Preparation Of Dissociated Cells And Characteristics Of Neoblast-Expressed Gene Pvmilio In Planarian

Planarians are famous model animals for studying regeneration and longevity mechanism, as theypossess neoblasts, a population of pluripotent adult stem cells able to self-renew and give rise to other cellsor tissues in asexual planarians. Isolating, marking and locating of the neoblasts is the base to study thebiological characteristics of planarian stem cells.To get the optimal cell-dissociated method for planarian, three key factors–trypsin, digestiontemperature and centrifugation speed–were tested in planarian Dugesia japonica and Polycelisyangchengensis. The planarian tissues were digested for15min by0.25%of trypsin (pH=7.2) at20℃in1.5mL of microtube,then centrifugated at1000rpm at4℃for3minto collect precipitation. The intatctcells and fewer cell pieces were seen in the suspended preciptation. Results showed that this method wassuitable for preparing dissociated cells in planarian.For marking neoblasts in planarian Polycelis yangchengensis, the full-length cDNA of pumilio, onespecific neoblast-expressed genes, was first isolated from planarian P. yangchengensis using RACEtechnology and named Pypum. The full-length cDNA of Pypum consists of2048bp, containing an openreading frame of1803bp according to Kozak rules and encoding600amino acid (molecular weight66.2kDa and isoelectric point7.4). It contains a5`untranslated regions (5`UTR) of92bp and3`UTR of153bp.Pumilio homology domain (PUM-HD) of368amino acids (212--579), located in the carboxyl terminus,contains two flanking region and8repeat regions. Compared with D. japonica pumilio gene, the homologyin nucleotide and predicted amino acids are66.8%and76.2%, respectively;70.9%and84.9%, respectively,in Pum-HD. Phylogenetic tree based on Pum-HD indicated that P. yanchengesis, D. japonica, Clonorchissinensis and Schistosoma mansoni construt one monophyly, implying that they have close relationships. In conclusion, this study established the cell-disassociated method for planarian and cloned thefull-length cDNA of pumilio gene of P. yangchengensis, which are the foundation for screening neoblastsand other biological researchs on neoblasts.