Cloning, Analyzing And Expression Of Pax-6 Gene From Tadpole Of Bufo Raddei Strauch In Lens Regeneration

After lentectomy through the pupillary hole, the epithelial cells at dorsal iris in Anuran tadpole of Bufo raddei Strauch can undergo transdifferentiation to regenerate a new lens. The morphological changes of lens formation from different regeneration stages were observed with both microcope and transmission electron microscope.The results shown that: (1) Bufo raddei Strauch tadpole was capable of lens regeneration, which originated from the epithelial cells at dorsal iris margin by depigmentation. (2) Depigmentation of Bufo raddei Strauch during lens regeneration was by two methods: First, pigment granules initially dispersed all over the cytoplasm were moved towards the periphery of the cell, and then were directly taken up by the amoeboid cells. Second, pigment granules in a cell were first crowded in a mass, surrounded by a membranous structure in the cytoplasm, which were eventually discharged as a mass from the cell. (3) During lens regeneration in the iris epithelial cells of Bufo raddei Strauch; profiles of the mitochondria became complicated ribosome manifold, and the endoplasmic reticulum was cisternal dilation. During the process of differentiation of lens fiber, the cell nucleolus became little, and chromatin shrinked.PAX6 is a member of the family of highly conserved paired-box and homeobox genes. Expression of the Pax6 gene in ocular tissues is essential for the structural development of the eye and ocular cell differentiation in vertebrates. The embryos of Bufo raddei Strauch at 18 stage of development were used in this study. Designing the degenerated primers and the primers of 3'RACE ( Rapid Ampliphication of cDNA Ends ) and 5' RACE, following the PCR cycles and RECE method, we obtained the Full-length cDNA clones of pax-6gene. The process of bioinformatics includes genome location, ORF finding, anticipating function domain and so on. Sequence analysis of this Full-length pax-6 cDNA fragment of Bufo raddei Strauch showed a 2026-bp sequence. The pax-6 sequence was identical 85% and 84% similar to the pax-6 sequence of Xenopus laevis and Homo sapiens, and shown a high homology to the other species. Analysis of its expression during eyes and lens development suggested that it may have important function in evolution.To study gene expression of Pax-6 in the different stages of lens development and lens regeneration in Bufo raddei Strauch model. The expression of Pax-6 gene in the different stages was demonstrated by RT-PCR (Real - Time PCR): (1) The mRNA of Pax-6 gene expression was positive in Bufo raddei Strauch embryo at 16 stage; and the gene expression was also detected at 20 stage. (2) In the lens regeneration of Bufo raddei Strauch, The mRNA of Pax-6 gene expression was positive at 7 day, 30 day during lens regeneration. The gene of Pax-6 was positive during the different time of lens development and regeneration. This result indicated that normal expression of Pax-6 gene was vital for lens development and regeneration and playied a key role in regulation during the process of differentiation of lens fiber and profiles of the cells.Present study would give a better understanding of the molecular mechanisam of pax-6 gene and proteins involved in the lens regeneration and repair of central nervous system.