| The eyespots morphogenesis and the transcripts expression of transcripts of eye-related genes in the genus Dugesia and Polycelis during eyespots regeneration were studied in this paper using whole-mount in situ hybridization, quantitative Real-time PCR, RACE technique, paraffin sections and bioinformatics. The results were as follow:1. Investigation of eyes morphogenesis in two species D.japonica and P. yangchengensis. Results showed that the eyes of D.japonica were first appeared about at 4 days after amputation, a semilunar eyecup were distinctly observed at 5 days, and the shape and size of new eyes were close to the intact planarians about 15-16 days in the dark at 22℃. then in P. yangchengensis, the regenerated tissue could be observed at 7 days after amputations in the dark at 14℃. The eyes were first appeared about 14-15 days after amputation and the regenerative rate was the fasest about 20-27 days.2. Selection of the most stable endogenous reference genes during planarian head regeneration. Three housekeeping genes,18s RNA, ACTB and DjEF2, were identified and established expression levels by real-time RT-PCR. The data were analyzed by GeNorm and NormFinder softwares. Results showed that ACTB exhibited the lowest variability and the most stable expressed control gene in the planarian head regeneration.3. Cloning and sequencing the full-length of three genes. Djopsin from D. japonica, Psix-1 and Pβ-actin from P. yangchengensis were cloned using RACE technology and sequenced. Three phylogenetic trees were constructed according to the deduced amino acid sequences of Djopsin and Psix-1, and nucleotide sequence of Pβ-actin by the neighbor-joining method, respectively. Results indicated that eyespots morphogenesis, recovery of photophobic response, and quantitative expression levels of Djopsin during eyes regeneration were corresponding, which implied that Djopsin were closely related to the functional recovery and maintenance of the planarian eyes. At the same time, the temporal and spatial expression of Psix-1 was studied by whole-mount in situ hybridization and quantitative real-time PCR in intact planarians and regenerative fragment, the results of whole-mount in situ hybridization showed that Psix-1 were expressed in both regenerating fragments and eyes of intact adults; the transcripts of Psix-1 was up-regulated at 13 and 21 days after amputations and in these days the eyes precursor cells were divised and the eyes were formed. So we speculated that Psix-1 played a key role in the eye differentiation (?) the functional maintenance of visual system.4. Analysis of nine eye-related genes in planarian D.japonica by quantitative real-time PCR. Nine (?) were selected, Djeya, Djsix-1, Eye53, DjotxA, Djnos, Djpax6, Djopsin, Djnetrin and 1020HH. According to their expression levels at different time, these genes could be divided into three groups:(1) early expression group, including Djeya, Djsix-1, Eye53, Djnos and DjotxA. which expression levels were significant increase at 1-3 days after amputation; (2) medium-term expression group, only including one gene, Djpax6, which expression value reached the peak on day 5; and (3) the late, including Djopsin, Djnetrin and 1020HH, which transcripts gradually increased with the eye regeneration. The data suggested that these genes consisted of the eye genetic network to control the eyes regeneration and development. In the genetic network, the single gene could play different function at different regeneration stage, the various genes could be distinctt function or the synergies. The corresponding relationship existed between eyespots morphogenesis and quantitative expression of these genes. |
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