绿鳍马面鲀CYP17-I基因克隆及其在繁殖周期中的表达

Molecular cloning and expression patterns of the cytochrome CYP17-I gene during the reproductive cycle in Navodon septentrionalis

为探究CYP17-I基因在绿鳍马面鲀繁殖周期中的作用及其与性激素水平变化的相关性,实验首先通过SmartTMRace技术克隆得到了绿鳍马面鲀CYP17-I c DNA全长序列1 580 bp,编码511个氨基酸。通过半定量RT-PCR技术检测了CYP17-I基因的时空表达,并采用放射免疫测定法检测雄鱼和雌鱼血清睾酮(T)和雌二醇(E2)的含量变化。结果发现,CYP17-I在卵巢、精巢和脑中表达量较高,其次为胃、心脏、脾脏、肾脏,在垂体和鳃中也检测到了微弱表达。在周期表达中发现,无论在卵巢还是精巢中,CYP17-I基因的表达水平与血清T含量密切相关,呈明显周期性变化,最高值均出现在5月份的卵细胞或精子成熟期,而与血清E2含量无显著的相关性。研究表明,CYP17-I在鱼类繁殖周期中对雄性激素睾酮的生成起关键的调控作用。

As a key steroidogenic enzyme, cytochrome P450c17 （CYP17,17a-hydroxylase/17,20-1yase） plays a critical role in the production of sex steroid hormone in many teleost. In order to evaluate the functions of P450c17-I during the gonad development and the relationship between its expression and sex steroid hormone levels, we first cloned and characterized the full-length cDNA of P450c17-I in Navodon septentrionalis. The full-length cDNA of CYP17-I is 1 580 bp, encoding 511 amino acids. The analysis of the structure shows that CYP17-1 contains relatively conserved sequence. Then RT-PCR method was used to check the expression of the CYP17-I in different tissues. The result shows that the higher expressions are in ovary, testis and brain, there is relatively low expression in heart, spleen, kidney, and there is much lower expression in pituitary and gill. In addition, the method of 125I labeled radioimmunoassay （RIA） is applied to examine the changes of serum testosterone （ T）, 17 β-Estradiol （ E2 ） levels during the reproductive cycle. The results show that the female fish has the highest level of E2 at the early stage of vitellogenesis and it decreases along with spawning. In the male fish the level of E2was not significantly different in the reproductive cycle. But the level of T in both male and female fish was developmental stage-dependent and the highest level of T appear at 5th month. RT-PCR showed that the expression patterns of CYP17-1 were developmental stage-dependent,which is consistent with the changes of T in male fish and female fish. And the highest expressions of CYP17-I are found in the gonad of both genders before the pre-ovulation phase in the reproductive season. But there is no obvious relationship between E2 and CYPI7-1 expressions. These suggest that CYP17-I plays a critical role in the production of male testosterone hormone in N. septentrionalis.