The Effect Of DjMDF In Dugesia Japonica Muscle Regeneration

Dugesia japonica is the phylum Platyhelminthes, class Turbellaria. Among themetazoans, planarians possess powerful regenerative abilities. There is one classicalexample to confirm these abilities: Morgan’s1898experimention demonstrate that asmall planarian fragment form the1/279th of the intact planarians was able toregenerate a complete planarian. Planarians are a simple organism. In addition topowerful regenerative abilities, they possess the characters of three tissue layers,bilateral symmetry, cephalization, So they play an important role in researching theevolution of metazoans. Over the past century, planarian has become the classicbiological model to answer the question of metazoans and human beingdevelopmental biology. In particular, the study of planarians regeneration mechanismis very important to build the moble of metazoans regeneration mechanism.In this paper, we study the expression of DjMDF in planarians. First of all, weinduced gene silencing of DjMDF by RNA interference (RNAi). Firstly, DjMDF wascloned by PCR amplification, then we connect DjMDF PCR fragments and PPR-240interference vectors producing DjMDF-PPR240recombinat vectors. DjMDF-PPR240recombinat vectors transformed into HT115strain by using an heat stress reaction.Thetwo sides of restriction enzyme cutting site of PPR-240vector have T7RNApolymerase promoters.When the producing DjMDF-PPR240recombinat vectors wereintroduced into competent HT115strain, which lacking RNase III, T7RNApolymerase promoters could be induced to express DjMDF-dsRNA in the presence ofIsopropyl β-D-1-Thiogalactopyranoside (IPTG). So there are lots of dsRNAsexpressed and stored for a long time in HT115cells. Then we resuspendeddsRNA-expressing HT115cells and mixed homogenized liver, ultra-low gellingtemperature agarose, producing planarian artificial food. Finally, we induce genesilencing of DjMDF by feeling artificial food in planarian. In situ hybridization was used as detected the effect of gene silencing afteringestion of bacterially expressed dsRNA in regenerating animals. At the beginning ofregeneration, DjMDF expression gradually rise over time. Until regeneration36hours,DjMDF expression reach the peek. Soon afterwards, DjMDF expression graduallyreduces. The tendency of DjMDF expression is similar in both normal planarians andgene silencing planarians, but the expression level of DjMDF in gene silencingplanarianis lower than normal planarians. Whole-mount in situ hybridizationdemonstrated that feeling the planarian artificial food induce gene silencing ofDjMDF. However, the expression level of DjMDF in gene silencing planarianisreduces but eliminates completely.In transfection experiment, DjMDF and mouse MyoD were cloned by PCRamplification, then we connect DjMDF or MyoD PCR fragments and PPR-240interference vectors producing DjMDF-PPR240recombinat vectors or MyoD-PPR240recombinat vectors. Recombinat vectors and vsv-g、 δ8.91vectors wereintroduced into host cells producing lentivirus containing DjMDF or MyoD PCRfragments.Then lentivirus containing DjMDF or MyoD PCR fragments weretransfected with Fugene-HD to293T cells.Finally, we detect the expression level ofGFP in293T cells by Fluorescence detection technology and FASC technology, anddetect the expression level of DjMDF or MyoD in293T cells by Western-Blot. Theresult of Western-Blot demonstrated that Mody but not DjMDF was expressed in293T, because of species diversity and tRNA diversity.In this paper, we study the expression and the function of DjMDF in planarianregeneration，which is helpful for our understanding of the molecular mechanisms ofregeneration in invertebrate.