Expression And Phylogenetic Analysis Of J-inx-like Gene From Planarian Dugesia Japonica

Dugesia japonica(Platyhelminthes, Tricladida) is an invertebrate animal which firstly appears bilateral symmetry and mesoderm. Dugesia japonica is well known for its extraordinary regenerative ability, and platyhelminthes are excellent models for the study of stem cell biology, regeneration, evolutionary and development. INX gene family is the existence form of gap junction proteins in invertebrates. It is an extracellular matrix and is expressed spatially and temporally during embryogenesis, tissue remodeling and repair. The roles of INX in embryonic development, tumorigenesis and chromosomal genetic diseases were uncovered successively. In this study, bioinformatical method was used to investigate the biological evolution of INX gene, and a serial of molecular biology methods, such as Northern blotting, whole mount in situ hybridization and RT-PCR, were used to analyze the structure, expression and function of the gene, in order to further understand the mechanism of regulation and regeneration from the molecular level. L4440-DjInx-like interference vector was also constructed to speculate the potential biological functions of Djlnx-like gene in planarians during different stages.B929, which is belonged to innexin gene family confirmed by BLASTP, was isolated from the cDNA library of Dugesia japonica. We named it Djlnx-like gene as its encoded protein contained a conserved domain of INX protein. The largest ORF of its cDNA consisted by 501bp and encoded a protein of 166 amino acids. Phylogenetic analysis of the protein indicated that Djlnx-like was at the base of the clade in the invertebrates group. Pannexin gene family in vertebrate and innexin gene family in invertebrate were clustered respectively. This result further confirmed that Djlnx-like gene was coordinated with innexin gene family. We assumed that Djlnx-like gene might be a novel Inx gene.Methods such as whole mount in situ hybridization were performed to investigate in vivo expression of the gene in planarian. Embryo in situ hybridization signals were located in the mesoderm, and transferred to the parenchymal tissue during the differentiation of mesoderm. The signals were located in parenchymal tissue in adult planarian, and had continuity with embryo in situ hybridization results. In regeneration planarian, the result was consistent with the adult planarian. Furthermore, in interfered regeneration and non-regeneration worms, the results were similar to the previouse ones. RNAi analysis also confirmed that Djlnx-like gene play a role in the regeneration prosess and lead to the mutation of eyes. Djlnx-like gene may play a role in the regeneration prosess of nervous system and in the formation of the eye.