Molecular Cloning And Whole-mount In Situ Hybridization Of DjMDF Gene In Dugesia Japonica

Dugesia japonica belongs to the phylum Platyhelminthes, class Turbellaria. Planarianspossess derivatives of all three germ layer: ectoderm, mesoderm, and endoderm. Planarianshave robust regenerative ability. Each part can regenerate all missing body parts whetherlongitudinal cutting, angle cutting or crosscut. Morgan discovered that a segmentcorresponding to 1 279th of a planarian could successfully regenerate into a new worm.Freshwater planarians have been used to study regenerative mechanism, cytodifferentiationand pattern formation because of their remarkable regenerative capacity. The study ofplanarian regeneration concentrate on nerve system regeneration, body axis rebuilding andendoblasts’characters maintains. The study of planarian myogenesis, especially the molecularregulation mechanism, is poor. However there are a lot of researches concerning vertebrate,XenoPus, Drosophila, C.elegans, Amphioxus. All the researches indicate that MyoD familyplays an important role in muscle formation.In this paper, we blast the sequence of MyoD family conserved domain by MegAlignsoftware. The gene specify primer pairs used for experiment are designed on the basis of blastresult. We get the conserved sequence of Dujesia japonica myogenic determinatefactor(DjMDF) by the molecular method-polymerase chain reaction. Rapid amplification ofcDNA ends (RACE) is used for the amplification of DjMDF in base of the known part, inorder to get the full-length cDNA sequence. We prognosised the amino acid sequence ofDjMDF by biology software. As well as studied their amino acid sequence alignment andphylogenetic analysis between Dujesia japonica MDF and XenoPus, Drosophila, C.elegans,Amphioxus, vertebrate MyoD family and so on. On the base of known DjMDF full-lengthsequence, we designed the primer pairs used for probe template amplification, whichcontained BamHI and HindⅢcutting site. With the method of in vitro transcription, we gainedthe antisense probe and sense probe used for in situ hybridization and confirm theconcentration of probe and antibody by dot blotting. Then we studied the expression pattern ofDjMDF in different regeneration time by whole-mount in situ hybridization. The regenerationtime contains: 0h, 24h, 36h, 48h, 72h, 5day, 7day, and 2week. Studies have shown that DjMDF participate in myogenesis and play a role in muscle formation to some degree.The full length of MDF cDNA exhibited 1558bp and contains a 29bp 5’-untranslatedregion (UTR), a 1422bp open reading frame (ORF) and a 107bp 3’-UTR. We deuced theamino acid sequence of DjMDF according to the open reading frame. The open reading frame(ORF) encoding a protein of 474 amino acids, and there are a conserved basic helix-loop-helixdomain between position 230 and 366. In the amino acids, position 230-314 is the basicdomain. Helix-loop-helix domain is between position 315 and 366.The sequence and phylogenetic analysis of DjMDF and some other known MyoD familyshow that they are highly similar. A sequence alignment of MyoD family and predictedDjMDF are performed at the amino acid level. The deduced amino acid sequence fromDjMDF was highly similar to MyoD family. All of these genes contain a conserveddomain-basic helix-loop-helix (bHLH). MyoD family forms homodimer itself or heterodimerswith others, binding on the promoter of target gene to regulate myogenesis related geneexpression. According to this, we deduced that DjMDF participated in planarian muscleregeneration and played a role in muscle formation, too.Semi quantitative PCR demonstrated that, after amputation, DjMDF mRNA transcriptswere highly abundant in regenerating planarian. At the beginning of regeneration, DjMDFexpression gradually rise over time and reach the peek when regeneration 36 hours. Soonafterwards, the expression gradually reduces. Yet the expression level is higher than intactplanarians. Whole-mount in situ hybridization demonstrated that DjMDF mainly expressed incells or tissues which are in process of myogenesis. In regenerating tail, the signal was firstfound in mesenchymal cell, along with regeneration and pharynx formation, the signalgradually focus back to the region which will develop a pharynx. In other words, the signalsfocus on the pharynx region little by little. In regenerating head, the signal is similar to theregenerating tail. While in the regenerating trunk, signal was first found in pharynx.Planarians is really the beginning of three germ layer animals, possess mesoderm and allderivatives of it. It’s very important for muscle origin and evolution, to study the regulatemechanism of myogenesis in planarian, even for mesoderm origin and differentiation duringanimal evolution. This paper clone Dujesia japonica myogenic determinant factor (MDF)full-length coda, and studied its amino acid sequence alignment and phylogenetic analysis,then research DjMDF gene expression and functions in planarian regeneration.