Cloning And Expression Study Of Runt-1,Runt-2 And HnRNPA2B1 From Dugesia Japonica

In this paper, RACE, whole-mount in situ hybridization, the real-time PCR and bioinformatics technologies were used to study the Dugesia japonica, cloned the full-length cDNA sequence of DjRunt-1, Dj Runt-2 and DjhnRNPA2B1 genes related to regeneration and wound of planarian and analyzed the expression results of these three genes. The results are as follows:1. The full-length cDNA of DjRunt-1, DjRunt-2 and DjhnRNPA2B1 were cloned using the RACE technology and the phylogenetic trees were constructed based on amino acid sequence predicted from these three genes by neighbor-joining method. The full-length cDNA of DjRunt-1 gene consists of 1029 bp and the open reading frame(ORF), 969 bp,encodes a polypeptide of 322 amino acid residues(aa). The full-length c DNA of DjRunt-2 gene consists of 765 bp and its ORF, 576 bp, encodes a polypeptide of 191 aa. The full-length cDNA of DjhnRNPA2B1 gene consists of 1142 bp and its ORF, 825 bp, encodes a polypeptide of 274 aa. Among them, the protein Runt-1 and Runt-2 contain the same "RD" domain and the hnRNPA2B1 protein contains a "RBD" domain. Phylogenetic analysis showed that these three genes had an original phylogenetic position and were very conservative in the evolutionary process.2. The ExPASy, TMHMM and other online bioinformatics softwares were used to analyze the characteristics of the amino acid sequences from the three genes. Results showed that: DjRunt-1 has a 37.55 kDa molecular weight and the isoelectric point is 9.59; DjRunt-2 has a 21.55 kDa molecular weight and the isoelectric point is 9.36; DjhnRNPA2B1 has a 31.4 kDa molecular weight and the isoelectric point is 8.35. These proteins were all hydrophilic with there multiple phosphorylation sites, but without signal peptide and transmembrane region.3. The spatial and temporal expression of the DjRunt-1, DjRunt-2 and Djhn RNPA2B1 genes were analyzed by the whole-mount in situ hybridization and the RT-PCR. The results showed that: DjRunt-1 gene extensively expressed outside the regenerating blastema; RT-PCR showed that DjRunt-1 gene was up-regulated at 1 and 3 days of regeneration and reached a peak at 1 day; DjRunt-2 was also extensively expressed inside the regenerating blastema like DjRunt-1 gene besides expressed outside the regenerating blastema during 5 and 7d regeneration, and RT-PCR showed that DjRunt-2 was up-regulated only after cutting and reached to the peak at 1 day, similar with the DjRunt-1. The results suggest that these two genes could promote the wound healing and induce the proliferation and differentiation of stem cells in Dugesia japonica. The DjhnRNPA2B1 gene was expressed in the testis in immature or sexual maturity planarians, expressed in the regenerating blastema in all the stages and the hybridization signals in 3 and 5 days of regeneration are the strongest among them. The results of the RT-PCR showed that DjhnRNPA2B1 gene was up-regulated at 0, 5 days and reached the peak at 0 day, which implied that Djhn RNPA2B1 genes was induced by the wound signals to regulate the cell proliferation and differentiation and DjhnRNPA2B1 gene could be a multifunctional gene which participated in the reproductive system and the development of the nervous system of planarian.