Studies On The Regeneration Of The Freshwater Planarian Eyespots And The Correlation Between Eyespots Regeneration And Djnos Expression

In this experiment,we use the Dugesia japonica and Polycelis yangchengensis as materials to seek after the most appropriate tempreture for its regeneration. the result shows that the most approapriate tempreture for the Dugesia japonica is 22℃,and that of the Polycelis yangchengensis is 14℃.Basing on this result, Specimens, which were 0-11 days of Dugesia japonica regeneration in the dark at 22℃and 0,7,13,14,15,17,18,19,20,23,26,29,33,35,38 days of Polycelis yangchengensis in the dark at 14℃,were made of paraffin and stained with hematoxylinand eosin,observed that the eyespots regeneration of two planarian.The result shows that the eyespots regeneration of the Dugesia japonica is of a much faster speed than that of Polycelis yangchengensis.On the first day the blaste of the Dugesia japonica ,what is observable and on the third day pigment can be seen . On the fourth, the eyespots can be easily seen.It takes at least 11days to complete the process of regeneration.While for the Polycelis yangchengensis ,it is on the 7th day that blaste can be easily seen. Small eyespots can be seen on the 15th day ,on the 18th day the number of eyespots visible increases considerably. And it took at least 38 days to finish this regeneration process.Apart from the differences,the intensity of what is reproduced of the two dffers as well, what is newly reprodced of the Dugesia japonica being relatively tight,which bears an obvious diffence from its original version,while that of the Polycelis yangchengensis is of little difference from its original one.To select the most stable endogenous reference genes in planarian head regeneration study, three housekeeping genes, 18SrRNA, ACTB and DjEF2, were identified and established expression levels by real-time RT-PCR. Examination of raw Ct values demonstrated that ACTB exhibited the lowest variability (CV= 8.9%). The data were analyzed by GeNorm and NormFinder software. Results showed that ACTB was the most stable expressed control gene with the M value 0.975 (by GeNorm) and stability value 0.021 (by NormFinder) in the planarian head regeneration study.We think ACTB is the best reference genes in planarian head regeneration study.To determine the expression levels of Djnos transcripts during head regeneration, real-time RT-PCR was performed on different time points after amputation. The results showed Djnos transcripts were up-regulated on the third day of regeneration, the critical period of eye precursor cells differentiation ,which implied that Djnos could also participated in the process of proliferation of eye precursor cells during eyes regeneration in Dugesia japonica. Djnos expression values gradually increased and reached the peak on day 12 when the primordial germ cells had formed, which could be related to the proliferation and maintenance of the germline stem cells. Djnos transcripts were expressed in asexual and sexual adults planarian and were more higher in the later.