Interaction Between Pax6and Pax6Variant During The Eye Development In Bufo Raddei Strauch

A novel Paired box6(Pax6) mutation called Paired box6variant (Pax6variant) was found in Bufo raddei Strauch, the aim of this study was to explore the relationship between Pax6variant and Pax6in Bufo raddei Strauch. The ORFs of Pax6and Pax6variant were cloned by RT-PCR; for yeast2-hybrid system,four plasmids were constructed:Pax6-pGAD-T7, Pax6-variant-pGBK-T7, Pax6-pGBK-T7and Pax6-variantpGAD-T7; co-immunoprecipitation was used to analyze the relationship between Pax6variant and Pax6in vivo using20stage Bufo raddei Strauch tadpole. Comparing nucleotide sequence of two genes, the homolog of ORF is high, the deduced amino acid sequences of PST region differs. The yeast2-hybrid system results showed that yeast Y190which expressed Pax6and Pax6variant proteins can grow in culture medium which lacked His, and the color of the colony changed, the results proved the interaction between Pax6and Pax6variant in yeast Y190. From Co-immunoprecipitation, the interaction in vivo was found. By3D-PSSM software, we found that Pax-6had two a-helix more than Pax-6variant protein in PST region.Pax6plays an important role in the eyes development, based on the experimental results, we suspected that the interaction between Pax6and Pax6variant may play a important role during the eye development of Bufo raddei Strauch, Pax6variant may influence the eye development by negatively regulating Pax6. Previous researches in other animals had suggested that Pax6mutations were always found in the PST region and caused disease, however, Pax6variant which was found in Bufo raddei Strauch maybe function during embryonic eye development, the molecular evolution of pax6mainly happened in the PST structure domain which was the protein binding area, and the changes of PST in Different Species were various, and the speed of evolution was quick, while the other two DNA binding domains were highly conserved, the evolution speed was relatively slow. Pax6variant and Pax6formed a combination, change in the normal structure or number of Pax6, results in the functional change of Pax6, the regulation of Pax6variant maybe plays an important part in the normal development of eyes.