布氏田鼠GnRH基因克隆及不同组织和发育阶段的基因表达特征

摘要

促性腺激素释放激素(GnRH)由GnRH编码,是调节动物繁殖活动的核心神经内分泌物质.布氏出鼠Lasiopodomys brandtii是我国内蒙古东部草原区的害鼠之一,具有明显季节性繁殖特征,但其繁殖调控机制仍未完全明确.本研究克隆了来自布氏田鼠下丘脑的GnRH cDNA序列,使用实时荧光定量技术检测了不同组织、不同年龄阶段GnRH mRNA水平.结果表明,克隆获得GnRH cDNA序列497 bp,包含开放阅读框273 bp,编码90个氨基酸和1个终止密码子的GnRH前体.DNA序列比对和氨基酸序列同源性分析表明,布氏田鼠下丘脑GnRH基因属于I型,与橙腹田鼠Microtus ochrogaste GnRH1相似性最高.GnRH mRNA在下丘脑、垂体、睾丸、肾上腺、肠、膀胱均有表达.雄鼠血清睾酮水平和雌鼠血清雌二醇水平在出生后8周、36周、80周处于高水平,显著高于4周龄鼠,但除80周龄鼠下丘脑GnRH表达量处于高水平,8周龄、36周龄鼠GnRH表达量与4周龄鼠差异均无统计学意义.推测4周龄鼠下丘脑GnRH受GnRH调节剂的中心抑制,而8周龄、36周龄鼠下丘脑GnRH受性类固醇介导的反馈抑制调控,当年龄增加至80周龄,性类固醇介导的反馈抑制消失或者应答时间延长.本研究结果为探究布氏田鼠繁殖调控规律提供了更多基础资料.%Gonadotrophin releasing hormone (GnRH) plays an important role in regulating the reproductive activity of animals.Brandt's vole (Lasperodomys brandtii) is one of the pests in the grassland of eastern Inner Mongolia,China,with obvious seasonal reproduction and its reproductive regulation mechanism is not completely clear.In this study,the cDNA of GnRH gene was cloned from the hypothalamus of L.brandtii,and the mRNA levels of GnRH in different tissues and different ages were measured using qPCR.The results showed that the cloned GnRH cDNA sequence contained an open reading frame of 273 bp,encoding a GnRH precursor of 90 amino acids.Alignment of homologous DNA and amino acid sequences showed that the GnRH of L.brandtii belonged to type I sharing the highest homology with GnRH1 of Microtus ochrogaste.The expression of GnRH gene was detected in hypothalamus,pituitary,testis,adrenal gland,intestine and bladder.The levels of serum testosterone in male voles of 8-week/36-week/80-week were significantly higher than those of 4-week,and the levels of serum estradiol in female voles of 8-week/36-week/80-week were significantly higher than those of 4-week.The highest expression of GnRH was detected in hypothalamus of 80-week voles,and there was no significant difference between 8-week/ 36-week voles and 4-week voles.We speculated that the GnRH expression of 4-week vole was mainly moderated by the central inhibition,while the GnRH expression of 8-week/36-week vole was mainly moderated by the feedback suppression of steroid.As the age reaching 80 weeks,sex steroid-mediated feedback suppression declined or response time was prolonged,and leading to the high GnRH mRNA level in this stage.These results provided basic data for exploring the reproductive regulation in L.brandtii.