| Penaeus monodon has an important role in aquaculture,but now the disease is an obstacle for the development of aquaculture farming,e.g.kinds of bacteria and virus,and the outbreak of the disease has caused great losses to shrimp aquaculture industry.At the same time,shrimp is favored by people because of its advantages,so the prospects of market is broad.This requires a new method in the disease and including prevention and treatment.The study focusd on the immune defense mechanism and transcriptional responses of immune-related genes.This may facilitate a better understanding of shrimp immune defense and recognition mechanisms and support the sustainable development of better disease management strategies in the crustacean farming industry.JNK signaling pathway,C-JUN,ATF-2 genes are activated under different environmental stresses,including osmotic shock,ultraviolet radiation,heat shock,oxidative stress,bacterial and viral,and protein inhibitors,chemotherapy Agents,apoptosis,immune effectors and proinflammatory cytokines.In this study,SMART-RACE technique was used to clone the full-length sequence of PmJNK,PmC-JUN and PmATF-2.The response mechanism of Gram-negative bacteria and Gram-positive bacteria was verified by qRT-PCR.The genes of JNK,C-JUN and ATF-2 and antimicrobial peptides were analyzed were treated with RNA interference.To test whether recombinant expression JNK,C-JUN and ATF-2 correspond to protein expression.The following results were obtained.In this study,three full-length of PmJNK,PmC-JUN and PmATF-2 cDNA sequence were cloned from P.monodon.The full-length Pm JNK gene contained 2645 base pairs(bp)and was organized into a 5'untranslated region(UTR)of 147 bp,an open reading frame(ORF)of 1263 bp and a 3'UTR of 1235 bp.The1263 bp open reading frame coded for a 421 amino acid(aa)protein with a calculated molecular mass of 47.5 kDa and a basic isoelectric point of pH 6.47.The full-length PmC-JUN gene contained 1875 bp and was organized into a 5'untranslated region(UTR)of 134 bp,an open reading frame(ORF)of 879 bp and a 3'UTR of 844 bp.The 879 bp open reading frame coded for a 293 amino acid(aa)protein with a calculated molecular mass of 32.58 kDa and a basic isoelectric point of pH 7.79.The full-length PmATF-2 gene contained 2291 bp and was organized into a 5'untranslated region(UTR)of 312 bp,an open reading frame(ORF)of 1581 bp and a 3'UTR of 1581 bp.The 1581 bp open reading frame coded for a 527 amino acid(aa)protein with a calculated molecular mass of 55.6 kDa and a basic isoelectric point of pH 7.11.QRT-PCR analysis indicted that the relative expression of PmJNK in the hemolymph,gill were significantly higher other tissues.The relative expression of PmC-JUN and PmATF-2 in the gill were significantly higher other tissues,the relative expression of PmC-JUN and PmATF-2 in hemolymph and hepatopancreas were significantly higher.The technology of RNAi was used to determine the effects of PmJNK,PmC-JUN and PmATF-2 gene expression.We performed preliminary study of the interplay of these genes in vitro.The gene silencing rate of PmJNK in haemolymph was lower than that of hepatopancreas and gills.We also found that the expression of JNK,C-JUN and ATF-2 is downstream,and the decrease trend of C-JUN and ATF-2 was significantly lower than that of target gene(P <0.05),and C-JUN decline trend is higher than the ATF-2 gene decline trend after injection of dsRNA-JNK.The target genes C-JUN and ATF-2 were silenced respectively,and the silencing rate of the two genes was higher in the gills.The expression of C-JUN was decreased after silencing target gene C-JUN,but we found that the expression of JNK and ATF-2 almost no change at all.After injection of dsRNA-ATF-2,ATF-2 expression was decreased,JNK and C-JUN did not change significantly.In this study,the expression of PmJNK was studied using the qRT-PCR method after injection of Vibrio harveyi and Streptococcus agalactia.As a result,the expression of PmJNK,PmC-JUN and PmATF-2 were significantly up-regulated under different stimuli.And the expression of AMP and PEN were significantly up-regulated at different exposure time.In situ hybridization(ISH),PmJNK,PmC-JUN and PmATF-2 positive signals began to increase compared with PBS control group after infection with Helicobacter pylori and Lactobacillus lactis.In this study,target gene silencing was performed on JNK,C-JUN and ATF-2,.After infected Vibrio harveyi and Streptococcus agalactia.The gene of expression was detected in hemolymph,hepatopancreas and gills comparing with the control group.It was also found that the expression of antimicrobial peptides was also significantly decreased.The levels of JNK,C-JUN and ATF-2 were Genes have different effects,and JNK on C-JUN and ATF-2 more obvious impact.The other two genes have different effects,after injection of dsRNA-JNK,dsRNA-C-JUN,dsRNA-ATF-2,respectively.The expression of signal pathway,C-JUN and ATF-2 protein in vitro was investigated by in vitro recombinant protein technique.The success of recombinant protein in vitro was studied by JNK signal pathway,C-JUN and ATF-2 gene.Shrimp immune response to the regulation of the function laid the foundation.To test whether recombinant expression Pm JNK,PmC-JUN,PmATF-2 correspond to protein expression.The protein in prokaryotes was verified via Western blot.Above all,it furthermore provides an evidence that will allow the study of these genes throughout the immunity and antibacterial effect of shrimp,and warrants further investigation into these potentially important gene. |
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