The Regulation Of Immune-related Genes Expression In Toll-like Receptors Silenced Macrobrachium Rosenbergii And Partial Cloning Of MyD88 Gene

Macrobrachium rosenbergii,also known as the Malaysian prawn,has now become one of the important farmed shrimps in China.It is native to Southeast Asia and mainly survives in freshwater or brackish water.It has the advantages of rapid growth and high nutritional value.However,the long-term disease problem has greatly restricted the development of M.rosenbergii industry,and the shrimp is mainly relying on innate immunity to resist disease invasion.Therefore,studying its innate immune mechanism can provide a reference for the prevention and control of M.rosenbergii disease.This study explored the physiological functions of three Toll-like receptors(TLRs)of M.rosenbergii,which provided a basis for the immune research of M.rosenbergii.The specific research is as follows:(1)The effects of three types of M.rosenbergii Toll-like receptors(MrTLRs)on the expression of immune-related genes in prawns were investigated in the present study,and these results will contribute to the better understanding of the innate immune regulation in M.rosenbergii.In the present study,RNA interference assays about three types of MrTLRs were conducted using specific short-interfering RNAs(siRNAs)with doses of 0.4,0.8,and 1.2?g/g,and the relative expression levels of three Toll-like receptor genes in gills at 0 h,6 h,12 h,24 h,and 48 h after siRNA injection were analyzed with relative quantitative real-time PCR.The result showed that 1.2 ?g/g was the optimal dose of siRNA for RNA interference assay of MrTLRs in the present study.In the RNA interference assay of MrTLR1,the expression of MyD88(Myeloid differentiation factor 88),Crustin and anti-lipopolysaccharide factor(ALF)in MrTLR1-silenced prawns obviously declined at 24 h or 48 h post injection,whereas the MrTLR2 expression was significantly upregulated.Furthermore,the expression levels of MyD88 and Crustin significantly decreased in MrTLR2-silenced prawns,and the silencing of MrTLR3 induced remarkable upregulation of the expression of immune deficiency(IMD)and ALF in M.rosenbergii.Additionally,there was no significant difference of prophenoloxidase(pro PO)expression between MrTLRs-silenced groups and scrambled-siRNA group.These results suggested that MrTLR1 and MrTLR2 might be involved in the regulation ofantibacterial peptides expression,and MrTLR3 exhibited regulatory effects on the Toll signaling pathway as well as IMD signaling pathway in M.rosenbergii.(2)In the experiment,the three TLR genes of MrTLR1,MrTLR2 and MrTLR3 were silenced by injecting siRNA into M.rosenbergii.WSSV or Vibrio alginolyticus infection was injected into M.rosenbergii at 24 h after RNAi.After 24 h and 48 h,the relative expression of immune-related genes in gill was analyzed.The results showed that under the infection of WSSV and V.alginolyticus,the relative expression of MyD88 in the control group increased significantly,but after silencing MrTLR1,MyD88 The expression level was significantly suppressed,and the same situation also occurred in the silence of MrTLR2.Only silenced MrTLR3 had no significant effect on the expression of MyD88;after silenced MrTLR1,the expression of ALF at 48 h after infection with WSSV was significantly lower than that of the control group,and Crustin at 24 h Both at 48 h and 48 h were significantly lower than the control group,and after injection of V.alginolyticus,the expression levels of ALF and Crustin were not significantly inhibited compared to the control group;after silencing MrTLR2,the expression level of Crustin also appeared a similar situation.The expression of IMD after MrTLR was significantly higher than that of the control group after infection with WSSV or V.alginolyticus;after injection of WSSV,the silence of MrTLR1 group Product mortality was significantly higher than other groups,up to 57%;and V.alginolyticus infection,silence MrTLR2 cumulative mortality was significantly higher than other groups,up to 53%.The results show that MrTLR1 and MrTLR2 can regulate the expression of MyD88 and antimicrobial peptides;three TLRs are associated with IMD signaling pathways;it is speculated that MrTLR1 plays a major role in viral immune defense,and MrTLR2 is more effective against bacterial infections.(3)The molecular cloning technique was used to obtain the fragment sequence of M.rosenbergii MyD88 gene.Sequence analysis and phylogenetic analysis were carried out to analyze and determine the expression of MyD88 gene in various tissues and organs of M.rosenbergii.The results showed that the MyD88 gene was expressed in the muscle,heart,stomach,eye stem,gill,hepatopancreas,intestine,and hemolymph tissues of M.rosenbergii.Three times the expression level may be because gills and hemolymph are the main immune organs of shrimp,and MyD88 as an important immune gene is expressed in the immune organs higher.This will lay a foundation for the laboratory to further study the physiological function of M.rosenbergii MyD88 gene.