b-Boule基因5′调控序列的克隆与睾丸组织DMR甲基化分析

doi:10.3864/j.issn.0578-1752.2011.18.017

中国农业科学 ›› 2011, Vol. 44 ›› Issue (18): 3859-3867.doi: 10.3864/j.issn.0578-1752.2011.18.017

b-Boule基因5′调控序列的克隆与睾丸组织DMR甲基化分析

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

1.南京农业大学动物科技学院
2.东南大学发育与疾病相关基因教育部重点实验室
3.中国农业大学农业生物技术国家重点实验室

收稿日期:2010-08-27 修回日期:2011-02-10 出版日期:2011-09-15 发布日期:2011-03-09
通讯作者: 通信作者李齐发，E-mail：liqifa@njau.edu.cn
作者简介:李明桂，E-mail：liminggui@yahoo.com.cn。徐洪涛与李明桂为同等贡献作者，E-mail：xhtzh@126.com。
基金资助:
国家自然科学基金（30500360）、农业生物技术国家重点实验室开放课题（2009SKLAB07-2）

Cloning and Testicular DMR Methylation Analysis of b-Boule Gene 5′-Flanking Regulation Regions

LI Ming-Gui, XU Hong-Tao, LI Yin-Xia, YU Sha-Li, ZHAO Xing-Bo, PAN Zeng-Xiang, ZHU Xiang, XIE Zhuang, LI Qi-Fa

1.南京农业大学动物科技学院
2.东南大学发育与疾病相关基因教育部重点实验室
3.中国农业大学农业生物技术国家重点实验室

Received:2010-08-27 Revised:2011-02-10 Online:2011-09-15 Published:2011-03-09
Contact: QiFa Li

摘要/Abstract

摘要： 【目的】研究b-Boule基因5′调控序列的序列特征，以及牦牛、黄牛与犏牛睾丸组织b-Boule基因DMR甲基化状态的差异，为揭示b-Boule基因的表达调控和犏牛雄性不育的表观遗传机制提供依据。【方法】采用PCR扩增和克隆测序技术获得牦牛b-Boule基因5′调控序列，利用生物信息学方法分析b-Boule基因5′调控序列的序列特征，采用亚硫酸氢钠测序法检测牦牛、黄牛与犏牛睾丸组织中b-Boule基因DMR的甲基化状态。【结果】b-Boule基因5′调控序列长度为1 352 bp，核心启动子区含有SP1等甲基化敏感位点，5′端存在一个CpG岛。犏牛b-Boule基因DMR的甲基化水平（17.78%）高于牦牛（7.50%）和黄牛（6.94%）（P＜0.01），特别是CpG位点33—35的甲基化水平差异更明显。【结论】犏牛b-Boule基因DMR的甲基化水平高于牦牛和黄牛，结合前期mRNA表达水平和组织学观察结果，认为DMR甲基化在b-Boule基因的表达调控中发挥关键作用，犏牛b-Boule基因可能是通过DMR区的高甲基化抑制其mRNA表达来阻滞精子发生减数分裂过程。

关键词: 牦牛, 犏牛, b-Boule基因, 5′调控序列, 甲基化

Abstract: 【Objective】 In order to reveal b-Boule gene expression and regulation, and to supply evidence for apparent genetic mechanism of cattle-yak male sterile, the 5′-flanking regulatory sequence of b-Boule, and the difference of b-Boule gene DMR methylation status in testes among cattle, yaks and cattle-yaks were studied. 【Method】 The 5′-flanking regulatory sequence of b-Boule in yak was cloned and sequenced by the PCR amplification, the 5´-flanking regulatory sequence of b-Boule was analyzed using bioinformatics, and the DMR methylation status of b-Boule gene in testes among cattle, yaks and cattle-yaks were detected using sodium bisulfite sequencing. 【Result】 The 5′-flanking regulatory sequence length of b-Boule is 1 352 bp, and there is a CpG island. The core promoter region contains SP1 methylation-sensitive site. The methylation level of b-Boule gene DMR in cattle-yaks (17.78%) was significantly greater than that in cattle (6.94%) and yaks (7.50%) (P＜0.01). The difference of the methylation level is obviously, especially the CpG site’s 33-35. 【Conclusion】 The methylation level of yak b-Boule gene DMR is significantly greater than that of cattle-yak. Combined with the previous study results about mRNA expression and histological observation, it was speculated that the DMR methylation played a key role in the expression and regulation of b-Boule, and it might arrest meiotic process of spermatogenesis by its mRNA expression inhibition through the high methylation level of b-Boule gene DMR in cattle-yak.

Key words: yak, cattle-yak, b-Boule gene, 5′regulation sequence, methylation

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[J]. 中国农业科学, 2011, 44(18): 3859-3867.

LI Ming-Gui, XU Hong-Tao, LI Yin-Xia, YU Sha-Li, ZHAO Xing-Bo, PAN Zeng-Xiang, ZHU Xiang, XIE Zhuang, LI Qi-Fa. Cloning and Testicular DMR Methylation Analysis of b-Boule Gene 5′-Flanking Regulation Regions[J]. Scientia Agricultura Sinica, 2011, 44(18): 3859-3867.

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b-Boule基因5′调控序列的克隆与睾丸组织DMR甲基化分析

Cloning and Testicular DMR Methylation Analysis of b-Boule Gene 5′-Flanking Regulation Regions

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