Cloning of Gonadotropin-inhibitory Hormone Receptor Gene NPPFR1 in Rabbit (Oryctolagus cuniculus) and Its Expression Analysis in HPT Axis
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
Abstract：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 50 μg 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 3 712 bp, encoded 432 amino acids, its protein was a 7-transmembrane polypeptide distributed in the plasma membrane, which was composed of α-helix, random curl, and β-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 50 μg, and the testicular NPPFR1 expression levels in the 50 μg GnIH-injected group were higher than those in the 5 μg 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.
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