| In this paper,based on the EST sequence of Penaeus monodon constructed in the laboratory,three genes of the MAPK signaling pathway,ERK branch pathway:PmMAPKK,JNK branch pathway: PmMKK4 and PmMKK7,were screened out from the differentially expressed genes of the salinity stress transcriptomes.In this study,three genes,PmMAPKK,PmMKK4 and PmMKK7,were selected from the differentially expressed genes of the salinity stress transcriptomes based on the EST sequence of the lab-constructed Penaeus monodon.The full-length cDNA of these three genes was obtained by rapid amplification of cDNA terminal(RACE)cloning,and the expression of these three genes in different tissues of P.monodon was analyzed by real-time fluorescence quantification.At the same time,the expression changes of these three genes after microbial stimulation and 96-hour acute low-salt stress experiment were studied,and the effects of these three genes on the low-salt tolerance of P.monodon and the expression changes of related signaling pathways were respectively tracked by RNA interference technology.On the basis of cDNA sequence,the intron sequence of PmMKK4 gene was amplified.By resequencing in three wild populations,the SNP sites related to low salt tolerance were screened,and the correlation analysis between genotype and traits was further conducted in the low-salt stress extreme populations.The full length of P.monodon Mitogen-activated protein kinase kinase(PmMAPKK)cDNA was 3665 bp and the open reading frame(ORF)was 1224 bp,encoding408-aa(amino acid)protein.The predicted molecular weight(Mw)of PmMAPKK amino acids was 45.14 k Da and the theoretical isoelectric point(p I)was 6.29,with 37 phosphorylation sites and 2 glycosylation sites.PmMAPKK has the highest homology with Litopenaeus vannamei according to the analysis of multiple sequences.PmMAPKK was expressed in all the 14 tissues detected in P.monodon,among which the expression level was the highest in the ovary and the lowest in the testis.The expression of PmMAPKK was the highest in the early development of P.monodon.From zoea1 stage,the expression level gradually decreased.The change of hepatopancreatic tissue was more obvious than that of gill tissue.After infection with Staphylococcus aureus and Vibrio anguillarum,the expression of PmMAPKK was significantly higher than that of PBS group(injected with sterilized phosphate buffer saline)(p < 0.05).After infection with Vibrio harveyi,its expression level was significantly lower than that of the PBS group(p < 0.05).We also found that the expression of PmMAPKK in hepatopancreatine and gill tissues changed significantly after low salt stress(p < 0.05).When ds MAPKK was injected,the expression of PmMAPKK gene was decreased,and the mortality rate was increased under low salt stress.The ERK,JNK and p38 signaling pathways are also activated to varying degrees.The full length of P.monodon Mitogen-activated protein kinase kinase(PmMKK7)cDNA length of 2710 bp,including 14 bp 5’ non-coding region(UTR),1295 bp’s 3’UTR,1401-bp open reading frame(open reading frame,ORF),encoding 466 amino acids.The molecular weight was predicted to be 55.33 k D,and the theoretical isoelectric point was 8.97.There were 13 phosphorylation sites and 3 glycosylation sites,belonging to n-linked glycosylation.PmMKK7 contains 261 amino acid conserved kinase domains,including a conserved serine/threonine protein kinase(S＿TKc)region,which is a potential site for MKKK phosphorylation.The results of multiple sequence alignment and phylogenetic tree analysis showed that PmMKK7 and L.vannamei MKK7 genes were clustered in one branch and had the highest homology: 99.14%.The quantitative expression results showed that PmMKK7 gene was expressed in every tissue of P.monodon,among which the highest expression was in the muscle and the lowest expression was in the stomach.After the injection test,both the infected S.aureus group and V.anguillarum group were significantly higher than the control group in hepatopancreatine and gill tissues(p<0.05).In the V.harveyi group,the expression levels in both tissues were significantly lower than those in the control group.After salinity drops sharply to 17 and 3,the expression of PmMKK7 in hepatopancreas and gill tissues was significantly up-regulated.After the knockdown of PmMKK7 gene,the mortality rate of P.monodon under low salt stress was significantly increased,and the expression of the downstream gene Pm JNK of its MAPK signaling pathway was inhibited in each group.It suggests that MAPK signal transduction pathway may play an important role in the immune defense and salinity stress of P.monodon.Mitogen-activated protein kinase kinase 4 of P.monodon(PmMKK4)cDNA was1582 bp,its 5’ and 3’ end non-coding region(UTR)of 567 bp and 157 bp respectively.Open reading frame(ORF)long 858 bp,encoding 285 amino acids.It was predicted that the molecular weight was 32.87 k D,the theoretical isoelectric point was 6.46,and it contained 22 phosphorylation sites and glycosylated sites.PmMKK4 contains A conserved serine/threonine protein kinase(S-I-A-K-T)region,which is the characteristic kinase domain for MKKK phosphorylation.Through homology analysis,PmMKK4 gene has the highest homology with Fenneropenaeus chinensis and L.vannamei.The results of real-time quantitative PCR showed that the expression level in PmMKK4 muscle tissue was the highest,the expression level in the eye stalk nerve was the lowest,and the expression level in the thoracic nerve,intestines,gills and lymphatic tissues was also high.The quantitative results after bacterial infection showed that after S.aureus infection,PmMKK4 in liver,pancreas and gill tissues were up-regulated,significantly down-regulated in both tissues after V.harveyi infection,and continuously down-regulated in both tissues after V.anguillarum infection.The results of the acute low-salt stress experiment showed that PmMKK4 gene in the gill tissue and hepatopandiatic tissue of P.monodon was activated after the stress,causing different degrees of up regulation.After ds MKK4 injection,the expression level of PmMKK4 gene was decreased,which increased the mortality rate of P.monodon under salinity stress.According to the obtained cDNA sequence of PmMKK4,primers were designed to clone part of its intron sequence.Ten SNPS were screened from the cloned genome sequence,one of which was on the exon and nine on the intron.Two groups of low-salt extreme populations were used as the template,and four of them were selected for further correlation analysis.The correlation results showed that there was no significant difference between the four selected SNP sites in the low-salt and low-salt tolerant populations. |
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