牦牛TSHB、DIO2和DIO3基因的克隆及其在繁殖轴的表达研究
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摘要
为了解TSHB-DIO2/DIO3系统对牦牛季节性发情的调控机制,以牦牛为研究对象,运用RT-PCR方法克隆牦牛TSHB、DIO2、DIO3基因的cDNA序列,结合GenBank已公布的普通牛TSHB、DIO2、DIO3基因序列,构建遗传进化树,采用荧光定量PCR技术检测其在牦牛下丘脑、垂体、卵巢、输卵管、子宫中的表达水平。结果表明,牦牛TSHB、DIO2、DIO3基因均与普通牛有最近的分子进化关系;TSHB基因CDS区为417 bp,在垂体中的表达水平高于其他组织,差异极显著(P<0.01);DIO2基因cDNA为951 bp,在所检测的组织中均有表达,且在子宫中的表达水平高于其他组织,差异极显著(P<0.01);DIO3基因cDNA为873 bp,在子宫组织中未检测到表达,在卵巢中的表达水平高于垂体、下丘脑和输卵管,差异极显著(P<0.01)。提示TSHBDIO2/DIO3可能参与牦牛繁殖调控,旨为揭示牦牛季节性繁殖分子调控机制提供参考。
In order to study the regulation mechanism of TSHB-DIO2/DIO3 system on seasonal breeding of the yak,RT-PCR was used to analyze the cDNA sequences of gene TSHB,DIO2,and DIO3,and the phylogenetic tree was constructed based on the sequences of bovine gene TSHB,DIO2,and DIO3 in the GenBank. Further,their mRNA expression levels in hypothalamus,pituitary,ovary,oviduct and uterus were analyzed by real-time PCR. These results indicated that there was very close evolutionary relationship in yak's gene TSHB,DIO2,and DIO3 between a yak and a bovine. The coding region of gene TSHB was 417 bp,and its expression level in pituitary was significantly higher than that in other tissues(P < 0.01). The cDNA sequence of DIO2 gene was 951 bp and expressed in all tissues,but it was significantly higher in the uterus than that in the other four tissues(P < 0.01). The cDNA sequences of DIO3 gene was 873 bp and was not detected in the uterus,the expression of which in the ovary was significantly higher than that in the pituitary,hypothalamus and oviduct(P< 0.01). This study suggests that the TSHB-DIO2/DIO3 may be involved in the regulation of yak reproduction,which may provide a reference for preliminarily revealing the molecular regulation mechanism of yak seasonal breeding.
In order to study the regulation mechanism of TSHB-DIO2/DIO3 system on seasonal breeding of the yak,RT-PCR was used to analyze the cDNA sequences of gene TSHB,DIO2,and DIO3,and the phylogenetic tree was constructed based on the sequences of bovine gene TSHB,DIO2,and DIO3 in the GenBank. Further,their mRNA expression levels in hypothalamus,pituitary,ovary,oviduct and uterus were analyzed by real-time PCR. These results indicated that there was very close evolutionary relationship in yak's gene TSHB,DIO2,and DIO3 between a yak and a bovine. The coding region of gene TSHB was 417 bp,and its expression level in pituitary was significantly higher than that in other tissues(P < 0.01). The cDNA sequence of DIO2 gene was 951 bp and expressed in all tissues,but it was significantly higher in the uterus than that in the other four tissues(P < 0.01). The cDNA sequences of DIO3 gene was 873 bp and was not detected in the uterus,the expression of which in the ovary was significantly higher than that in the pituitary,hypothalamus and oviduct(P< 0.01). This study suggests that the TSHB-DIO2/DIO3 may be involved in the regulation of yak reproduction,which may provide a reference for preliminarily revealing the molecular regulation mechanism of yak seasonal breeding.
引文
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[14]Steinbrenner H, Speckmann B, Klotz LO. Selenoproteins:Antioxidant selenoenzymes and beyond[J]. Arch Biochem Biophys, 2016, 595:113-119.
[15]周小萍.硒及氧化应激诱导剂对血管内皮细胞中几种硒蛋白m RNA表达水平影响的研究[D].武汉:华中科技大学,2007.
[16]SáenzdemieraC,MoneckeS,Bartzen-SprauerJ,etal.A circannual clock drives expression of genes central for seasonal reproduction[J]. Curr Biol, 2014, 24(13):1500-1506.
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[18]Perfito N, Guardado D, Williams TD, et al. Social cues regulate reciprocal switching of hypothalamic Dio2/Dio3 and the transition into final follicle maturation in European starlings(Sturnus vulgaris)[J]. Endocrinology, 2015, 156(2):694-706.
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[20]Ernst DK, Bentley GE. Neural and neuroendocrine processing of a non-photic cue in an opportunistically breeding songbird[J]. J Exp Biol, 2016, 219(6):783-789.
[21]Detti L, Uhlmann RA, Fletcher NM, et al. Endometrial signaling pathways during ovarian stimulation for assisted reproduction technology[J]. Fertil Steril, 2013, 100(3):889-94.
[22]黄冬维,等.山羊繁殖季节性相关基因DIO2与DIO3的表达分析[J].农业生物技术学报, 2016, 24(10):1536-1543.
[23]Xu M, Sulkowski ZL, Parekh P, et al. Effects of perinatal lipopolysaccharide(LPS)exposure on the developing rat brain;modeling the effect of maternal infection on the developing human CNS[J]. Cerebellum, 2013, 12(4):572-586.
[24]Bastian TW, Anderson JA, Fretham SJ, et al. Fetal and neonatal iron deficiency reduces thyroid hormone-responsive gene mRNA levels in the neonatal rat hippocampus and cerebral cortex[J].Endocrinology, 2012, 153(11):5668-5680.
[25]Tuncozcan E, Ullmann TM, et al. Low dose thyroxine attenuates autism associated adverse effects of fetal alcohol in male offspring’s social behavior and hippocampal gene expression[J]. Alcohol Clin Exp Res, 2013, 37(11):1986-1995.
[26]Janssen R, Zuidwijk MJ, et al. MicroRNA 214 is a potential regulator of thyroid hormone levels in the mouse heart following myocardial infarction, by targeting the thyroid-hormone-inactivating enzyme deiodinase type III[J]. Front Endocrinol, 2016, 7:22.
[27]Deng WB, Liang XH, Liu JL, et al. Regulation and function of deiodinases during decidualization in female mice[J].Endocrinology, 2014, 155(7):2704-2717.
[28]Kagami M, Matsuoka K, et al. Paternal uniparental disomy 14 and related disorders:Placental gene expression analyses and histological examinations[J]. Epigenetics, 2012, 7(10):1142-1150.
[2]Lomet D, CogniéJ, Chesneau D, et al. The impact of thyroid hormone in seasonal breeding has a restricted transcriptional signature[J].Cell Mol Life Sci, 2017, 75(5):1-15.
[3]Nakao N, et al. Thyrotrophin in the pars tuberalis triggers photoperiodic response[J]. Nature, 2008, 452(7185):317-322.
[4]Hanon EA, et al. Ancestral TSH mechanism signals summer in a photoperiodic mammal[J]. Curr Biol, 2008, 18(15):1147-1152.
[5]Yoshimura T. Thyroid hormone and seasonalregulationof reproduction[J]. Front Neuroen, 2013, 34(3):157-166.
[6]Ikegami K, Yoshimura T. Circadian clocks and the measurement of daylength in seasonal reproduction[J]. Molecular&Cellular Endocrinology, 2012, 349(1):76-81.
[7]孟庆辉,陈永杏,董红敏,等.牦牛分布特点及其种群数量[J].家畜生态学报, 2017, 38(3):80-85.
[8]Zi XD. Reproduction in female yaks(Bos grunniens)and opportunities for improvement[J]. Theriogenology, 2003, 59(5):1303-1312.
[9]Bockmann J, B?ckers TM, Winter C, et al. Thyrotropin expression in hypophyseal pars tuberalis-specific cells is 3, 5, 3’-triiodothyronine,thyrotropin-releasing hormone, and pit-1 independent[J].Endocrinology, 1997, 138(3):1019-1028.
[10]黄冬维,曹贵玲,储明星,等.山羊促甲状腺素β亚基基因(TSHB)cDNA克隆与组织表达研究[J].安徽农业大学学报,2012, 39(6):847-853.
[11]Goulart-Silva F, Souza PBD. T3 rapidly modulates TSHβmRNA stability and translational rate in the pituitary of hypothyroid rats[J]. Mol Cell Endocrinol, 2011, 332(1-2):277-282.
[12]刘浩,臧晓怡,等.促甲状腺激素β基因剪接变体在BALB C小鼠不同组织中的表达[J].天津医药, 2011, 39(3):239-242.
[13]Majumdar G, Trivedi AK, Gupta NJ, et al. Circadian synchronization determines critical day length for seasonal responses[J]. Physiol Behav, 2015, 147:282-290.
[14]Steinbrenner H, Speckmann B, Klotz LO. Selenoproteins:Antioxidant selenoenzymes and beyond[J]. Arch Biochem Biophys, 2016, 595:113-119.
[15]周小萍.硒及氧化应激诱导剂对血管内皮细胞中几种硒蛋白m RNA表达水平影响的研究[D].武汉:华中科技大学,2007.
[16]SáenzdemieraC,MoneckeS,Bartzen-SprauerJ,etal.A circannual clock drives expression of genes central for seasonal reproduction[J]. Curr Biol, 2014, 24(13):1500-1506.
[17]Tavolaro FM, Thomson LM, Ross AW, et al. Photoperiodic effects on seasonal physiology, reproductive status and hypothalamic gene expression in young male F344 rats[J]. J Neuroendocrinol,2015, 27(2):79-87.
[18]Perfito N, Guardado D, Williams TD, et al. Social cues regulate reciprocal switching of hypothalamic Dio2/Dio3 and the transition into final follicle maturation in European starlings(Sturnus vulgaris)[J]. Endocrinology, 2015, 156(2):694-706.
[19]Srivastava A, Trivedi N, Malik S, et al. Molecular basis of photoperiodic control of reproductive cycle in a subtropical songbird, the Indian weaver bird(Ploceus philippinus)[J]. Gen Comp Endocrinol, 2015, 220(1):41-45.
[20]Ernst DK, Bentley GE. Neural and neuroendocrine processing of a non-photic cue in an opportunistically breeding songbird[J]. J Exp Biol, 2016, 219(6):783-789.
[21]Detti L, Uhlmann RA, Fletcher NM, et al. Endometrial signaling pathways during ovarian stimulation for assisted reproduction technology[J]. Fertil Steril, 2013, 100(3):889-94.
[22]黄冬维,等.山羊繁殖季节性相关基因DIO2与DIO3的表达分析[J].农业生物技术学报, 2016, 24(10):1536-1543.
[23]Xu M, Sulkowski ZL, Parekh P, et al. Effects of perinatal lipopolysaccharide(LPS)exposure on the developing rat brain;modeling the effect of maternal infection on the developing human CNS[J]. Cerebellum, 2013, 12(4):572-586.
[24]Bastian TW, Anderson JA, Fretham SJ, et al. Fetal and neonatal iron deficiency reduces thyroid hormone-responsive gene mRNA levels in the neonatal rat hippocampus and cerebral cortex[J].Endocrinology, 2012, 153(11):5668-5680.
[25]Tuncozcan E, Ullmann TM, et al. Low dose thyroxine attenuates autism associated adverse effects of fetal alcohol in male offspring’s social behavior and hippocampal gene expression[J]. Alcohol Clin Exp Res, 2013, 37(11):1986-1995.
[26]Janssen R, Zuidwijk MJ, et al. MicroRNA 214 is a potential regulator of thyroid hormone levels in the mouse heart following myocardial infarction, by targeting the thyroid-hormone-inactivating enzyme deiodinase type III[J]. Front Endocrinol, 2016, 7:22.
[27]Deng WB, Liang XH, Liu JL, et al. Regulation and function of deiodinases during decidualization in female mice[J].Endocrinology, 2014, 155(7):2704-2717.
[28]Kagami M, Matsuoka K, et al. Paternal uniparental disomy 14 and related disorders:Placental gene expression analyses and histological examinations[J]. Epigenetics, 2012, 7(10):1142-1150.