拟穴青蟹Spfox-1基因及调控其表达的lncRNA的鉴定与分析

Identification and Analysis of Spfox-1 Gene and Its Regulation-Related lncRNA in Mud Crab Scylla paramamosain

为探究Spfox-1基因及存在靶向关系的lncRNA在拟穴青蟹性腺发育和幼体发育过程中的作用,从其成熟雌雄性腺转录组中得到开放阅读框全长为1410 bp的Spfox-1序列,该序列编码的蛋白包含1个RRM结构域,进一步预测获得1个可能与Spfox-1基因存在靶向关系的lncRNA HX26820。多重比较和进化树分析表明,Spfox-1的RRM结构域高度保守且与其他节肢动物的fox-1亲缘关系较近。实时荧光定量PCR结果显示:Spfox-1基因在成熟青蟹各组织器官中均有不同程度的表达,其中在卵巢和雄性脑神经节中表达量最高。在性腺不同发育阶段,Spfox-1基因在卵黄发生期的表达量显著高于卵巢其他发育阶段,而在精巢发育过程中,则表达量持续下降。在幼体不同发育过程中,Spfox-1基因的表达量从溞状幼体Ⅴ期到仔蟹Ⅰ期表达量呈逐渐上升趋势。lncRNA HX26820在卵巢发育过程中表达量持续下降,在幼体发育过程中只在溞状幼体Ⅴ期有较高的表达量。研究表明Spfox-1和lncRNA HX26820可能参与卵巢发育和幼体发育。过表达试验显示,lncRNA HX26820能够抑制Spfox-1基因的表达,推测其可能对Spfox-1在性腺及幼体发育中的功能具有负调控作用。本试验结果可为甲壳动物lncRNA调控机制的研究奠定基础。

In order to explore the role of Spfox-1 gene and lncRNA with targeting relationship in gonadal development and larval development of mud crab Scylla paramamosain,Spfox-1 sequences with a 1410 bp open reading frame were obtained from its mature male and female gonadal transcriptome.The protein encoded by this sequence contained an RRM domain,which further predicts the acquisition of an lncRNA HX26820 that may have a targeting relationship with Spfox-1.Multiple comparisons and phylogenetic tree analysis revealed that the RRM domain of Spfox-1 was highly conserved and closely related to that of other arthropods.Real-time fluorescent quantitative PCR results showed that Spfox-1 was expressed in various tissues/organs of mature mud crab to varying extent,with the maximal expression in the ovary and brain ganglion in male.There was significantly higher expression of Spfox-1 at the vitellogenesis stage than that at other stages of ovarian development during gonadal development,while during the development of the testis,the expression continued to be declined.The expression of Spfox-1 was shown to be gradually increased from zoeaⅤstage to larvalⅠstage during larval development.However,the expression of lncRNA HX26820 with targeted relationship with Spfox-1 continued to decline during ovarian development,with high expression in only Z5.The results indicate that Spfox-1 and lncRNA HX26820 may participate in the process of ovarian development and larval development.The overexpression experiment showed that lncRNA HX26820 inhibited the expression of Spfox-1,suggesting that it may have a negative regulatory effect on the function of Spfox-1 in gonadal and larval development.It is the first time to investigate the correlation of gene and lncRNA in a cell line in mud crab,which laid a certain foundation for the study of regulation mechanism of lncRNA in crustaceans.