金钱鱼Sox9 cDNA克隆及其表达分析

c DNA Cloning and m RNA Expression Analysis of Sox9 in Scatophagus argus

为了解Sox9基因在金钱鱼（Scatophagus argus）性腺分化中的作用,本研究利用c DNA末端快速扩增法（RACE）克隆了金钱鱼Sox9基因c DNA全长序列,同时研究了投喂芳香化酶抑制剂来曲唑（LE）（50 mg/kg）后该基因的表达及其性腺的组织学变化。金钱鱼Sox9 c DNA全长2 759 bp,包括5′非翻译区（5′-UTR）31 bp、3′非翻译区（3′-UTR）1 288 bp和开放阅读框（ORF）1 440 bp,编码479个氨基酸。该氨基酸序列104-172位为HMG保守盒,在该盒内存在一个特征性基序,两个核定位信号NLS及一个富含亮氨酸的核输出信号NES。该序列与赤点石斑鱼（Epinephelus akaara）的相似性最高,为96.0%,与非洲爪蟾（Xenopus laevis）、人（Homo sapiens）、原鸡（Gallus gallus）、小家鼠（Mus musculus）等物种的相似性为61.5%-76.1%。Real-time PCR显示,金钱鱼Sox9基因在脑、鳍、精巢中表达量较高。组织学研究表明,芳香化酶抑制剂来曲唑能有效诱导金钱鱼发生不同程度的性逆转,性腺中卵母细胞退化,精原细胞增殖。Real-time PCR结果显示,金钱鱼Sox9基因在来曲唑处理20 d后开始不断上升,于处理后第40天时达到最高值,在第60天时表达量迅速下降。上述结果表明,金钱鱼Sox9基因高度保守,可能在金钱鱼性腺雄性化过程中起重要作用。

In order to elucidate the role of Sox9 in gonadal differentiation of Scatophagus argus, the full length eDNA of Sox9 was cloned from testis of S. argus using RT-PCR and rapid amplification of cDNA ends （RACE）. To investigate effect of aromatase inhibitor letrozole （LE） on Sox9 gene expression and gonadal development, juveniles were fed with diets containing LE 50 mg/kg, and gonadal development was observed histologically and the expression of Sox9 was measured with quantitative real-time PCR. RACE results （Fig. l） showed that full length eDNA of 2 759 bp contained a 31 bp 5＇-untranslated region （5＇-UTR）, a 1 288 bp 3＇-untranslated region （Y-UTR） and a 1 440 bp open reading frame （ORF） encoding 479 amino acids. A HMG （high mobility group） box containing a specific motif, two nuclear localization signal （NLS） sequences and a nuclear export signal （NES） was found in the deduced amino acid sequence of SOX9 （Fig. 1）. The amino acid sequence had a high similarity to SOX9 of other species, and the percent identity compared with Epinephelus akaara, Xenopus laevis, Homo sapiens, Gallus gallus, and Mus musculus was 61.5% - 96.0%. The expression levels of Sox9 mRNA in different tissues were analyzed by quantitative real-time PCR. The highest expression level was detected in the brain, intestine and fin （Fig. 3）. Histological results showed that sex reversal could be induced by LE, and the degenerating oocytes and proliferation of sperlnatogonia were observed in gonad from LE-treated S. argus （Fig.4）. The expression level of Sox9 mRNA was up-regulated significantly at 20 d, reached its peak at 40 d of treatment with LE but was down-regulated markedly at 60 d （Fig. 5）. These results suggest that Sox9 is highly conserved. Masculinization can be induced by LE treatment and Sox9 may be one of the important factors initiating the masculinization orS. argus during sex reversval.