Function Analysis Of HSP70 Family Related Genes From Dugesia Japonica

Dugesia japonica belongs to Platyhelminthes Turbellaria,plays an important role in the evolution of animals,and is well known for its extraordinary regenerative ability.But the mechanism of regeneration is still unknown.In this study,the key genes of HSP70 family,Djhsp70 a,Djhsp70b,Djhsp70 c and Djhsp70 d were analysis by Real-time PCR technology,whole-mount in situ hybridization,and RNA interference technique in D.japonica.The results were as follow:(1)First,planarians under temperature,cutting and ionic liquids treatments,the results showed that under temperature below or above 20 ?,cutting damage and ionic liquid treatments,the expressions of four genes were significantly up-regulated.(2)Bioinformatics analysis of Djhsp70 a gene showed that the DjHSP70 a was a stable non secretory hydrophilic protein with a molecular weight of 45.60 KDa,which had a number of phosphorylation sites and glycosylation sites.The protein contained a heat shock protein 70 tag 3 and a highly conserved EEVD sequence at the end.The results of in situ hybridization showed that the Djhsp70 a was widely expressed in whole and regenerating planarians.The whole planarian had strong hybridization signals on both sides of the body.The hybridization signals were also found at the base of bud regeneration of planarian and the regeneration of the 5th day and 7th day were the strongest.After Djhsp70 a gene interference,2/3 of the number of planarias appeared pharyngeal swelling,systemic collapse,head incision,surface crack,and uneven body color deepened.The rate of regeneration in 3/5 of the number of planarias was significantly slower than the control.The expression of Djhsp70 a gene interference detected by Real-time PCR found that the expressions of Djhsp70 a,Djpiwi-1,Djpiwi-b and Djh2 b all decreased significantly.The results suggested that Djhsp70 a gene in planarian regeneration process of cell proliferation and differentiation played a key role.(3)The molecular weight of Dj HSP70 b protein was a hydrophilic protein that contained3 eukaryotic cytoplasmic HSP70 family tags and a highly conserved EEVD sequence.The results of in situ hybridization showed that the Djhsp70 b gene in theplanarian head edge and both sides of the body hadstrong hybridization signals.After Djhsp70 b gene interference,3/5of the number of overall planarias showed gaps in the dorsal midline,pharynx fractured or notched tail.The 4/5 of the number of regeneration of planarians became slow.The rate of regeneration from the tail was significantly lower than that of the control,and there was no eyespots while on the 5th day.The expression of Djhsp70 b gene interference detected by Real-time PCR found that the expressions of Djhsp70 b and Djpiwi-1 were significantly reduced.These results suggested that the gene played an important role in stem cell maintenance and tissue differentiation,such as focal differentiation.(4)The DjHSP70 c protein was a stable hydrophilic protein that contained 3 eukaryotic cytoplasmic HSP70 family tags and a highly conserved EEVD sequence.The results of in situ hybridization showed that Djhsp70 c gene was widely expressed in whole and regenerating planarians,which had strong hybridization signals in the whole planarian head edge,on both sides of the body,and at the base of bud regeneration of planarian.After Djhsp70 c gene interference,7/10 of the number of planarians showed obvious damage before swallowing and tail,until the fracture occurred at the site of injury in the later stage.The 4/5 of the number of planarians stopped regeneration.The expression of Djhsp70 c gene interference detected by qReal-time PCR found that the expression of Djhsp70 c,Djpiwi-1 and the Djpiwi-b all decreased significantly.These results suggested that the Djhsp70 cplayed an important role in planarian for stem cell maintenance and differentiation.And the gene was a critical regulator of regeneration in planarian.(5)The Dj HSP70 d protein was a hydrophilic protein with multiple phosphorylation sites and glycosylation sites.The protein contained a heat shock protein 70 tag 3.The results of whole mount in situ hybridization showed: Djhsp70 d was widely expressed in whole and regeneration planarian,which two sides of the body in the whole planarian,and close to theskin had strong hybridization signals.The hybridization signal of the 5thday was the strongest,the hybridization signal of the 7thday weakened Djhsp70 d gene RNA interference showed that half of the number of planarians had back midline ulcers,tail had obvious damage,and until the late injury occurred fracture.The expression of Djhsp70 d gene interference detected by Real-time PCR found that the expression of Djhsp70 d,Djpiwi-1 and the Djpiwi-b all decreased significantly.The results show that Djhsp70 d gene in planariainvolved in stem cell maintenance,could participate in the related protein of endoplasmic reticulum degradation pathway.In a word,the four genes had a protective function in cells under stress conditions in the planarian.In addition,the four genes regulated stem cell genes to maintain homeostasis in the body.Djhsp70 a,Djhsp70b and Djhsp70 c had effects on the differentiation and proliferation of stem cells,especially Djhsp70 c was a key regulatory factor required for the regeneration process.