Biological Characteristics And Functions Of Pax6 Variant In The Lens Development Of Bufo Raddei Strauch

Pax6 is one of many transcription factors during the development of tissues including central nervous system and endocrine glands of vertebrates and invertebrates,which also regulates eye development in animals ranging from jellyfish to Drosophila to humans.There are several reported isoforms of the Pax6 protein,including Pax6, Pax6ΔPD and Pax6(5a).Previous studies have shown that different isoforms activate different sets of downstream targets during development.We made use of the tadpole of Bufo raddei Strauch at stage 20(gills blood circulation stage),and new gene--Pax6 variant(Genebank:FJ175151)was amplified by Rapid Amplification of cDNA Ends(RACE) methods.Bioinformatic analysis was carried out, such as removal of vector,ORF finding,translating coding domain,analysing homology and so on.Compared with Pax6 and Pax6 variant,the difference is in the 3' end of PST domain and whose similarity reaches 53.1%.Highly conservativeness in paired domain and paired-like homeodomain implies that Pax6 variant plays a crucial role in fundamental developmental processes.The different sequences of Bufo raddei Strauch Pax6 and Pax6 variant were amplified by reverse transcription polymerase chain reaction(RT-PCR),and were subcloned into the pMD18-T vector.Then positive clones were selected for nucleotide sequencing. Pax6 and Pax6 variant genes were inserted into pGEX-4T-3 to yield the recombinant cloning vector pGEX-Pax6 and pGEX-Pax6 variant.After recombinant cloning vectors were transformed to Rosetta(DE3),fusion proteins GST-Pax6 and GST-Pax6 variant soluble proteins induced by adding 1.0 mM IPTG,can be purified by GST via affinity chromatography using GSTrap^TM FF columns,white rabbits by intramuscular injection and several subcutaneous injection on the back of rabbits homogenized proteins with Freund adjuvant.Sensitivity and specificity for the antisera(Polyclonal antibodies)by ELISA shows that the antibody titers both reach to 1:100 000.Besides, Western Blot assay shows the transcription factors of Pax6 and Pax6 variant were present in Bufo raddei Strauch embryonic developmental processes.From the gills blood circulation stage to the opercular closure stage,Pax6 is present,while Pax6 variant only exists in gills blood circulation stage.The truncated PST domain of Pax6 variant lose their transactivation ability,whose DNA binding ability was remain,which resulted in Pax6 repression with dominant negative effects that competed for target DNA-binding sites with the Pax6.Our results laid the foundation for the mechanism of Pax6 variant gene and Pax6 variant protein in the process of the amphibian lens development,and provided the basis for future studies into the functions of Pax6 isoforms.