Mediated TRIAP1 In Response To Vibrio Harveyi Stress In Penaeus Monodon

Penaeus monodon has characteristics including excellent taste,nutrient-rich and fast growth,and is one of the most commercially valuable species in Southeast Asia and southern China.In recent years,due to the increasing environmental pressure,the disease-causing microbes have become more and more serious damage,and the breeding industry of Penaeus monodon has been severely hit.Among them,the Vibrio harveyi is a pathogen that can infect crustaceans,fish and shellfish which widely distribute in marine.The Vibrio harveyi causes animal diseases such as exophthalmos,chronic skin ulcers and sepsis easily.There are investigations found that pathogenic bacteria infection can cause apoptosis in penaeus monodon,leading to cell death.miRNA is an important factor regulating gene expression and plays an important role in the regulation of apoptosis.Tumor protein p53 regulated inhibitor of apoptosis 1(TRIAP1)can participate in vivo immune response by inhibiting apoptosis,and its gene expression can be regulated by miRNAs.In this study,the full-length cDNA of TRIAP1 gene of Penaeus monodon was cloned by RACE.The expression pattern of TRIAP1 gene(PmTRIAP1)in different tissues was determined.The miRNAs targeting TRIAP1 were screened and verified by dual luciferase reporter assay.Use qRT-PCR and Western Blot to analysis the effects of miR-454 and miR-145 on the regulation of TRIAP1 gene expression under Vibrio harveyi challenge;and to investigate the relationship between apoptosis induced by Vibrio harveyi、miR-454 、miR-145 and PmTRIAP1.The specific research content is as follows:(1)The full-length cDNA sequence of TRIAP1 in Penaeus monodon has a total of 2522 bp,including 11 bp 5’-UTR,2289 bp 3’-UTR,222 bp ORF encoding 73 amino acids with a molecular weight 8.6 kDa.The theoretical isoelectric point(PI)is 4.9.PmTRIAP1 was expressed in all tissues examined,with the highest expression in blood cells,18.58-fold higher than in ovary.The expression in hepatopancreas and intestine,4.37-fold and 3.49-fold higher than in ovary,respectively.The lowest expression of PmTRIAP1 is expressed in the thoracic nerve.The results of sequence alignment showed that the similarity between PmTRIAP1 and Diaphorina citri TRIAP1 was 61%,which was highly conservative in evolution.(2)Screening of miRNAs: In order to find miRNAs that bind to PmTRIAP1-3’UTR,16 miRNAs were predicted to regulate TRIAP1 using Mireap,Miranda,and Targetscan software.When penaeus monodon was stimulated by Vibrio harveyi,miR-145 and miR-454 were significantly up-regulated at 6 h,24 h,36 h,and 72 h.The relative expression of PmTRIAP1 reached the highest level at 48 h.So the expression of miRNA was negatively correlated with the expression of PmTRIAP1.The dual luciferase reporter showed that miR-454 could regulate the expression of luciferase in the 3’UTR of PmTRIAP1 gene(p=0.0047);however,no significant change in luciferase activity was detected after mutation at the binding site.miR-145 regulates the expression of luciferase with the PmTRIAP1 3’UTR;however,no significant change in luciferase activity was detected after mutation at the binding site.miR-145 and miR-454 regulate the expression of luciferase with PmTRIAP1 3’UTR(3)Overexpression of miR-145 decreased the relative expression of PmTRIAP1 at 24 h and 48 h,respectively,by 0.67 and 0.81 fold;overexpression of miR-454 decreased the expression of PmTRIAP1 at 24 h,a decrease of 0.62 fold;silencing miR-145 can up-regulate PmTRIAP1 expression,which is significantly higher than the control group by 2.2 times and 1.43 times at 24 h and 48 h,respectively.The results of apoptosis assay showed that the apoptosis rate of penaeus monodon was increased at 48 h after overexpression of miR-145.After silencing miR-145,the apoptosis of penaeus monodon was significantly decreased at 48 h.miRNAs mediate apoptosis by modulating the transcriptional level of PmTRIAP1.(4)The role of miR-454 and miR-145-regulated PmTRIAP1 in apoptosis induced by Vibrio harveyi.The results of qRT-PCR showed that the relative expression of PmTRIAP1 over-expressing miR-145(miR-145-agomir)was up to 24 h with(V.harveyi+NCM)after being stimulated by Vibrio harveyi compared with the group,the expression of miR-454 was over-expressed,and the relative expression of PmTRIAP1 was down-regulated compared with the(V.harveyi+NCM)group at 6 h and 24 h after injection.The apoptosis test results showed that the cells of penaeus monodon showed apoptosis was significantly down-regulated at 24 h and 48 h compared with the control group;the relative expression of PmTRIAP1 in silencing miR-145(miR-145-antagomir)at 24 h was higher than that in the control group(V.harveyi+NCI).Significantly upregulated,while silencing miR-454 was up-regulated compared with the(V.harveyi+NCM)control group at 6 h and 24 h after injection,and apoptosis results showed that the penaeus monodon was at 24 h,48 h The degree of apoptosis was significantly higher than that of the control group.Under Vibrio harveyi challenge,the expression of TRIAP1 gene was disturbed,and the level of apoptosis was significantly up-regulated compared with the control group.All the results indicated that miR-145 and miR-454-regulated apoptosis-related gene TRIAP1 regulate the apoptosis of penaeus monodon under Vibrio harveyi challenge.