DjRock2参与东亚三角涡虫再生调节

DjRock2 involved in regeneration activities in Dugesia japonica

利用体外转录的方法合成DjRock2基因的RNA探针,检测RNA探针效率后,对成体及再生过程中的涡虫进行原位杂交试验;将DjRock2基因插入到L4440载体中,构建L4440-DjRock2干扰载体,合成dsRNA并微注射涡虫,利用RT-PCR和Western blot技术检测干扰后再生过程中DjRock2 mRNA和蛋白表达的下调作用。结果表明:DjRock2基因在成熟涡虫中枢神经系统中特异性表达,再生过程中伤口处胚基位置有阳性信号;干扰成体涡虫在再生第3天,头部、中部、尾部都有胚基的形成,但是第5天开始伤口处细胞凋亡,成体干细胞没有完成正常的分化,头部、中部、尾部再生都受到抑制,不能正常完成再生,甚至死亡。RT-PCR技术检测到RNA干扰后DjRock2mRNA的表达显著下降。Western blot检测到干扰后DjRock2蛋白的表达下调。所构建的L4440-DjRock2干扰载体干扰效率高,表型变化明显,从基因和蛋白方面进行分析,为进一步研究Rock2基因的功能奠定了基础。

In vitro transcription of RNA synthesized DjRock2 gene probe, the detection efficiency of RNA probe, and regeneration of adult planarians in situ hybridization.Then insert DjRock2 gene to the vector L4440, construct L4440-DjRock2 interference vector, synthetic dsRNA and microinjection planarian, using RT-PCR and Western blot technique to detect interference regeneration process expressing down effect on DjRock2 mRNA and protein.The results showed that：DjRock2 genes specifically expressed in mature planarian central nervous system regeneration blastema wound posi-tion has a positive signal;interfere adult planarian regeneration of the first three days at the head, middle and tail of the embryo group has, but the first five days at the beginning of the wound apoptosis, adult stem cells do not complete the normal differentiation of the head , middle and tail regeneration is inhibited, not the normal completion of regeneration, and even death.RT-PCR technique significantly decreased DjRock2 mRNA expression was detected after RNA interfer-ence.Upon detection of interference Western blot protein expression DjRock2 down.Constructed L4440-DjRock2 high interference carrier interference efficiency, phenotypic changes significantly, the analysis of gene and protein, laid the foundation for further research Rock2 gene function.