| The Pax gene family encodes a class of evolutionarily conserved transcription factors, which not only play an essential role in regulating cell proliferation, self-renewal and the coordination of specific differentiation programs during embryonic development, but also play a key role in regulating the body’s physiological and pathological damage repair in tumorigenesis. In1986, the first Pax gene Paired was found in Drosophila melanogaster, Since then a variety of Pax genes were found in the Homo sapiens, nematostella vectensis, Acropora millepora, Trididemnum solidum, oyster and other organisms, thus attracting the attention of scientific community.In this parper, Pax genes of horseshoe crab Tachpleus tridentatus were studied and Pax6gene of deep-sea animals Ridgeia piscesae living in deep-sea hydrothermal areas was preliminary studied. The detailed results were as follows:1. Based on embryonic Tachpleus tridentatus transcriptome sequencing data, we obtained totally five Pax genes with complete CDS using3’RACE and5’RACE. According to the pax gene sequence comparison to other organisms,, we named these Pax genes as Pax1/9a, Paxl/9b, Pax2/5/8a, Pax6a, Pax6b, Pax2/5/8c, Pax3/7a, Pax2/5/8b, Pax6c and Pax6d. In addition, we cloned one Pax6gene containing complete CDS based on the R.piscesae transcriptome sequence.2. In order to study the evolutional relationship between Pax gene family among organisms, the phylogenetic tree was constructed by the animo acid sequences comparison of Pax genes of H. Sapiens, D.rerio, T,’tridentatus, R.piscesae, Drosophila, and C.elegans, It was found that Pax gene family of T.tridentatus were highly homologous to Pax gene family of Drosophila and Pax6gene of R.piscesae were highly homologous to the Pax6gene of H.Sapiens and D.rerio.3. In order to study the function of the Pax genes, we used model organism Drosophila to study Pax gene family function of T.tridentatus. The above mentioned eight Pax genes of T.tridentatus were inserted into pUAST vector and microinjected into the fresh embryos of Drosophila respectively to obtain transgenic flies. Using the UAS/GAL4system, eight transgenic flies were respectively hybridized with ey-GAL4, GMR-GAL4, en-GAL4, da-GAL4and dpp-GAL4Drosophila strains to obtain Drosophila overexpressing Pax genes of T. tridentatus in different tissues, and found abnormal formation of the eyes, legs and wings. The UAS-Tt Pax1/9b/ey-GAL4, UAS-Tt Pax2/5/8b/ey-GAL4, UAS-to the conTt Pax6b/ey-GAL4and UAS-Tt Pax6d/ey-GAL4Drosophila eye were smaller as compared trol; the wing of the UAS-Tt Pax6b/GMR-GAL4was curly wing; the UAS-Tt Pax2/5/8b/GMR-GAL4Drosophila legs had ectopic eyes, UAS-Tt Pax6d/dpp-GAL4leg tarsus was severely shortened leg tarsus;in a word, overexpression of T.tridentatus Pax genes except T.tridentatus Paxl/9a in Drosophila could affect the normal development process of Drosophila. In addition, we use the same method as above constructed transgenic fly of Pax6gene of tubeworms. Using the UAS/GAL4system, the transgenic fly was respectively hybridized with ey-GAL4, GMR-GAL4, en-GAL4, da-GAL4and dpp-GAL4Drosophila strains; found that UAS-Rp Pax6/ey-GAL4Drosophila eye was missing, the middle Drosophila eye of UAS/e-Rp Pax6/GMR-GAL4was missing and the eye surface was smooth; while UAS-Rp Pax6n-GAL4, UAS-Rp Pax6/dpp-GAL4and UAS-Rp Pax6/da-GAL4Drosophila were dead in the embryonic period.4. To explore the possible molecular mechanisms that the Pax6gene of R.piscesae and T.tridentatus overexpressed in Drosophila eye caused the abnormal eye, we selected Drosophila Pax6(ey) downstream target genes, sine oculis (so) and rhodopsinl (rh1) promoter, to carry out luciferase reporter assay. In293T cells, Pax6c and Pax6d of T.tridentatus can activate the promoter of the so gene but did not activate the rhl gene promoter; Pax6a and Pax6b of T.tridentatus and Pax6of R.piscesae could not activate the promoter of so and rh1in293T cells. It is possible that the abnormal eye caused the Pax6c and Pax6b gene of T.tridentatus may be due to competitive binding of the Pax6c and Pax6b protein of T.tridentatus and the ey protein of Drosophila to the so promoter. Moreover, we replaced the C-terminus of Tachpleus tridentatus Pax6b gene and R.piscesae Pax6gene 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 Tachpleus tridentatus Pax6b and R.piscesae Pax6binding to the promoter of so and rh1in293T cells by the luciferase reporter gene assay.Pax gene family encodes conserved protein that extremely widespread in animal, and play a key role in regulating the body damage repair,tumor development and other physiological and pathological process. Our study helps to understand the role of marine animals Pax genes during development. |
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