Functional Analysis Of Pax6 Genes From The Deep Sea Tubeworm Ridgeia Piscesae And Horseshoe Crab Tachpleus Tridentatus

Pax6 gene was first cloned in mouse and human,which encodes a transcription factor in both vertebrates and invertebrates are conservative and in the eyes of animals,thymus,and in the development of the central nervous system play an important role.Pax6 gene can regulate the expression of multiple downstream genes,and Pax6 mutation can lead to a variety of symptoms,such as aniridia,ectopia pupil,keratitis,nerve developmental disorders,optic nerve hypoplasia etc.,mouse microphthalmia(small eye),the fly eye missing(eyeless),and so on.Functional analysis of Pax6 genes on the tube worms Ridgeia piscesae and Tachpleus tridentatus.The main results were as follows:1: Sequence analysis: Pax6 genes sequences of Ridgeia piscesae and Tachpleus tridentatus have been cloned and affirmed are right,and alignment with the Pax6 gene of Drosophila.And by the phylogenetic tree of Pax6 protein sequence about zebrafish,Drosophila,Tachpleus tridentatus,worm and Ridgeia piscesae shows that:The Pax6 gene of Ridgeia piscesae and the ey/toy gene of Drosophila have high homology,There is high homology of Pax6 genes of Tachpleus tridentatus Pax6a?Pax6b with Pax6 genes of Drosophila ey/toy,and there is high homology of Pax6 genes of Tachpleus tridentatus Pax6c?Pax6d with Pax genes of Drosophila eyg/toe.2: Functional analysis of the Ridgeia piscesae Pax6 : In order to study the function of Pax6 gene,we make full use of Drosophila as a research tool.We have obtained a transgenic fly of Ridgeia piscesae Pax6 gene by using micro injection technology.According to the UAS/GAL4 system in Drosophila to study the function of Pax6 gene.And we found some abnormal phenotypes.We dissected the UAS-RpPax6/ey-GAL4 third instar larval stage of larval Drosophila imaginal disc and found that the eye imaginal disc almost completely missing,and eye imaginal disc is develops into the head an important organization,further proof of the Ridgeia piscesae Pax6 gene in Drosophila over expression can affect the development of theeyes and head.3.To explore the possible molecular mechanisms that the Pax6 gene of R.piscesae overexpressed in Drosophila eye caused the abnormal eye,we selected Drosophila Pax6(ey)downstream target genes,sine oculis(so)promoter,to carry out luciferase reporter assay.In 293 T cells,Pax6 of R.piscesae could not activate the promoter of so in 293 T cells.Moreover,we replaced the C-terminus of R.piscesae Pax6 gene with the C-terminus of Drosophila ey gene and performed luciferase assay.The C-terminal protein of ey Drosophila promoted the N-terminal protein of R.piscesae Pax6 binding to the promoter of so in 293 T cells by the luciferase reporter gene assay.4.In order to study the reason of RpPax6 during the development of Drosophila caused the apoptosis,we make full use of Drosophila model,selection of R.piscesae Pax6 transgenic flies and temperature sensitive flies da-GAL4,tub-GAL80 cross,first step: under the environment of 18 ° C cultivated and then at 29 ° C incubator.Dissected the offspring three instars with acridine orange staining and found that RpPax6 indeed caused apoptosis of Drosophila.To rescue the R.pPax6/ey-Gal4 missing most of the head phenotype,we selected the genotype of UAS-P35 flies,successively with transgenic flies UAS-RpPax6 and ey-Gal4 cross.Generation 6% of y w;UAS-P35 / +;UAS-RpPax6 / ey-Gal4 have head structure,but no eyes,and the remaining 94% have no head phenotype.5,Using the same method of Tachpleus tridentatus Pax6 transgenic flies,and eventually found the flies in the eyes,legs and other structures appear abnormal phenotypes.Illustrates that the Tachpleus tridentatus Pax6 in Drosophila overexpress could affect the normal development process of Drosophila.The Pax6 gene encodes a transcription factor essential for eye development in a wide range of animal phyla.In the regulation of T.tridentatus and deep-sea hydrothermal vents R.piscesae Pax6 function research contributes to understanding of marine animals of the Pax6 gene in the development process of role,and our study helps to understand the role of marine animals Pax6 genes during development.