Cloning And Expression Analysis Of Djβ-actin Gene During Regeneration Of Dugesia Japonica

Planaria, belongs to the phylum Platyhelminthes, class Turbellaria. Freshwaterplanaria has long been regarded as the animal kingdom from the second layer radialsymmetry evolution to three layer on both sides of the symmetrical typicalrepresentative.It is the first time appear symmetry, the three layer on both sides of theanimal. Planaria have strong regeneration ability, that the cutting down of any part ofthe body can regenerate a complete individual. Eukaryotic cytoskeleton, especially ofactin microfilament, plays a very important role in cell movement. Regenerationprocess is involved in a series and a variety of embryonic morphogenesisphenomenon, which related to the differentiation. In the process of embryo formation,cytoskeleton plays a fundamental role.Based on Dugesia ryukyuensis and Dugesia tigrina beta-actin cDNAsequences,we designed gene specific primer, and use PCR amplification to get part ofthe Djbeta-actin cDNA sequence, and then adopts the technology of the RACE for thefull-length cDNA sequence of Djbeta-actin.We use software to prognosised aminoacid sequence, and carry on the analysis of system evolution. Based on the full-lengthcDNA sequence of Djbeta-actin,we designed primers for probes of whole-mount insitu hybridization to detect the Djbeta-actin expression in complete planaria and indifferent regeneration time planaria. Then we use a technique called RNA interferenceto construct vector of Djbeta-actin RNA interference in order to further analysis therole of the Djbeta-actin gene in the regeneration process.The full-length cDNA of Djbeta-actin is1243bp, including10bp of5’-untranslated region (5’ UTR),102bp of the3’-untranslated region (3’ UTR) and1131bp of open reading frame (ORF).The open reading frame encoding376aminoacids which molecular weight is41.7KD. Its functional protein is composed of371amino acids, which is between position6and376. We use software to analysisDjbeta-actin amino acid sequence,which showed that the species of beta-actin aminoacid sequence is highly conserved in the process of evolution. From theevolutionary tree,we can see that Dugesia japonica and Dugesia ryukyuensis is in the same branch, and with some mammals and invertebrates, fish in different branches.Whole-mount in situ hybridization results showed that in the complete planariaDjbeta-actin expressed in whole body. In the cutting head, the middle section and thetail, Djbeta-actin expression was deceted near the truncation, where the blastemaformation. Along with the regeneration process, Djbeta-actin expression quantityincrease gradually, when the regeneration reach to48hours, Djbeta-actin expressedreach the peak in lesions. More than48hours, Djbeta-actin gene expression bylesions gradually spread to the whole body.When the regeneration reach to7day,Djbeta-actin gene expression return to normal planaria levels. Thus we infer thatDjbeta-actin involved in the planaria regenerate, and played a role.Because of unknown planaria intake for interference food and interference foodrelease and distribution of dsRNA,we found that interference efficiency is not high, soin this study we found that the interference effect is not obvious. The mechanism andmethods of the RNA interference for planaria has yet to be improved, and we willcontinue research of RNA interference in future experiments.