三角鲂foxl2基因克隆和时空表达特征及EE2对其表达的影响

Cloning and spatio-temporal expression analysis of foxl2 gene and the influence of EE2 on its expression in Megalobrama terminalis

为探究foxl2基因(forkhead transcription factor gene 2)在三角鲂(Megalobrama terminalis,M.terminalis)性腺发育过程中的功能,该研究克隆了三角鲂foxl2基因的开放阅读框(open reading frame, ORF),分析了其编码蛋白的结构特征,检测了其在胚胎发育不同时期和成体不同组织中的表达情况,并研究了腹腔注射性激素17α-乙炔基雌二醇(17α-ethinylestradiol, EE2)对foxl2表达的影响。结果表明:三角鲂foxl2基因ORF为921 bp,共编码306个氨基酸,具有保守的叉头(forkhead, FH)结构域,与斑马鱼(Danio rerio)FOXL2的同源性较高达到96%。三角鲂FOXL2蛋白与鲤科鱼类亲缘关系较近,而与人类和小鼠相距较远。qRT-PCR结果显示,foxl2 mRNA在三角鲂整个胚胎发育过程中均表达,在受精后24、48、60 h表达量较高(P<0.05);在卵巢中的表达量显著高于精巢和其他组织(P<0.05),在肌肉、皮肤、脾、肾和肠组织中弱表达或不表达,表达具有组织特异性。腹腔注射EE2对三角鲂foxl2 mRNA表达调控结果显示,在脑组织中,雌鱼foxl2 mRNA在注射24 h达到峰值且显著高于对照组和雄鱼(P<0.05);在性腺组织中,foxl2 mRNA在注射24 h后的卵巢和精巢中表达量最高,且在36 h的卵巢中持续高表达(P<0.05),表明EE2能够明显促进三角鲂脑和卵巢foxl2 mRNA的表达。研究表明,foxl2基因在三角鲂性腺中呈性别二态性表达,推测其在卵巢发育和功能维持方面起重要作用,foxl2可能是与性类固醇激素相互作用来调节三角鲂性腺发育和功能维持。该研究可为三角鲂性腺发育及性别调控机制提供基础资料。

To investigate the function of forkhead transcription factor gene 2(foxl 2)during gonadal development of M.terminalis,we cloned the open reading frame(ORF)of M.terminalis foxl 2 gene,analyzed the structural characteristics of the encoded protein,and detected its expression at different stages of embryonic development and in different adult tissues in this study.The effect of intraperitoneal injection of sex hormone 17α-ethinylestradiol(EE2)on foxl 2 expression was also studied.The results showed that the ORF of foxl 2 gene was 921 bp,encoding 306 amino acids,with a conserved forkhead(FH)domain,and had a 96%homology with zebrafish FOXL2.The FOXL2 protein of M.terminalis is closely related to cyprinid fish,but distantly related to humans and mice.The qRT-PCR results showed that foxl2 mRNA was expressed throughout embryonic development,and the expression was higher at 24,48,60 h of post-fertilization(P<0.05).The expression level in the ovary was significantly higher than that in the testis and other tissues(P<0.05),and weakly or not expressed in muscle,skin,spleen,kidney and intestine tissues.The expression was tissue-specific.The regulation of foxl 2 mRNA expression by EE2 injection showed that foxl 2 mRNA in female reached the peak at 24 h after injection and was significantly higher than that in the control and male(P<0.05).In the gonadal tissue,the expression of foxl 2 mRNA was highest at 24 h in the ovary and testis after injection,and continued to be high at 36 h in the ovary after injection(P<0.05),suggesting that EE2 could promote the expression of foxl 2 mRNA in the brain and ovary of M.terminalis.foxl 2 gene is the expression of sexual dimorphism in the gonads of M.terminalis,which may play an important role in the maintenance of ovarian development and function.foxl 2 may interact with sex steroid hormones to regulate the gonadal development and function of M.terminalis.This study can provide basic information for the gonadal development and sex regulation mechanism of M.terminalis.