三疣梭子蟹(Portunus trituberculatus)foxl2基因功能初探及相关miRNA分析

Preliminary functional study of foxl2 in Portunus trituberculatus and analysis of its related miRNA

foxl2在脊椎动物卵巢分化、发育和功能维持等方面具有重要作用,然而其在三疣梭子蟹(Portunus trituberculatus)卵巢发育中的功能尚不明确。本研究首先克隆了三疣梭子蟹foxl2(Ptfoxl2)基因cDNA全长序列,该基因5′和3′非编码区域(UTR)长度分别为701 bp和211 bp,开放阅读框的长度为1590 bp。基因表达分析结果显示,foxl2在三疣梭子蟹不同组织中均有表达,但在卵巢中表达量最高;其在卵巢发育不同时期的表达存在显著差异,在V期表达量最高;切除眼柄后,该基因的表达出现显著下降;干扰该基因表达后,卵巢vtg基因的表达显著上调。上述结果表明,foxl2可能在三疣梭子蟹卵巢发育调控中发挥重要功能,能够抑制卵巢组织中卵黄蛋白的合成。为进一步分析该基因的表达调控方式,利用生物信息学方法,预测了靶向foxl2的miRNA,并通过双荧光素酶报告基因检测实验,从细胞水平验证了这些miRNA对Ptfoxl2的调控作用;分析了其在卵巢发育不同时期以及切除眼柄后的表达模式。结果显示,共转染miR-9类似物和包含foxl23′UTR的pmirGLO质粒组,萤火虫酶与海肾荧光素酶活性比值出现显著下降,且在卵巢发育过程及切除眼柄后与foxl2表达模式相反。该结果初步证实miR-9可以从转录后水平调控三疣梭子蟹foxl2基因的表达。

The foxl2 plays essential roles in regulating ovarian differentiation,development and functional maintenance of vertebrate.However,its function in ovarian development of the swimming crab Portunus trituberculatus remains unknown.In the present study,we firstly cloned the full-length cDNA of P.trituberculatus foxl2(Ptfoxl2) which contained a 5′ untranslated region(UTR) of 701 bp,a 3′ UTR of 211 bp,and an open reading frame(ORF) 1 590 bp.Gene expression analysis in different tissues showed that Ptfoxl2 exhibited the highest expression in ovary.There were significant differences in its expression in different stages of ovarian development,and its expression was the highest in stage V.After eyestalk ablation,Ptfoxl2 expression decreased significantly.In addition,RNAi of Ptfoxl2 resulted in a significant reduction in vitellogenin expression of ovary.Taken together,these results indicated that Ptfoxl2 played an important role in the regulation of ovarian development of P.trituberculatus by inhibiting endogenous vitellogenesis in maturation stage.In order to further analyze the post-transcriptional regulation of Ptfoxl2,the miRNAs targeting Ptfoxl2 was predicted by bioinformatics analysis and the interaction between the miRNAs and Ptfoxl2 was verified using the dual luciferase reporter assay.The results showed that the relative luciferase activity was significantly reduced after co-transfection of miR-9 mimics and pmirGLO-Ptfoxl2-3′ UTR.Expression analysis showed that miR-9 exhibited opposite pattern at ovarian maturation stage and after eyestalk ablation.Collectively,the results demonstrated that miR-9 can regulate Ptfoxl2 expression in the swimming crab.