Clonging And Functions Of Thymosin β4 In The Pearl Oyster Pinctada Fucata

Pinctada fucata is a significant species for the Chinese pearl farming industry.Surgical insertion of mantle pieces and beads for pearl culture is a very important part.This is closely related to the production and quality of pearls.Surgery will create a wound between the foot and the gonad of P.fucata.The process of surgery usually uses red mercury for simple disinfection of mantle pieces.There are essentially no drugs that promote wound healing and inhibit bacterial growth in the wound.Moreover,there are few studies on the molecular mechanism of wound healing in P.fucata.Thymosinβ4 is a multifunctional protein that inhibits bacterial growth,inhibits ROS production,promotes wound healing,promotes cell proliferation and cell migration.In this study,a cDNA of thymosinβ4 was obtained from the transcriptome of P.fucata and named Pfthymosinβ4.To study the potential of Pfthymosinβ4 in pearl culture.First,qRT-PCR was performed on each tissue of P.fucata to initially understand the distribution of Pfthymosinβ4.After immunostimulating P.fucata,the transcription level of Pfthymosinβ4 in hemolymph was detected.To determine whether it is involved in the immune defense process.In vitro antibacterial activity of Pfthymosinβ4 was examined.The relationship between it and heavy metals and H₂O₂ was tested.To understand the pathway and ability of Pfthymosinβ4 to promote wound healing,this article conducted a study on Pfthymosinβ4 transcription levels in hemolymph and wound tissue after surgery,effect of Pfthymosinβ4 on the proliferation and migration of MOVAS,effects of injection and silencing of Pfthymosinβ4 on the number of circulating blood cells in the body and whether Pfthymosinβ4 can promote the healing of wounds.In addition,the transcriptional level of Pfthymosinβ4 during the development of five larvae stages was studied.The main findings are as follows:（1）The thymosinβ4 cDNA was obtained in the transcriptome and named Pfthymosinβ4.The open reading frame of Pfthymosinβ4 is 126 bp,which encodes 41 amino acids.The 5’UTR is 41 bp,3’UTR is 94 bp,and its molecular weight is 4781.43 D.PI is 6.19.It contains the characteristic sequence LKKTETQEKNPL and the sequence LKKTET bound to actin monomers.Pfthymosinβ4 lacks glycosylation sites,signal peptides,transmembrane domains,and transmembrane helices,but it has 7 phosphorylation sites.Thymosinβ4 similarity is very high in different species.In the phylogenetic analysis,it is first grouped with shellfish and fish,and then it is grouped with terrestrial animals and amphibians.In the expression analysis of various tissues,Pfthymosinβ4 expressed the highest amount in hemolymph,followed by foot and gill,and the expression of other tissues was very low and the difference was not significant.In the five seedling developmental stages of P.fucata,the transcriptional level of Pfthymosinβ4 was 2.23-fold up-regulated during metamorphosis.It is speculated that it is involved in the abnormal attachment process of P.fucata.（2）After three hours of the injection of Vibrio anguillarum,Pfthymosinβ4 began to upregulate and reached the highest level at 72 hours and began to decline.After 3 hours of Vibrio alginolyticus injection,the expression of Pfthymosinβ4 was up-regulated,but the difference was not significant compared with the control group.The highest relative expression level was at 12 hours.After the injection of Streptococcus agalactiae,the expression of Pfthymosinβ4 was up-regulated twice at 6 and 48 hours,respectively.After3 to 6 hours of injection of LPS,the relative expression of Pfthymosinβ4 was significantly up-regulated and peaked at 18 hours.From the experimental results,it can be concluded that Pfthymosinβ4 participates in the immune defense process of P.fucata.In addition,the recombinant protein Pfthymosinβ4 has antibacterial activity in vitro.It displays different in vitro antibacterial sensitivities against different bacteria.It has strong antibacterial activity against Aeromonas hydrophila,Vibrio coralliilyticus,V.anguillarum,Vibrio vulnificus,and V.alginolyticus,but has poor antibacterial activity against Vibrio parahaemolyticus,Staphylococcus aureus and Vibrio harveyi.In addition,overexpression of Pfthymosinβ4 in E.coli enhanced the tolerance of E.coli to Cu²⁺,Zn²⁺,Cd²⁺,and H₂O₂.（3）After surgery,the relative expression of Pfthymosinβ4 in the hemolymph continued to increase from 0 to 6 hours,then began to decline and returned to normal.However,its relative expression in wound tissue was initially downregulated but not significant,and it was upregulated at 6 hours and then returned to normal.Pfthymosinβ4is involved in the recovery process after surgery.Pfthymosinβ4 can enhance the proliferation of MOVAS,and the more protein,the stronger the ability to promote cell proliferation.In addition,Pfthymosinβ4 can also enhance the migration ability of MOVAS.P.fucata increased the number of circulating hemolymph after injection of Pfthymosinβ4 protein.The number of circulating hemolymphs of P.fucata decreased after silencing Pfthymosinβ4.Injection of prokaryotic expressed proteins can accelerate the healing of P.fucata wounds.Injecting chemically synthesized proteins has no obvious effect.In summary,Pfthymosinβ4 has antibacterial activity in vitro,enhances cell resistance to heavy metal and H₂O₂,enhances cell proliferation and migration,and promotes wound healing.It has the potential to reduce wound infection rates and accelerate wound healing after surgery.However,further studies are needed to apply thymosinβ4 in cultivating pearls.In addition,Pfthymosinβ4 can promote cell proliferation and migration.During the formation of the pearl sac,the cells of the mantle piece will proliferate and migrate.Therefore,Pfthymosinβ4 also has the potential to speed up the formation of pearl sac.