| 摘要: |
| 【目的】研究生物钟基因调控牦牛睾酮分泌的分子机制，为生物钟靶向调控牦牛繁殖力提供理论依据。【方法】于清晨08:00左右（ZT0）和下午20:00左右（ZT12）采集雄性牦牛的血液和睾丸，用ELISA检测牦牛血清睾酮含量是否存在昼夜节律性表达；用IHC法检测生物钟蛋白Bmal1在睾丸中的表达定位；采用普通PCR法检测生物钟基因（Bmal1、Dbp、Nr1d1、Per1）在牦牛睾丸中的表达；采用实时荧光定量PCR法检测生物钟基因、类固醇合成基因(StAR、Hsd3b1、Hsd17b3、Cyp11a1）和核受体基因（Sf1、Nr4a1、Lhcgr、Nr0b1）在牦牛睾丸中的时空表达规律；用蛋白免疫印迹（WB）检测生物钟蛋白Bmal1和类固醇合成蛋白StAR在牦牛睾丸中的表达变化；用生物信息学预测牦牛睾酮合成相关因子启动子区域生物钟作用元件。【结果】牦牛体内睾酮含量存在昼夜节律性表达，ZT0时的含量极显著低于ZT12（P＜0.001）；生物钟蛋白Bmal1在牦牛睾丸中存在节律性表达，主要分布在睾丸间质细胞中；4个生物钟基因、类固醇合成基因Hsd17b3和Cyp11a1及核受体基因Nr4a1、Lhcgr和Nr0b1在牦牛睾丸中均存在节律性表达；类固醇合成蛋白StAR在牦牛睾丸中有表达，但无节律性；牦牛类固醇合成相关基因启动子区域存在与生物钟基因结合的E-box、RORE和D-box元件。【结论】牦牛睾丸生物钟基因通过调控类固醇合成相关基因的表达，影响睾酮的分泌水平。 |
| 关键词: 生物钟 牦牛 睾酮合成 类固醇合成基因 核受体基因 |
| DOI： |
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| 基金项目:青海大学青年科研基金项目（2021-QNY-8）；青海省科技厅自然科学基金青年项目（2022-ZJ-934Q）；青海省科技厅科技援青合作专项（2020-QY-217） |
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| Mechanism of circadian clock gene regulating testosterone secretion in yak |
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GAO Lei1,HONG Jin1,ZHANG Liping,HONG Jin,ZHANG Liping,et al
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| Abstract: |
| 【Objective】This study investigated the molecular mechanism of circadian clock gene regulating testosterone secretion in yak to provide basis for improving fecundity by circadian clock targeted regulation.【Method】The blood and testes of male yaks were collected around 08:00 a.m.(ZT0) and 20:00 p.m.(ZT12).ELISA was used to detect circadian changes of serum testosterone contents in yak,and IHC was used to detect the expression and distribution of circadian clock protein of Bmal1 in yak testes.PCR was used to detect expressions of clock genes (Bmal1,Dbp,Nr1d1 and Per1) in yak testes,and RT-qPCR was used to detect rhythmic expressions of clock genes,steroidogenic genes (StAR,Hsd3b1,Hsd17b3 and Cyp11a1) and nuclear receptor genes (Sf1,Nr4a1,Lhcgr and Nr0b1) in yak testes.WB was used to detect expressions of circadian clock protein Bmal1 and steroidogenic protein StAR in yak testes,and bioinformatics was used to predict circadian clock elements in the promoter region of yak testosterone synthesis related factors.【Result】There existed a circadian rhythmicity of testosterone level in yak,and the content of ZT0 was significantly lower than that of ZT12 (P＜0.001).Circadian clock protein Bmal1,mainly in Leydig cells,was expressed rhythmically in yak testes.All circadian clock genes,steroidogenic genes of Hsd17b3 and Cyp11a1,and nuclear receptor genes of Nr4a1,Lhcgr and Nr0b1 were expressed rhythmically in yak testes.Steroidogenic protein of StAR was expressed without rhythm in yak testes.There were E-box,RORE and D-box elements combined with circadian clock genes in the promoter region of steroidogenesis related genes in yaks.【Conclusion】Circadian clock genes affected the level of testosterone secretion by regulating expression of steroidogenesis related genes in yaks. |
| Key words: circadian clock yak testosterone synthesis steroidogenic genes nuclear receptor genes |
