兔促性腺激素抑制激素受体基因NPFFR1的克隆及其在HPT轴的表达分析  

Cloning of Gonadotropin-inhibitory Hormone Receptor Gene NPPFR1 in Rabbit(Oryctolagus cuniculus)and Its Expression Analysis in HPT Axis

作　　者：桑雷[1] 高承芳[1] 孙世坤[1] 王锦祥[1] 陈冬金[1] 谢喜平[1] SANG Lei;GAO Cheng-Fang;SUN Shi-Kun;WANG Jin-Xiang;CHEN Dong-Jin;XIE Xi-Ping(Institute of Animal Husbandry and Veterinary Medicine/Fujian Key Laboratory of Animal Genetics and Breeding,Fujian Academy of Agricultural Sciences,Fuzhou 350013,China)

机构地区：[1]福建省农业科学院畜牧兽医研究所/福建省畜禽遗传育种重点实验室,福州350013

出　　处：《农业生物技术学报》2024年第5期1102-1110,共9页Journal of Agricultural Biotechnology

基　　金：国家兔产业体系福州综合试验站项目(CARS-43-G-5);福建省自然科学基金(2021J01486);福建省科技计划公益类专项(2021R10260014,2022R1026003);福建省农业科学院科技创新团队建设项目(CXTD2021006-2-2)。

摘　　要：神经肽FF受体1(neuropeptide FF receptor 1,NPFFR1)是促性腺激素抑制激素(gonadotropininhibitory hormone,GnIH)的主要受体,在调控动物生殖生理方面起着重要作用。为了解NPFFR1基因的功能,本研究以闽西南黑兔(Oryctolagus cuniculus)为研究对象,采用cDNA末端快速克隆(rapid amplification of cDNA ends,RACE)技术克隆了NPFFR1基因(GenBank No.OP791886),并对其进行了生物信息学和时空表达谱分析;此外,公兔腹腔注射0、0.5、5和50μg GnIH多肽,利用q PCR方法检测下丘脑-垂体-睾丸(hypothalamic-pituitary-testicular,HPT)轴中NPFFR1基因的表达水平。生物信息学分析表明,兔NPFFR1全长3712 bp,编码432个氨基酸;其翻译的蛋白由α螺旋、无规卷曲和β折叠组成,是镶嵌于质膜的7次跨膜蛋白,与高原鼠兔(Ochotona curzoniae)和北美鼠兔(Ochotona princeps)NPFFR1高度同源。qPCR结果表明,NPFFR1基因在HPT轴中均有表达,在下丘脑的表达量显著高于垂体和睾丸(P<0.05)。出生后,90日龄兔下丘脑NPFFR1基因表达量显著低于11、30、60和150日龄(P<0.05),30和120日龄兔垂体NPFFR1基因表达量显著低于11和90日龄(P<0.05),30日龄兔睾丸NPFFR1基因表达量显著低于11和90日龄。兔腹腔注射5~50μg GnIH多肽后,下丘脑NPFFR1基因表达量呈现剂量依赖性增加;50μg剂量组睾丸NPFFR1基因表达量显著大于5μg剂量组(P<0.05);注射4种剂量GnIH多肽都未引起垂体NPFFR1表达量发生改变。本研究为深入探讨兔NPFFR1在生殖生理方面的作用机制提供基础资料。Neuropeptide FF receptor 1(NPFFR1)is a primary receptor to the gonadotropin-inhibitory hormone(GnIH),which plays an obvious effect on the controls of animal reproductive physiology.To investigate the function of NPFFR1,the NPFFR1 gene(GeneBank No.OP791886)was cloned from the Minxinan Black rabbit(Oryctolagus cuniculus)by rapid amplification of cDNA ends(RACE)method,and the bioinformatics and spatiotemporal expression profiles were analyzed.Moreover,the male rabbits were intraperitoneally injected with 0,0.5,5,or 50μg GnIH-related peptides,and the NPFFR1 mRNA level in the hypothalamic-pituitary-testicular(HPT)axis was detected by qPCR.The results of bioinformatics analysis showed that the full CDS sequence of rabbit NPFFR1 gene was 3712 bp,encoded 432 amino acids,its protein was a 7-transmembrane polypeptide distributed in the plasma membrane,which was composed ofα-helix,random curl,andβ-fold,and highly homologous with Ochotona curzoniae and Ochotona princeps.qPCR analysis showed that NPFFR1 expressed in all tissues of the HPT axis,and the expression level in the hypothalamus was significantly higher than that in the pituitary gland and testis(P<0.05).After being born,the hypothalamic mRNA levels of NPFFR1 gene in 90-day-old rabbits were lower than those in 11-,30-,60-or 150-day-old rabbits(P<0.05),the pituitary mRNA level of NPFFR1 gene in 30-or 120-day-old rabbits were lower than those in 11-or 90-day-old rabbits(P<0.05),and the testicular mRNA levels of NPFFR1 gene in 30-day-old rabbits were lower than those in 11-or 90-day-old rabbits(P<0.05).The hypothalamic mRNA levels of NPFFR1 increased in a dose-dependent manner after the rabbits administered with GnIH using a dose from 5 to 50μg,and the testicular NPPFR1 expression levels in the 50μg GnIH-injected group were higher than those in the 5μg group(P<0.05);there were no significant differences in the mRNA levels of pituitary NPFFR1 among the 4 dose-groups.This study provides a theoretical support and reference in research on the function of rabbit NPFFR1

关 键 词：兔 促性腺激素抑制激素(GnIH) 神经肽FF受体1基因(NPFFR1) 克隆 基因表达 

分 类 号：S829.1[农业科学—畜牧学]