四川裂腹鱼脑型芳香化酶Cyp19b基因组织分布及温度对其表达水平的影响

Tissue Distribution of Brain Aromatase Gene Cyp19b in Schizothorax kozlovi and Temperature Effects on Its Expression

为研究脑型芳香化酶基因Cyp19b在四川裂腹鱼(Schizothorax kozlovi)早期性别分化中的作用,采用RACE方法从四川裂腹鱼脑中扩增得到该基因c DNA全长序列,并应用荧光定量RT-PCR技术测定该基因m RNA的相对表达量,探讨该基因在不同规格四川裂腹鱼鳃、脑、心、肝、脾、肾、肌肉、精巢、卵巢组织中的表达差异,以及温度对其早期仔鱼阶段该基因表达的影响。四川裂腹鱼Cyp19b基因的c DNA全长序列共3 021 bp,共编码507个氨基酸,属于脑型芳香化酶基因;四川裂腹鱼Cyp19b基因编码的氨基酸序列与其他鱼类脑型芳香化酶基因编码的氨基酸序列同源性可达70%以上,而与性腺型芳香化酶基因编码的氨基酸序列同源性较低,为64%左右;四川裂腹鱼Cyp19b基因仅在脑组织中表达,具有比较高的组织特异性,且随着个体的增长,逐渐呈现显著的雌雄差异;对不同温度处理12日龄仔鱼6 d后,低温(10℃和14℃)能显著促进Cyp19b基因表达量的升高,但高温(26℃)却对其表达量无显著影响。由此推测,脑型芳香化酶基因Cyp19b可能在低温导致四川裂腹鱼雌性化过程中发挥着重要作用。

Previous studies by authors indicated that low temperature could result in more females of Schizothorax kozlovi during the early developmental stage.In order to elucidate the role of Cyp19 b in sex differentiation of S.kozlovi,the full length c DNA of Cyp19 b was cloned from the brain of S.kozlovi by using rapid amplification of c DNA ends（RACE） method.Relative m RNA expression levels of Cyp19 b were determined by using quantitative real-time PCR（RT-PCR）.Then the differences of Cyp19 b m RNA expression levels in gills,brain,heart,liver,spleen,kidney,muscle,testis and ovary among different body-sized groups were discussed,while temperature effects on its expression during the larval stage of S.kozlovi were evaluated.The full length c DNA of Cyp19 b was 3 021 bp,which encoded 507 amino acids and belonged to brain aromatase gene; the deduced amino acid sequence of Cyp19 b of S.kozlovi had more than 70% identity compared with brain aromatase genes of other teleost fishes,but only around 64% when compared with gonadal aromatase genes of other teleost fishes.The m RNA expression of Cyp19 b of S.kozlovi only appeared in brain but not in other tissues,presenting relatively high tissue specificity.One-Way Analysis of Variance（ANOVA） revealed that the m RNA expression levels of Cyp19 b were not significantly different between females and males in small body-sized samples（mean standard length was 12 cm）,but gradually showed significant difference between females and males in large body-sized samples（mean standard length was 26 cm）（Fig.3）.Larvae at 12 days of hatching were cultured in five different temperature groups,and ANOVA revealed that six days later the m RNA expression levels of Cyp19 b were significantly increased by low temperature（10℃ and 14℃）,but not influenced by high temperature（26℃）（Fig.4）.The present results suggest that brain aromatase gene Cyp19 b may play important roles in the feminization of S.kozlovi,which is affected by low temperature during the early developmental stage.