| Salinity is the most common abiotic factor affecting the culture of Apostichopus japonicas,which is the main factor limiting in the stable development of the sea cucumber industry.In order to improve the economic benefits of sea cucumber breeding,it is indispensable to study the adaptation mechanism of sea cucumber in salt stress.MicroRNA(miRNA)is an endogenous non-coding small RNA of 18-25 nucleotides in length.In this study,the ginseng monocarboxylic acid transporter family 16a13 gene(SLC16A13),the monocarboxylate transporter family 6a8 gene(SLC6A8),the chitin receptor protein gene(FIBCD1),and the AMPA-type glutamate receptor 1 gene(Gria1)).Through the real-time fluorescence quantification technology,the relationship between the expression of four genes in salinity,intestinal and respiratory trees under different stress time,and the change of salinity were explored in order to enrich the physiological theory of sea cucumber and lay a foundation for healthy culture of sea cucumber..The research team constructed a small RNA library of 6 psu for 6 h in the tissues of the sea cucumber and performed high-throughput sequencing to obtain differentially expressed miRNAs and predicted the target genes.Based on the miRNAs and target genes related to salinity stress in high-throughput sequencing,three miRNAs(miR-2008,miR-278-3p,miR-92a)and corresponding target genes were selected.Through the analysis of its expression pattern in vitro and in the sea cucumber,the role of miRNA and target genes in the salinity stress of sea cucumber was obtained,which provided data support for the salinity adaptation mechanism of sea cucumber.The research results are as follows:(1)Four genes were selected from the expression profile of transcriptome database after salinity stress,including: monocarboxylate transporter family 16a13 gene(SLC16a13),monocarboxylate transporter family 6a8 gene(SLC6a8),carapace The receptor protein gene(FIBCD1),the AMPA-type glutamate receptor 1 gene(Grial).qRT-PCR was used to detect the expression of intestinal,respiratory and body cavity cells at 1.5,3,6,12,24,48 and 72 h after the low salt stress.The results showed that the SLC16a13,SLC6a8,FIBCD1,and Grial genes had tissue differences in different tissues at the same stress time point,and the expression levels of the same tissue at different stress time points were sequential.(2)The miR-2008 mimic(agomir)was imported into the primary cultured sea cucumber cells,and the expression profiles of miR-2008 and the target gene PLEKHA3 were detected by real-time PCR.The interaction between miR-2008 and the target gene PLEKHA3 was verified during salinity stress.The expression levels of miR-2008 and the target gene PLEKHA3 were different at different stress time points between the negative control agomir NC group and the agomir 2008 treatment group.The expression level of miR-2008 was significantly up-regulated in 0 h(no salinity stress)and 6 h in the body cavity cells of the sea cucumber.The expression of target gene PLEKHA3 was down-regulated,which was significantly different from the control group agomir NC(P<0.05).The miR-2008 and target gene PLEKHA3 exhibits a negative regulatory relationship in coelomocyte in control group.The expression of miR-2008 and target gene PLEKHA3 was not significantly different from that of control group agomir NC in 24 h body cavity cells(P>0.05).In the 48 h body cavity cells,the expression of miR-2008 was down-regulated,which was significantly different from the control group agomir NC(P<0.05).The expression level of PLEKHA3 was down-regulated,which was significantly different from the control group agomir NC(P< 0.05).(3)After 24 hours of injection of miR-2008 mimic(agomir 2008),the sea cucumbers were subjected to low salt stress.The results showed that the expression levels of miR-2008 and PLEKHA3 were different at different salt stress time points.At low salt stress stage,the expression of miR-2008 was up-regulated,the expression of PLEKHA3 was down-regulated,and the relationship between miR-2008 and target gene PLEKHA3 was negatively regulated.(4)After 24 hours of injection interference reagent(SIPLEKHA3),the sea cucumbers were performed under low salt stress.The expression of the target genes PLEKHA3 and miR-2008 was measured by RT-PCR.The results showed that the expression of PLEKHA3 at 24 h and 48 h after low salt stress was significantly different from other time points(P<0.05).The expression level of miR-2008 at 48 h was significantly different from that of 0 h(P<0.05).In the target gene interference group(SI),the expression level of PLEKHA3 was significantly higher than that at other time points under low salt stress for 6 h.The expression level of miR-2008 at low salt stress time was significantly different from that at 0 h(P<0.05).The expression level of target gene PLEKHA3 was down-regulated at 0 h,and the expression level of miR-2008 was significantly down-regulated.The expression of PLEKHA3 was not significantly different from that of the control group,and the expression of miR-2008 was up-regulated,which was significantly different from the control group(P<0.05).The expression of PLEKHA3 was down-regulated at 24 h,compared with the control group.miR-2008 was significantly down-regulated at ???h.The expression of PLEKHA3 was not significantly different at 48 h,and miR-2008 was significantly up-regulated.(5)The miR-92 a mimetic(agomir 92a)and the inhibitor(antagomir 92a)were introduced into the body cavity cells of the sea cucumber for 24 h and then subjected to low salt stress.The results showed that miR-92 a mimics,inhibitors(antagomir 92a)and negative control sequences were introduced into the body cavity cells of the sea cucumber,and the expression levels of miR-92 a and PLSCR2 were different at different salt stress time points.The expression of miR-92 a was up-regulated at 0 h after non-low salt stress at each time point,and the difference was significant(P<0.01).The time points were up-regulated,and the difference was significant compared with the control group(P<0.05).The target gene PLSCR2 was down-regulated at 24 h and 48 h,and the difference was significant compared with the control group(P<0.05).The expression level of miR-92 a was down-regulated at each time point,and the expression level of target gene PLSCR2 was down-regulated at 0 and 24 h.(6)The miR-278-3p mimetic(agomir)and inhibitor(antagomir)were imported into the body cavity cells of the sea cucumber for 24 h and then subjected to low salt stress.The results showed that miR-278-3p mimics,antagomir and negative control sequences were imported into the body cavity cells of the sea cucumber,and the expression levels of miR-278-3p and Htr2 b were different at different salt stress time points.The expression of miR-278-3p was up-regulated at each time point,and the target gene Htr2 b was up-regulated at each time point.miR-278-3p was down-regulated at 0 h(control group),up-regulated at 24 h,and the target gene Htr2 b was down-regulated at 0 h(control group),at 6hwhile was up-regulated at 48 h. |
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