斑鳢叉头框转录因子基因Foxl2的克隆表达及性类固醇激素刺激下的表达响应

Molecular cloning,expression and response of Foxl2 gene induced by sex steroid hormones in blotched snakehead Channa maculata

为探究叉头框转录因子Foxl2基因(forkhead transcription factor gene 2)在斑鳢Channa maculata性腺发育过程中的作用,采用RT-PCR和RACE技术克隆出斑鳢Foxl2基因,利用qRT-PCR解析Foxl2基因在雌雄斑鳢1龄成鱼组织、不同发育时期性腺中的表达情况,以及外源性激素诱导后雌雄斑鳢性腺中Foxl2的表达变化,通过酶联免疫法测定性腺不同发育时期雌雄斑鳢血清中的雌性激素(estrogen,E)总含量,并利用免疫组化技术检测性腺中Foxl2蛋白表达的细胞类型。结果表明:克隆获得斑鳢Foxl2 cDNA序列全长为1939 bp,开放阅读框(ORF)为921 bp,共编码306个氨基酸;组织表达分析显示,Foxl2在卵巢中表达量最高且极显著高于精巢(P<0.01),但在鳃和脑组织中雄鱼表达量显著高于雌鱼(P<0.05);性腺发育时期表达分析显示,Foxl2在卵巢中表达量高于精巢,且在孵化出膜后105 d时,卵巢中表达量达到最高,精巢中表达量最低,两者表达量存在极显著性差异(P<0.01),血清中雌激素总含量变化与之相似;免疫组织化学检测显示,Foxl2蛋白表达于斑鳢卵巢成熟卵母细胞周围的颗粒细胞,精巢中未检测到Foxl2表达信号;经外源性类固醇激素17α-乙炔基雌二醇(17α-ethynylestradio,EE2)和17α-甲睾酮(17α-methyltestosterone,MT)处理后,卵巢中Foxl2的表达均受到抑制,但精巢中表达水平被EE2上调,被MT下调。研究表明,Foxl2基因在斑鳢性腺中呈现明显的性别二态性表达,推测其参与卵巢的发育和维持,通过外源激素诱导,Foxl2可能响应性类固醇激素的调节,本研究从mRNA和蛋白层面初步揭示了Foxl2在斑鳢性腺发育中的重要作用,为深入研究其性别分化机制及性别控制技术提供了科学参考。

In order to explore the role of Foxl2 gene(forkhead transcription factor gene 2)in gonadal development in blotched snakehead Channa maculata,the Foxl2 gene was cloned by qRT-PCR and RACE(rapid amplification of cDNA ends)techniques in this study.The expression levels of Foxl2 qRT-PCR were detected in different adult tissues including gill,liver,spleen,intestine,middle kidney,muscle,head kidney,ovary,testis,heart,hypothalamus,and brain of XX female and XY male individuals and in the developmental stages of gonads.The expression changes were also analyzed in Foxl2 in gonads of female and male blotched snakehead exposed to exogenous sex hormone treatment(17α-ethynylestradio(EE2)and 17α-methyltestosterone(MT)),and the total contents of serum estrogen(E)were determined in males and females at developmental stages by ELISA.Furthermore,the cell types that Foxl2 protein were expressed in the gonads were detected by immunohistochemistry.The results showed that the full-length cDNA sequence of Foxl2 was found to be 1939 bp,including 921 bp open reading frame(ORF),encoding 306 amino acids.Tissue expression analysis revealed that the maximal expression levels of Foxl2 was observed in ovaries of 105 d old blotch snakehead post hatching,with significantly different Foxl2 expression levels in ovaries and testis(P<0.01).There was higher expression of Foxl2 in gills and brain in male than that in female individuals(P<0.05).During gonadal development,the transcription of Foxl2 were higher in ovaries than that in testis,with the maximal Foxl2 levels in ovaries of 105 d blotch snakehead post hatching,and with the minimal in testis at that time(P<0.01).The total contents of serum estrogen were similar to the Foxl2 expression during gonadal development.Immunohistochemical detection showed that Foxl2 protein was expressed in granulosa cells around mature oocytes of ovaries,and no signal was detected in testis in blotch snakehead.The expression of Foxl2 was inhibited in ovaries of blotch snakehead exposed to exogenous sex steroid hormone EE2 and MT,but the up-regulated expression level in testis exposed to EE2 and down-regulated expression level in testis exposed to MT.The findings indicated that Foxl2 had obvious sexual dimorphism in the gonads of blotch snakehead,suggesting that Foxl2 be involved in the development and maintenance of ovary.The exogenous sex hormone treatment indicated that Foxl2 was responsive for the regulation of sex steroid hormones.The Foxl2 played an important role in gonadal development of blotch snakehead from mRNA and protein levels,which laid a foundation for the further understanding of the mechanism of sex differentiation and provided a scientific basis for the sex control technology of blotch snakehead.