团头鲂促肾上腺皮质激素释放激素受体基因序列特征及表达分析

Molecular cloning, characterization, and regulation of Megalobrama amblycephala corticotropin-releasing hormone receptor expression

促肾上腺皮质激素释放激素受体(corticotropin-releasing hormone receptor,CRHR)是鱼类下丘脑–垂体–头肾调控轴上的重要应激调节因子。本研究通过c DNA末端快速扩增(RACE)技术成功克隆出团头鲂(Megalobrama amblycephala)CRHR1 m RNA全长序列,应用生物信息学方法对其序列特征进行解析;同时采用荧光定量PCR技术分析了团头鲂CRHR1的组织分布图谱及外源性皮质醇注射模拟应激处理下团头鲂CRHR1 m RNA的表达变化。研究结果表明,CRHR1 m RNA序列开放阅读框(open reading frame,ORF)为1290 bp,编码429个氨基酸。团头鲂CRHR1氨基酸序列与鲤科鱼类的CRHR1氨基酸序列同源性最高;该受体具有7次横跨膜结构和氨基端激素受体结构域。CRHR1在垂体中表达量最高,其次为下丘脑,在心脏、肝、脾等组织中表达丰度较低。经外源性皮质醇注射后,实验组血糖和血清皮质醇显著高于对照组,在处理2 h后达到峰值;实验组血清ACTH水平与对照组差异总体不显著,但呈现先升高后下降。应激处理后,CRHR1转录水平在4种组织中的表达变化存在差异;垂体中CRHR1在早期出现明显的表达抑制,在下丘脑中则呈现先缓慢升高后缓慢下降的趋势,而心脏和头肾中CRHR1在早期则表现出表达迅速上调而后缓慢下降的趋势。本研究进一步丰富了CRHR在鱼类研究方面的基础资料,为鱼类应激调控提供了理论基础。

Corticotropin-releasing hormone （CRH） and its receptor （CRHR） play important roles in the hormonal response to stress by regulating the release of adrenocorticotropin （ACTH） from the hypothalamic-pituitary-in-terrenal axis and consequent secretion of glucocorticoids. In this report,Megalobrama amblycephalaCRHR1 cDNA was cloned by rapid amplification of cDNA ends-polymerase chain reaction （RACE-PCR） analysis and characterized by bioinformatics analysis. The expression profile ofM. amblycephala CRHR1 in different tissues was analyzed by quantitative real-time PCR and changes in expression with stress were detected following a cor-tisol injection. Serum glucose, cortisol, and ACTH levels were monitored after the injection. The full-lengthM. amblycephala CRHR1 mRNA contained a 1290-bp open reading frame and encoded 429 amino acids. The amino acid sequence alignment showed thatM. amblycephala CRHR1 had 99% sequence identity withCarassius auratus CRHR1. The tissue expression analysis revealed thatM. amblycephala CRHR1was expressed at high levels in the pituitary and hypothalamus, but lower levels were detected in liver, kidney, muscle, skin, and heart. Serum glucose plateaued at 2–24 h, serum cortisol peaked at 12 h, and serum ACTH and mRNA expression in different tissues did not change after the cortisol injection. A significant decrease in pituitary CRHR1 expression was detected imme-diately after the cortisol injection, whereas it increased significantly in the head kidney and heart. The change in hypothalamic CRHR1 followed a unimodal pattern of increasing slowly at the early stage and then decreasing slowly at the end. In conclusion, we cloned the full-lengthM. amblycephala CRHR1 mRNA and detected changes in CRHR1 expression after a cortisol injection.