| Due to its strong regeneration ability,planarian has become a model organism for studying evolution,regeneration and regulation of stem cell differentiation.B-cell lymphoma-2(abbreviated as Bcl-2),is a proto-oncogene.Bcl-2 gene is one of the most extensively studied apoptosis regulatory genes in the study of apoptosis.In the present study,full-length sequences of DjBcl2-1 and DjBcl2-3 genes are cloned by RT-PCR(reverse transcription PCR)and RACE(rapid-amplification of c DNA ends)from Dugesia japonica.The full-length DjBcl2-1 gene is 848 bp,it contains an open reading frame of 558 bp,which encodes a protein of 185 amino acids with a predicted molecular mass of approximate 21.86 k Da.Its 5’UTR is 178 bp and 3’UTR is 112 bp.The full-length DjBcl2-3 gene is 851 bp,it contains an open reading frame of 639 bp,which encodes a protein of 212 amino acids with a predicted molecular mass of approximate 24.33 k Da.Its5’UTR is 36 bp and 3’UTR is 176 bp.Sequence analysis using NCBI BLASTP identified that both of the proteins contain the four conserved BCL2 domains.Similarity analysis using clustal W2 showed that DJBCL2-1 has 66.1% similarity with the same genus of Schmidtea mediterranea BCL2-1,DJBCL2-3 has 58.5% similarity with the same genus of Schmidtea mediterranea BCL2-3.Phylogenetic analysis using MEGA7.0 revealed that DJBCL2-1,DJBCL2-3 and Schmidtea mediterranea BCL2-1,BCL2-3 are clustered into an independent sub-clade respectively,located in the above of Caenorhabditis elegans,it is in line with evolutionary status,further demonstrated that DjBcl2-1,DjBcl2-3 are the members of the Bcl-2 family.Whole mount in situ hybridization showed that DjBc12-1 and DjBcl2-3 genes are mainly expressed in the intestinal of Dugesia japonica.During the regenerating 3-5 days,the expression levels of the regeneration regions increased while the expression level in the intestinal branch near the eyespots decreased.Further studies by real-time PCR showed that the expression level of DjBcl2-1 decreased in the process of regeneration of planarian’s head,trunk and tail segments through 1-10 days.During the regeneration of the head segment,the expression level of DjBcl2-1 decreased significantly,and increased slightly on the third day.During the regeneration of the trunk and tail segments,the expression level of DjBcl2-1 also decreased significantly on 1-7 days,but both increased on the tenth day.The expression level of DjBcl2-3 gene also decreased in the regeneration process of the planarian’s head,trunk and tail.It decreased more in the trunk segment than in the head and tail segments.The expression level of DjBcl2-3 in the head,trunk and tail segments increased on the tenth day.After RNAi technology was used to knock down Djbcl2-1 and Djbcl2-3 genes,and then the rates of planarian’s movement were counted.Results showed that when the expression of DjBcl2-1 gene was knocked down,the movement rates of the planarian increased significantly on the third day.While the expression of DjBcl2-3 gene was knocked down,the movement rates of the planarian increased significantly on the third and fourth days.For the phenotypic characteristics,no obvious phenotypic changes were observated in the intact planarian.For the regenerating planarian,it was found that the regeneration rates of the head,trunk and tail segments were faster than the control groups.Prog is a cell division marker gene in the early stage of regenerating planarian.When DjBcl2-1 and DjBcl2-3 genes were knocked down by RNAi technology,whole mount in situ hybridization results showed that the expression of Prog was no significant change in the intact planarian,indicating that knocking down the expression of DjBcl2-1,DjBcl2-3genes has no significant effect on the expression of Prog.For the regenerating planarian,the expression level of Prog gene was higher than the control group,indicating that lower expressions of DjBcl2-1 and DjBcl2-3 genes promoted the expression of Prog gene in the regenerating planarian.However,Prog is a marker gene of cell division during the regeneration of planarian and Prog increase in expression can promote cell proliferation and then promote the regeneration of planarian.It indicated indirectly that lower expressions of DjBcl2-1 and DjBcl2-3 genes can promote the regeneration of planarian.Results of single cell gel electrophoresis showed that when the DjBcl2-1 and DjBcl2-3genes’ expressions were knocked down by RNAi technology,the cell damages were aggravated both in the intact planarian and the regenerating planarian.And the cell damages were serious particularly on the first,third and fifth days of regeneration,indicating that DjBcl2-1 and DjBcl2-3 genes can inhibit the apoptosis of planarian.Down-regulated expressions of the two genes can promote cell apoptosis,and then promote severe cell damage.The above experimental results indicate that DjBcl2-1 and DjBcl2-3 genes are involved in the regeneration of planarian and have the function of inhibiting apoptosis.The results of this study provide a reference for elucidating the molecular mechanisms of DjBcl2-1 and DjBcl2-3 genes during the regeneration of planarian. |
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