| Thymosin ?4,as a newly discovered important gene that can simultaneously inhibit bacterial growth and promote wound healing,has not been studied in P.monodon.In this study,thymosin ?4 gene of P.monodon(Pm?4)was cloned and its role in antibacterial immunity and wound healing was explored.The antibacterial activity of Pm?4 in vitro was studied by liquid medium method and enzyme-linked immunosorbent assay.By RNAi and overexpression techniques,the clearance effect of Pm?4 on V.parahaemolyticus in hemolymph and the effect of Pm?4 on the survival rate were clarified.The relationship between Pm?4 and key genes of wound healing(SOD and CAT)was explored through RNAi and overexpression.The effect of Pm?4 on wound healing was further confirmed by tissue sections.The mechanism of Pm?4 in innate immunity and wound healing of P.monodon was preliminarily revealed,which provided theoretical support for the infection and prevention of pathogens in the process of shrimp farming,and the repair of wounds.(1)The full length of Pm?4 c DNA sequence was obtained in the transcriptome.The ORF is 501 bp and encodes 166 amino acids.The predicted protein molecular weight is 18714.05 D and the isoelectric point is 5.75.The results of multiple sequence alignment and phylogenetic tree showed that Pm?4 of P.monodon was similar to thymosin ?4 of other prawns and it had the closed evolutionary relationship with Penaeus japonicas.Tissue distribution revealed that Pm?4 is abundantly expressed in immune organs such as hemolymph,lymphatic and hepatopancreas of P.monodon.(2)In this study,the antibacterial ability of Pm?4 was studied in vivo and in vitro.In vitro,in the enzyme-linked immunosorbent assay(ELISA)experiment,when the protein concentration reached from 64?g / m L to 128?g / m L,the binding activity of V.parahaemolyticus,V.vulnificus,V.harveyi and Vibrio anguillarum showed high binding activity.The liquid culture method has strong antibacterial activity against Vibrio vulnificus,V.parahaemolyticus,Streptococcus agalactiae and S.aureus.The results showed that the recombinant protein Pm?4 had broad-spectrum antibacterial activity and different sensitivity in vitro.In vivo,first of all,in the immunostimulation experiment,the expression levels of Pm?4 showed an upward trend,reached a peak and remained relatively high after the injection of Vibrio harveyi,Vibrio parahaemolyticus and Staphylococcus aureus.In situ hybridization and q RT-PCR experiments proved that the ds RNA successfully interfered with Pm?4.After injection of V.paralyticus,the hemolymph-coated plate method was used to estimate that the bacterial concentration of the interference group was significantly higher than that of the blank control group.At this time,the number of dead P.monodon was increased.The results of the overexpression group of recombinant protein Pm?4 were contrary to those of the interference group,indicating that Pm?4 participated in the immune defense process.(3)In order to study whether Pm?4 has the effect of wound healing and explore how to promote the specific mechanism of wound repair.Firstly,the relative expression of Pm?4 in hemolymph was analyzed after making the wound.It was found that Pm?4 peaked after 12 h and has an obvious upward trend.It was preliminarily speculated that Pm?4 is related to the recovery process of wound healing.Then the expression of wound healing genes was detected after 24 hours of silence and overexpression of Pm?4.After wounding,it was found that the expression levels of CAT and SOD genes were significantly reduced after interference and significantly increased after overexpression.It is suggested that Pm?4 may participate in the wound repair process by regulating the expression of antioxidant genes.(4)In order to deeply explore how Pm?4 participates in the wound repair process,the wound condition of muscle was observed by tissue section.The damaged muscle tissue changed and was infiltrated by blood cells,surrounded by melanin in a circle and then the outermost layer began to necrosis.In the interference group,necrosis began to occur earlier than the blank group.However,no necrotic area was observed in the overexpression group.It shows that Pm?4 can delay traumatic necrosis.It is fully proved that Pm?4 may promote wound repair by regulating the transcription of wound healing genes and delaying necrosis. |
PDF Full Text Request