E, Okano H. Molecular signatures to define spermatogenic cells in common marmoset (Callithrix jacchus). Reproduction, 2012, 143(5): 597-609.