Transcriptome Analysis Of The Regeneration Process Of Dugesia Japonica Based On High-throughput Sequencing

Dugesia japonica provides a good model for the research of regeneration mechanism due to its strong regeneration ability and short regeneration cycle.In this study,the Illumina Hi Seq^TM2000 sequencing technology is used on the transcriptome of the planarian which is intact worms and regenerated worms that were cut down the head after 0d,1d,3d,5d,and 7d.And the biological information of the sequencing results have been analyzed preliminary.According to the analysis results,the functions of the three genes,DjCAT,DjGST and DjSOD,with significantly increased expression levels in the early stage of regeneration are selected for analysis by RNAi and qPCR.The purpose of this research is to explore the physiological activities and regeneration mechanisms of Dugesia japonica in regeneration process.The main results are as follows:?1?Gene information of Dugesia japonica is enriched by transcriptome sequencing.A total of 101,183 unigenes are obtained in this test.Among them,30.78%of unigenes are matched against at least one database for functional prediction.Nine different expression genes are randomly selected for qPCR validation,and the results demonstrate the reliability of transcriptome data.Combining the KOG and GO databases to predict and classify the function of differential genes in different experimental groups.KEGG data has 8106 genes involved in 257 metabolic or signal pathways of planarian.The largest number of differential genes are obtained from 1 day of regeneration and 5 days of regeneration?839 and 239unigenes,respectively?,and the involved metabolic pathways are also abundant with the intact group and the regeneration 0 day group as the control group respectively.The active physiological and biochemical reactions in Dugesia japonica on the first day of regeneration played a key role in the repair and regeneration after injury.The fifth day of regeneration is a important functional recovery period when the up-regulated or down-regulated gene expression in the early stage of regeneration gradually returned to normal.Therefore,it is considered that 1 day and 5 days of regeneration are the critical periods of head regeneration.?2?The results of DjCAT expression during different regeneration periods show that DjCAT is upregulated during the entire regeneration process,reaching the highest value on the first day.RNAi results show that 3/5 of the tail of intact worms collapsed and the body shrunk and died.9/10 planarian is not able to be observed the regeneration of pharyngeal on the fifth day in tail regeneration.Two-fifths of worms have abnormal eye spots on the fifth day of head regeneration.The measurement results of blastema show that on the third day of regeneration,the lengths of the blastema of tail fragments are significantly larger than that in control group and return to normal on the seventh day.The results of the marker gene test show that after DjCAT interference,the sigma-neoblasts marker gene DjSoxp-1,cell cycle regulatory genes Djcyclin A and DjcyclinB,tail marker gene DjFrizzled,muscle fiber marker gene Djmhcb significantly increased,and the eye spot marker gene Djtyrosinase significantly downregulated.So we speculate that DjCAT?RNAi?promotes the proliferation of neoblast and interfered with the synthesis of eye pigments and the formation of pharynx.The expression of myosin genes and the maintenance of the tail are severely affected.?3?DjGST expression changes at different regeneration periods show that the expression of DjGST is upregulated during the entire regeneration process and reached the highest value on the third day of regeneration.The results of RNAi show that both the intact?4/20?and the regenerative worms?4/20?appear tail ulceration and body shrinkage.The measurement results of blastema show that the lengths of blastema of tail fragments are significantly smaller than that in control group on the 3rd and 4th day of regeneration.And the lengths of blastema of head fragments are significantly smaller than that of the control group on the 5th day of regeneration,and return to normal on the seventh day.The results of the marker gene assay show that after DjGST interference,the expression levels of the neoblasts marker genes DjpiwiA and Djpcna significantly downregulated,and the expression levels of the stem cell division marker genes DjAGAT-2 and DjNB21.11.e significantly upregulated.So we speculate that the proliferation of neoblast cells is inhibited after DjGST was interfered,while the early differentiation of neoblast is promoted.?4?DjSOD expression changes at different regeneration periods show that:compared with the intact group,the expression of Dj SOD increases at the early stage of regeneration and reaches the highest value on the first day,then gradually decreases on the third day and to the lowest value on the fifth day of regeneration.On the 7th day,it is significantly higher again.The results of RNAi show that 1/2 of intact worms appear tail ulceration and shrank in different parts of the body.The measurement results of blastema show that the growth rate of regenerative group is slow,and the lengths of blastema in head regeneration and tail regeneration are significantly smaller than that of the control group on the 3rd,4th,5th,and6th days of regeneration,and then gradually return to normal.In addition,2/5 of the head fragments lysis on the fifth day of regeneration.The results of marker gene detection show that after DjSOD interference,the expression levels of DjpiwiA and Djpcna significantly downregulated,while the expression levels of DjAGAT-2,DjNB21.11.e,Djmhcb,and DjFrizzled significantly upregulated.We speculate the proliferation of neoblast cells is inhibited after DjSOD was interfered,which promotes the early differentiation of neoblast cells and interferes with the normal expression of myosin genes and the maintenance of the tail homeostasis.In summary,the 1st and 5th days of regeneration after cutting are the critical periods for the initiation of regeneration and functional recovery,respectively.DjCAT,DjGST,and DjSOD influence the regeneration of planarian by regulating the proliferation of stem cells,and play an important role in the maintenance of normal tissue regeneration and homeostasis of planarian.