Cloning And Function Analysis Of Flotillins Gene And Critical Genes Involved Inneurotrophin Signaling Pathwayfrom Dugesia Japonica

Dugesia japonica belonging to platyhelminthes, plays an important role in the evolution of animals and is well known for its extraordinary regenerative ability. At present, although scores of genes have been identified associated with planarians regeneration, the mechanisms and functions of these genes are still unknown.In this study, the key genes of neurotrophin signaling pathway, Djras、Djshc、Djakt、Djmek1 and Djmek2 were cloned by RACE technique, and then their quantification, localization, and functional analysis were performed by Real-time PCR, whole-mount in situ hybridization, immunofluorescence and RNA interference technique.The results were as follow:(1) The full-length cDNA of Djras gene is 1219 bp, including an open reading frame(ORF) of 555 bp encoding a polypeptide of 184 amino acids, which displayed three conserved domains and a CaaX module. Phylogenetic analysis showed that the Ras proteins were highly conserved among species; the Djras gene was more ancestral than those of other ras genes from phylum Platyhelminthes. Real-time PCR results showed that the expression value of Djras gene exhibited a significant upregulation on the 1st and 7th day after amputation. Whole-mount in situ hybridization showed that Djras gene transcripts were expressed in cephalic ganglion peripheral region and intestinal branches, but the signals were stronger in blastemas. Djras gene interference significantly inhibited tail regeneration and delayed head regeneration of planarians. Above results showed that Djras gene played a critical role during planarians regeneration.(2) The full-length cDNA of Djshc gene is 1728 bp, including an open reading frame(ORF) of 750 bp encoding a polypeptide of 249 amino acids, which contained PTB and SH2 domains. Phylogenetic analysis showed that the Shc proteins were relatively conserved among species; the shc genes from phylum Platyhelminthes were ancestral in the history of evolution. Real-time PCR results showed that the expression levels of Djshc gene was upregulation on the 1st and 7th day after amputation. Whole-mount in situ hybridization showed that Djshc gene transcripts were expressed on both sides of planarians bodies and intestinal branches, but the signals were stronger in blastemas. RNA interference results showed that Djshc gene interference inhibited tail regeneration, and both intact planarians and head regeneration showed multi-eyespots abnormality. Above results showed that Djshc gene played an important role in regulating the eyespots differentiation and involved in the regeneration of planarians.(3) The full-length cDNA of Djakt gene is 2067 bp, including an open reading frame of 1758 bp encoding a polypeptide of 585 amino acids, which displayed three conserved domains. Phylogenetic analysis showed that the Akt proteins were highly conserved among species; the akt genes from platyhelminthes were the same origination. Real-time PCR results showed that the expression value of Djakt gene exhibited a significant upregulation on the 1st day after amputation. Whole-mount in situ hybridization showed that Djakt gene transcripts were strongly expressed in the blastemas and extended to the post-blastema with regeneration process. After the interference of Djakt gene, planarians tail regeneration was inhibited. Above results showed that Djakt gene involved in the regeneration and restore of planarians.(4) The full-length cDNAs of Djmek1/2 genes were firstly cloned from planarian D. japonica. The full-length cDNA of Djmek1 gene is 1721 bp, including an open reading frame(ORF) of 1419 bp encoding a polypeptide of 472 amino acids. The full-length cDNA of Djmek2 gene is 1743 bp, including an open reading frame(ORF) of 1278 bp encoding a polypeptide of 425 amino acids. Both DjMek1 and DjMek2 proteins contained a conserved serine/threonine protein kinase catalytic domain and a Ser-X-Ala-X-Ser motif. Phylogenetic analysis revealed that the amino acid sequences identity of DjMek1 and DjMek2 is 48 %; DjMek2 is more conserved than DjMek1 among species. Real-time PCR results showed that the expression levels of Djmek1 gene was significant upregulation on the 1st and downregulation on 5th day after amputation; Djmek2 gene significant upregulation on the 1st and 7th day after amputation. Whole-mount in situ hybridization showed that Djmek1/2 genes transcripts were expressed in intestinal branches and blastemas. The morphology of planarians had no significant changes whether Djmek1 or Djmek2 gene interference, and slower regenerating, crooked tail and tail incision were discovered after Djmek1/2 genes co-interference. Above results showed that the function of the two genes was similarly and complementary, and they played an important role in the regeneration of planarians.(5) Whole-mount in situ hybridization showed that Djflotillins transcripts were ubiquitously in intact planarians except the central nervous system, and strongly expressed in the blastemas of regenerating planarians. Djflotillin-1 gene interference resulted in morphological changes of the intact planarians, such as auricles contraction, blunt head and body folds. The morphological changes of Djflotillins co-interference were similar to that of Djflotillin-1 depletion planarians. However, Djflotillin-2 gene interference did not display any obvious morphological changesin the intact worms. Furthermore, the exercise capacity of planarians significantly reduced after Djflotillins genes interference. Immunofluorescence results showed that there was not any significant morphological defect in the CNS both of intact and regenerating planarians after Djflotillins silencing. Above results indicated that Djflotillins were associated with the regeneration of planarians, but not involved in the maintenance and regeneration of the planarians CNS; Djflotillins played roles in the modulation of actin cytoskeleton and may thus promoted planarians movement; Djflotillins depletion suppressed the endocytosis of planarians.