The Function Analyses Of Eya And Six Family Genes On Eyespot Development In Polycelis Yangchengensis

Polycelis yangchengensis is a species of Tricladida,Planariidae.It has strong regeneration ability and simple eye spot structure,each eye spot consists of only one pigment cell and one photoreceptor cell.Therefore,it is of great significance to study the eye spot regeneration of P.yangchengensis..So far,there have been few studies on eye spot regeneration and gene regulation of P.yangchengensis.In this study,we have selected two conserved gene families,Eya family and Six family,from the P.yangchengensis transcriptome that they can affect eye spot development.Through bioinformatics analysis,situ hybridization,RNAi and other technologies,we studied the expression pattern and related functions of gene Peya-1,Peya-2,Psin,Psix1,Psix2 in P.yangchengensis.Here was the results.Through bioinformatics,we analyzed the physicochemical properties and protein structure of the amino acid sequence deduced by genes Peya-1,Peya-2,Psin,Psix1,Psix2.The results show that,Peya-1and Peya-2 belong to the Eya family.The protein encoded by the Peya-1 gene is expressed as an unstable hydrophilic protein in the nucleus of eukaryotic cells.The protein encoded by the Peya-2 gene is expressed as a stable hydrophilic protein in the nucleus of eukaryotic cells.Both of them have the ED domain unique to the EYA family.Genes Psin,Psix1,Psix2 belongs to the Six family,and the proteins they encode are unstable hydrophilic proteins expressed in the nucleus of eukaryotic cells.They all have SD domain and HD domain unique to the SIX family.Here was the situ hybridization results.Genes Peya-1 and Peya-2 are mainly expressed in the parenchyma tissue and the area around the eye point.Genes Psin,Psix1 and Psix2 are mainly expressed in the body tissues of planarians,they also have a strong positive signal in the head.After RNA interference of genes Peya-1 and Peya-2 in P.yangchengensis.q RT-PCR detection was performed on the control and experimental groups of planarians on the 14days,21days and 38days after the RNA interference.The results show that,compared with the control group,the expression levels of Peya-1and Peya-2 in the experimental group were significantly reduced.Stereo microscope observation results show that,in the experimental group,the whole planarians had degenerated eye spots;the tail regenerative head group worms failed to regenerate the eye spots normally on the 14days;The number of regenerated eye spots was still less than that of the control group on 21days and 38days.By measuring the length of the bud base in the control group and the experimental group,there was no significant difference in the length of the regenerated bud base between the two groups.After RNA interference of genes Psin,Psix1 and Psix2 in P.yangchengensis.q RT-PCR detection was performed on the control and experimental groups of planarians on the 14days,21days and 38days after the RNA interference.The results showed that compared with the control group,the expression levels of Psin,Psix1 and Psix2 in the experimental group were all significantly reduced.The observation results of stereomicroscopy showed that compared with the control group,the whole planarian group in the experimental group showed the phenomenon of eye spot degeneration.In the tail regeneration head of planarians group,the eye spot regeneration of the experimental group was slow,or even no eye spot regeneration,compared with that of the control group on 14days.On the 21days and 38 days,although there were regenerative eye spots in the experimental group,the number was still less than that in the control group.There was no significant difference in bud length between the control group and the experimental group.Immunofluorescence results showed that the neuronal development was slow in the genes Peya-1 and Peya-2 gene interference groups on the 14days of regeneration.The number of H3P~+cells in the Psin,Psix1 and Psix2 genes interference group didn't change significantly on the 14days of regeneration.q RT-PCR results showed that,after RNA interference of Peya-1 and Peya-2,the expression levels of Psin,Psix1 and Psix2 were reduced..After Psin RNA interference,the expression level of Peya-1 was reduced.No changes in expression levels of other genes were detected after RNA interference of Psix1 and Psix2.The above results are summarized that genes Peya-1 and Peya-2 belong to the Eya gene family,genes Psin,Psix1 and Psix2 belong to the Six gene family.The Eya gene family and the Six gene family have synergistic effect in P.yangchengensis.They can affect the differentiation of the eye cells,but it has no obvious effect on cell proliferation and bud base regeneration in P.yangchengensis.