Function Analysis Of SoxB Family In Dugesia Japonica

Dugesia japonica is a member of Platyhelminthes Turbellaria,and occupies an important position in the evolution of animal systems.It has extremely strong damage repairing and regeneration capabilities.Whereas,its mechanism of regeneration is still not clear.In this experiment,the protein structure,expression pattern,and function of DjSoxB1,DjSoxB2 and DjSoxB3 are analyzed using bioinformatics,the whole-mount in situ hybridization,RNA interference,and real-time fluorescence PCR technique in D.japonica.The results are as follows:The bioinformatics analysis of the structure and evolutionary status of the amino acids deduced by DjSoxB1,DjSoxB2 and DjSoxB3 reveal that the three genes belong to the SoxB subfamily and deduced proteins are unstable hydrophilic proteins.DjSoxB1,DjSoxB2 and DjSoxB3 contain not only the Sox family highly conserved HMG-box region and the signature motif RPMNAFMVW,but also the eight characteristic sequence tags of SOXB subfamilies after the HMG-box region.Among them,the HMG-box region of DjSoxB2 is highly homologous to vertebrates and contains a part of SoxP domain.The whole in situ hybridization and qPCR results during regeneration showe that the DjSoxB1,DjSoxB2 and DjSoxB3 gene have strong hybridization signals on both sides of the whole planarian body and on the regenerating blastemas of the regeneration fragments,especially in the 3-5 days of regeneration,the hybridization signal are stronger,and this period is the time of the CNS formation and function recovery of the planarian.After DjSoxB1,DjSoxB2 and DjSoxB3 genes interference,the expression level of DjPC2 are significantly down-regulated,indicating that DjSoxB1,DjSoxB2 and DjSoxB3 participate in the formation of the nerve center of planarian.After DjSoxB1 and DjSoxB2 genes interference,the whole planarian worm do not have obvious phenotypic changes,but the regenerative blastemas of regenerative worms are significantly shorter in the 5-7 days than in the control group,indicating that the regeneration speed of the interference planarian are significantly slower than the control,and there are no distinct eye formation on the 5^th day of regeneration in the tail fragments.At the same time qPCR results showed that after the DjSoxB1 and DjSoxB2 genes interfered,the expression levels of the stem cell marker gene DjPiwiA,cell cycle regulatory genes DjcyclinA/B,Djcdk,and stem cell?neoblasts?early division progeny marker gene DjNB21.11.e are significantly reduced,the expression level of the eye maker gene Djovo is significantly increased at the late stage of DjSoxB1 interference,and the expression level of the eye maker gene Djtyrosinase at the late stage of DjSoxB2 interference is significantly increased,indicating that the DjSoxB1 and DjSoxB2 genes are involved in stem cells proliferation and tissue differentiation.During the process of regeneration of the tail fragments,the indentation of the neck and the small head are observed,and a heterocercal phenomenon appeared during the regeneration of the head fragments,indicating that the ratio of the regeneration part to the original body is out of balance.The results of qPCR show the expression of the dorsoventral axis marker gene DjnlgA is significantly down-regulated in the early stage of DjSoxB1interference,while the expression of DjnlgA and the anteroposterior axis marker gene Dj?-catenin-1 are significantly down-regulation in the early stage of DjSoxB2 interference,indicating that DjSoxB1 and DjSoxB2 participate in the construction of body polarity during the regeneration process.At the same time,the expression of Djcaspase3,a marker of apoptosis,is significantly upregulated after DjSoxB1 and DjSoxB2 interference,suggesting that DjSoxB1 and DjSoxB2 may be involved in the apoptosis of planarian.After disturbance of Dj SoxB3 gene,the worms show malformations at the auricle,the dissolution of the tail,and obvious movement disorders.There are no obvious regenerating blastemas on the 3^th day of regeneration.The regenerative blastemas are significantly shorter than the control group at 5-9 days of regeneration,indicating that regeneration speed is inhibited.In the interference group,the formation of eye is inhibited on the 5^th day and there is a small amount of planarian do not regenerate eye on the 7^th day in the tail fragments.The qPCR results show that the expression of DjPiwiA,Djpcna,DjcyclinA,DjcyclinB,Djcdk,DjNB21.11.e,late division progeny marker genes DjAGAT-2,the marker gene of zeta-NBs subgroup Djzfp,sigma-NBs subgroup marker gene DjSoxp1,and cell migration marker gene Djmmp are significantly down-regulated,the expression level of the eye maker gene Djtyrosinase is significantly up-regulated,indicating that DjSoxB3 plays an important role in stem cell proliferation and tissue differentiation.At the same time,as with DjSoxB1 and DjSoxB2,the DjSoxB3 gene has an indentation of the neck and small head in the tail fragments,and a heterocercal phenomenon appeared in the head fragments,indicating that the ratio of the regenerative part to the original body is disordered.The qPCR results also show that the expression leveles of DjnlgA,Dj?-catenin-1,and DjFrizzled maker gene are significantly down-regulated after DjSoxB3 interference,suggesting that DjSoxB3 participates in the construction of body polarity during regeneration.In addition,after DjSoxB3interference,the expression levels of muscle marker gene Djmhcb,ciliary marker genes DjIFT and DjLC1 are significantly down-regulated,and the speed of movement show that the movement speed of the DjSoxB3 interfered planarian is significantly slower than the control group,indicating that DjSoxB3 participates in the maintenance of the exercise system.The expression of Djcaspase3,a marker of apoptosis,is significantly up-regulated after DjSoxB3interference.Therefore,it is believed that DjSoxB3 participates in the apoptosis of the planarian.In addition,the DjSoxB3 interference group has a greater impact on the planarian phenotype,and at the same time,DjSoxB3 and DjSoxB2 expression levels are significantly reduced at the late DjSoxB3 interference.Therefore,it is believed that DjSoxB3 plays a more important role in the maintenance of organization and regeneration of planarian in the DjSoxB family,and is a key regulatory gene in the DjSoxB family.The results of co-interference between DjSoxB1 and DjSoxB2 show that head dissolving and deformity at the auricle are observed in addition to the phenotypes of DjSoxB1 and DjSoxB2 alone interference.In addition,qPCR test results show that DjSoxB1 does not affect the expression of DjSoxB2,DjSoxB2 only affects the expression of DjSoxB1 in the late phase of the DjSoxB2 interference,indicating that the whole planarian appearing in the co-interference results in head dissolving and the phenomenon of auricle malformation are caused by the superposition of DjSoxB1 and DjSoxB2.The results of co-interference between DjSoxB2 and DjSoxB3 show that in addition to the phenotypes of DjSoxB2 and DjSoxB3alone,there is multiple eyes in the whole planarian,and there is an inconsistency in the proportion of eye in the tail fragments;qPCR results show that DjSoxB2 and DjSoxB3 interact with each other,the results of the co-interference between DjSoxB2 and DjSoxB3 are caused by the synergy between DjSoxB2 and DjSoxB3.In summary,the DjSoxB1,DjSoxB2 and DjSoxB3 genes are widely expressed in D.japonica,and DjSoxBs genes are cross-functional and synergistic.In the regeneration process,they participate in the stem cell proliferation,tissue differentiation and construction of polarity,and play an important role in the early stage of regeneration.