GnIH基因克隆、表达及对幼龄公兔生殖激素的影响

doi:10.11843/j.issn.0366-6964.2023.01.019

摘要/Abstract

摘要： 旨在获得兔促性腺激素抑制素(gonadotropin-inhibitory hormone，GnIH)基因，检测其在不同组织和不同发育阶段下丘脑中的表达水平，研究其对幼龄公兔生殖激素分泌的影响。本研究以90日龄健康闽西南黑兔公兔为研究对象，采用RACE技术(rapid-amplification of cDNA ends)从下丘脑组织中克隆出GnIH基因，并对序列进行生物信息学分析；采用实时荧光定量PCR方法(real-time quantitative PCR，qPCR)检测GnIH基因mRNA在90日龄公兔不同组织表达水平(n=5)及11、30、60、90、120、150日龄公兔下丘脑中的表达水平(n=6)；连续10d向80日龄公兔分别注射0、0.5、5和50μg鹌鹑GnIH相关肽(n=10)。第11天早上采集耳静脉血液后，屠宰公兔，采集睾丸组织，分别检测血清中生殖激素浓度(GnRH、FSH、LH、INHB和T)和睾丸组织中睾酮合成相关酶基因(StarR、3β-HSD和p450scc)mRNA的表达水平。结果表明，兔GnIH基因cDNA全长904bp，包含5'UTR 41bp，CDS区606bp，3'UTR 227bp，编码201个氨基酸。该基因在90日龄公兔大部分组织中均有表达，其中在下丘脑中表达最高(P<0.001)；在11到150日龄公兔下丘脑中，90日龄的表达量最低(P<0.01)，90日龄后随着日龄增长其表达量极显著增加(P<0.001)。注射GnIH后，50μg剂量组幼龄公兔的血清睾酮浓度极显著低于对照组和0.5μg剂量组(P<0.001)，5μg剂量组睾酮浓度显著低于对照组和0.5μg剂量组(P<0.05)，50μg剂量组的LH浓度显著高于对照组(P<0.05)；此外，5μg组的p450scc mRNA表达量极显著高于其他处理组(P<0.001)，而其他组睾酮合成相关酶基因表达量则差异不显著(P>0.05)。结果提示，GnIH基因可能与LH和睾酮的分泌与合成有关，并参与公兔的生殖发育调控，长期注射高剂量GnIH相关肽会导致公兔对药物不敏感，其具体调控机制还需后续研究。

关键词: GnIH, 闽西南黑兔, 基因克隆, 基因表达, 生殖激素

Abstract: The purpose of this study was to obtain the sequence of the rabbit GnIH gene, detect its expression patterns in different tissues and different developmental stages in the hypothalamus, and investigate its effect on the secretion of reproductive hormones in young male rabbits. In this study, the 90-day-aged healthy male Minxinan black rabbits were used as experimental animals, the sequence of the GnIH gene was cloned by RACE (rapid-amplification of cDNA ends)and analyzed by bioinformatics methods; the relative expression levels of GnIH in different tissues of male rabbits aged 90 days (n=5) and in the hypothalamus aged 11, 30, 60, 90, 120 and 150 days (n=6) were determined by real-time quantitative PCR (qPCR); the male rabbits aged 80 days were chosen and intraperitoneally injected with 0, 0.5, 5 or 50 μg quail GnIH related peptides for 10 consecutive days, respectively (n=10). These rabbits were slaughtered and their testes were collected after ear vein blood sampling on the morning of the 11th day, and then, the concentrations of reproductive hormones (GnRH, FSH, LH, INHB, and T) in serums and the mRNA levels of testosterone synthesis related enzyme genes (StarR, 3β-HSD and p450scc) in testes were tested, respectively. A total length of 904 bp GnIH gene sequence was cloned successfully, including 41 bp of 5'UTR, 606 bp of CDS, and 227 bp of 3'UTR, encoding 201 amino acids. The GnIH gene expressed in most tissues and its mRNA level was higher in the hypothalamus than in other tissues (P<0.001); Aged from 11 to 150 days, its mRNA levels in hypothalamus reached the lowest point at 90 day-aged (P<0.01), and then significantly increased with increasing age(P<0.001). The young male rabbits were administrated at different doses of GnIH polypeptide. The results showed that the concentration of serum testosterone in group 50 μg was signicantly lower than those in control and 0.5 μg groups(P<0.001), the concentration of testosterone in group 5 μg was significantly lower than those both in control and 0.5 μg groups(P<0.05), and the concentration of LH in group 50 μg was significantly higher than that in control group (P<0.05); the mRNA level of p450scc gene in group 5 μg was significantly higher than those in the other groups(P<0.001), and there were no significant differences in the mRNA levels of other testosterone synthesis related enzyme genes among the GnIH administrating-groups and control group (P>0.05). These results indicated that the GnIH gene might be related to the synthesis and secretion of LH and testosterone, and participate in the regulation of reproductive development of male rabbits, chronic injection with a high dose of GnIH-related peptide leads to drug insensitivity of male rabbits, and the specific regulatory mechanism needs subsequent studies.

Key words: GnIH, Minxinan black rabbit, gene cloning, gene expression, reproductive hormone

中图分类号:

S829.1

桑雷, 陈冬金, 孙世坤, 高承芳, 王锦祥, 陈岩锋, 谢喜平. GnIH基因克隆、表达及对幼龄公兔生殖激素的影响[J]. 畜牧兽医学报, 2023, 54(1): 201-212.

SANG Lei, CHEN Dongjin, SUN Shikun, GAO Chengfang, WANG Jinxiang, CHEN Yanfeng, XIE Xiping. Cloning and Expression of GnIH Gene and Its Effect on Reproductive Hormones Secretion of Young Male Rabbits[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 201-212.

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