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为进一步提高半滑舌鳎(Cynoglossus semilaevis Günther)人工育苗技术水平,对半滑舌鳎新型膜孕激素受体(m PR-like)基因进行研究。采用同源克隆和末端快速扩增(RACE)方法,获得全长为2 002 bp的半滑舌鳎新型膜孕激素受体的c DNA序列;二级结构分析表明,该蛋白存在7个跨膜区域;三级结构分析表明该蛋白有多个结合位点。使用MEGA4.0临位相联法和Clustal X方法对m PR-like的氨基酸序列进行聚类分析和序列相似度分析,发现半滑舌鳎m PR-like与青鳉(Oryzias latipes)和三刺鱼(Gasterosteus aculeayus)的m PR-like聚为一支,相似度分别为68%和72%。应用实时定量PCR方法分析了m PR-like m RNA表达情况,发现m PR-like m RNA在性成熟雌性半滑舌鳎不同组织表达具有广泛性,但表达量存在差异,在脑、卵巢、心脏、鳃、脾和胃等组织表达丰富;在不同发育阶段卵母细胞中,m PR-like m RNA表达水平从II时相卵母细胞到V时相卵母细胞持续升高,而在VI时相卵母细胞的表达水平显著下降(P<0.05);在半滑舌鳎繁殖周期的脑和卵巢组织中,m PR-like m RNA的表达水平从性腺发育II期到V期持续升高,并且在V期达到最高峰(P<0.05),VI期表达量开始下降;在半滑舌鳎繁殖周期的垂体组织中,不同繁殖期m PR-like m RNA表达水平变化幅度不大,但在性腺发育V期时垂体中的表达量显著高于其他繁殖期(P<0.05)。放射免疫测定的结果表明,半滑舌鳎雌鱼血清中孕酮激素的含量变化在整个繁殖周期中差异显著(P<0.05),在性腺发育IV期含量迅速升高并在V期达到峰值。该基因在半滑舌鳎多种组织中的表达特征,预示其具有多种生理学作用;在卵母细胞和繁殖周期脑–垂体–卵巢的表达特征表明其参与卵母细胞成熟过程和生殖调控。
In order to further improve the technical level of artificial breeding of Cynoglossus semilaevis Günther, the m PR-like gene of the new type of soleus halluraxa was studied. The c DNA sequence of the progesterone receptor of the new half-smooth tongue pouch with a total length of 2 002 bp was obtained by means of homology cloning and rapid amplification of ends (RACE) method. The secondary structure analysis showed that there are 7 transmembrane regions in this protein; Structural analysis shows that the protein has multiple binding sites. The results of cluster analysis and sequence similarity analysis of m PR-like amino acid sequences using MEGA4.0 and Clustal X methods showed that the relationship between m PR-like and Oryzias latipes and Gasterosteus aculeayus ) M PR-like clustered together, the similarity was 68% and 72%. The expression of m PR-like m RNA was analyzed by real-time quantitative polymerase chain reaction (RT-PCR). The results showed that m PR-like m RNA was widely expressed in different tissues of sexual maturity female, but its expression was different. Spleen and stomach were abundant in oocytes. The expression of m PR-like m RNA in oocytes from different stages of development increased continuously from phase II to phase V oocytes, (P <0.05). The expression level of m PR-like m RNA in the brain and ovarian tissues during the reproductive cycle from the gonadal stage to the V stage continued to increase, and reached the peak in the V stage (P <0.05). The expression level of m PR-like m RNA in different reproductive stages of pituitary gland tissues did not change much, but in pituitary gonadal development period Significantly higher than other breeding periods (P <0.05). The results of radioimmunoassay showed that the content of progesterone in the serum of the female soleus was significantly different during the whole reproductive cycle (P <0.05), and rapidly increased in the stage of gonadal development and peaked in the V stage. The expression of this gene in many tissues of the soleus halluraxis indicates that it has multiple physiological functions. The expression characteristics of the gene in the oocyte and the reproductive cycle brain-pituitary-ovary indicate that it is involved in oocyte maturation and reproductive regulation.