日本三角涡虫细胞自噬基因Atg5的克隆和功能研究

Molecular Cloning and Functional Analysis of Autophagy-related Gene Atg5 in Planarian Dugesia japonica

细胞自噬相关基因Atg5在细胞自噬过程中发挥重要作用,其功能丧失损坏神经发生和轴突再生。涡虫是研究脑再生的良好模型,其头部切除后1周就能再生出1个新的脑结构。因此,研究Atg5基因在涡虫脑再生中的作用对探究自噬调控神经再生具有重要意义。本研究克隆了日本三角涡虫Atg5基因(DjAtg5)的全长cDNA序列,并利用RNAi技术研究了其在涡虫再生中作用。结果显示,DjAtg5 cDNA全长1014 bp,编码284个氨基酸。DjATG5含有ATG5蛋白质家族的Pfam结构域和高度保守的ATG5-ATG12相互结合位点。切割虫体后,DjAtg5表达显著增加。再生3~5天,其转录本主要在新再生的脑结构表达。但是,敲降DjAtg5并未引起涡虫脑再生异常,且对涡虫干细胞的增殖也未见明显影响。研究结果表明,DjAtg5参与涡虫脑结构的重新形成,但不是涡虫再生的重要调控因子。然而,自噬抑制剂3-MA能够阻止涡虫再生,说明细胞自噬机制对涡虫再生非常重要。

Autophagy-related gene 5(Atg5)plays an essential role in autophagy,the loss of its function impairs neurogenesis and axon regeneration.However,the biological function of Atg5 has not been characterized in planarian.Planarian is an ideal model for the study of brain regeneration.It can regenerate a new brain de novo in 1 week following amputation.To explore the role of Atg5 in planarian brain regeneration,we dissected the molecular characteristics of Atg5 in planarian Dugesia japonica(DjA tg5)and examined its function by RNAi.The full-length cDNA of DjA tg5 is 1014 bp encoding 284 amino acids.The deduced amino sequence of DjA tg5 contains the functional Pfam domain of ATG5 and highly conserved residues for ATG5-ATG12 interaction.After amputation,the transcrips of DjA tg5 are increased and mainly distributed in the newly regenerated brain on day 3-5 of regeneration.However,knockdown of DjA tg5 by RNAi does not impair the regeneration ability and brain structure reformation,nor affects the neoblasts proliferation.Our results suggest that DjA tg5 participates in re-formation of planarian brain structure following amputation,but it is not an important regulator for planarian regeneration.However,autophagy inhibitor 3-MA can block planarian regeneration,which suggests that autophagy is necessary for planarian regeneration.