| P.monodon,an economically important crustacean aquaculture species in South China and Southeast Asia,has been shown to develope resistance to oxidative damage under multiple conditions,such as invading pathogens,environmental toxicity,or another change in environmental factors.Vibrio Harveyi is a conditional pathogen that is widely found in the marine environment.It can infect crustaceans,fish and shellfish and other aquatic animals,and cause chronic skin ulcers,sepsis,and exophthalmos in diseased animals.The development of the shrimp aquaculture industry has also been severely affected.Autophagy can phagocytose bacteria or viruses and has important implications for cellular immunity.The regulation of gene expression plays a very important role in the interaction between bacteria and the host,and miRNA is an important factor in regulating gene expression.Therefore,it is of great significance to study the role of miRNA-regulated autophagy genes in the stimulation of Vibrio Harveyi.Partial sequences of PmATG5 and PmATG12 were obtained from the splicing transcriptome of P.monodon established in our laboratory.We use Primer 5 to design specific primers for ORF verification and the rapid amplification of cDNA ends（RACE）approach to get the cDNA of ATG genes 3’ UTR.The tissue distributions of ATG genes transcripts were analysed by quantitative real-time PCR（qRT-PCR）.Screen miRNAs that regulating ATG5 and ATG12 and use the 3’-UTR luciferase reporter assay to verify the relationship between miRNA and ATG5 and ATG12.We investigated the expression of two autophagy-related genes ATG5 and ATG12 under the stress of Vibrio harveyi,the injection of miR-7562-agomir or miR-7562-antagomir,the injection of Vibrio harveyi with miR-7562-agomir or miR-7562-antagomir.We used western blot to investigated the level of autophagy under the injection of miR-7562-agomir or miR-7562-antagomir,the injection of Vibrio harveyi with miR-7562-agomir or Vibrio harveyi with miR-7562-antagomir.The results of the studies are listed as follows:（1）The PmATG5 gene sequence cDNA contains a 114 bp 5’-UTR,a 1357 bp 3’-UTR,and an 810 open reading frame encoding 269 amino acids.The molecular weight of PmATG5 is 31.125 kDa and its theoretical isoelectric point is 5.66.The PmATG12 cDNA has a total length of 1101 bp and contains a 363 ORF,a 16 bp 5’-UTR and a 722 bp 3’-UTR.The open reading frame encodes 120 amino acids and the molecular weight of PmATG12 is 13.59 kDa and its theoretical isoelectric point is 6.12.PmATG5 and PmATG12 were expressed in all tissues of P.monodon,and the highest expression level of PmATG5 and PmATG12 was in muscle（p<0.05）.The expression patterns of both were similarly.The sequence alignment showed that the amino acid sequence of PmATG5 was 87% similar with the ATG5 of the crab（CAJ31266.1）,and the sequence of the PmATG12 was highly consistent with the silkworm（NP001135963.1,71%）.It shows that both are highly conservative in evolution.ATG5 and ATG12 have a very important role in the body.（2）miRNA screening: Three target softwares,mireap,miranda,and targetscan,were used to predict the target genes.The intersection of the predicted results of target genes obtained by the three softwares was used as the result of miRNA target gene prediction.Screen the ATG5 and ATG12 miRNAs from small RNA libraries of P.monodon in our laboratory.A total of 22 miRNAs regulated ATG5,24 miRNAs regulated ATG12,and three genes commonly used to regulate PmATG5 and PmATG12 were mir-7562,mir-8485,mir-278.The expression of miR-278 and miR-7562 were significantly down-regulated at 6h and 24 h,and the relative expression of PmATG5 and PmATG12 reached the highest level at 72 h after V.harveyi infection.The expression of miRNA was negatively correlated with the expression of PmATG5 and PmATG12.The 3’-UTR luciferase reporter assay results showed that miR-7562 could regulate the expression of luciferase with ATG5 3’UTR;after the mutation of the binding site,this regulatory relationship was weakened and decreased by 9.3%.It confirmed that miR-7562 may regulate luciferase expression through this binding site.miR-7562 can regulate the expression of luciferase with ATG12 3’UTR;and after the mutation at the binding site,this regulatory relationship was significantly attenuated and attenuated by 46.2%.Thus,it was confirmed that miR-7562 can regulate the expression of luciferase with ATG5 3’ UTR and luciferase of ATG12 3’ UTR.（3）miRNAs mediate autophagy by modulating the transcriptional levels of ATG5 and ATG12.Overexpression of miR-7562 could down-regulate the relative expression of PmATG5 at both 24 h and 48 h,which was about 0.67-fold and 0.81-fold of that of the control group,and the relative expression of PmATG12 was significantly down-regulated,which was about 0.62-fold that of the control group at 24 h.Silencing of miR-7562 increased the relative expression of PmATG5 and PmATG12,which was significantly higher than that of control at 2.2-and 1.43-fold at 24 h,respectively.After overexpression of miR-7562,the autophagy level of shrimp cells decreased at 48 h.After silencing miR-7562,the autophagy level of shrimp cells increased significantly at 48 h.（4）The role of miR-7562-regulated PmATG5 and PmATG12 in cell autophagy after V.harveyi infection.V.Harveyi injection and overexpressed miR-7562（miR-7562-agomir）showed that the relative expression of PmATG5 was significantly down-regulated in 24 h compared with（V.harveyi+NCM）control group,and the relative expression of PmATG12 was down-regulated compared with（V.harveyi+NCM）group in 6h and 24 h.Protein quantitative results showed that the cell autophagy of P.monodon was significantly lower than that of the control group in 48 h and 72 h.V.Harveyi injection and siliencing miR-7562（miR-7562-antagomir）showed that the relative expression of PmATG5 was significantly up-regulated in 24 h compared with（V.harveyi+NCI）control group,and the relative expression of PmATG12 was up-regulated compared with（V.harveyi+NCI）group in 6h and 24 h.Protein quantitative results showed that the degree of autophagy in 48 h and 72 h cells of P.monodon was significantly higher than that of the control group.Under the stimulation of V.harveyi,the level of autophagy was significantly lower than that of the control group,whether it interfered with the expression of the single gene of ATG5/ATG12 or interfered with the expression of ATG5 and ATG12 simultaneously.All the results showed that miR-7562 bridge V.harveyi infection and host autophagy in black tiger shrimp,Penaeus monodon. |
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