摘要
【目的】揭示novel miR-56对罗氏沼虾免疫相关基因的调节机制,为进一步探究miRNA参与罗氏沼虾先天免疫调节提供理论依据。【方法】以实时荧光定量PCR检测副溶血弧菌感染后罗氏沼虾鳃组织中的novel miR-56表达变化,采用RNAhybrid预测筛选出novel miR-56的潜在靶基因TLR1,通过双荧光素酶报告基因检测验证novel miR-56与靶基因TLR1的靶向关系;体外注射不同剂量(0.6、1.0和1.5μg/g)的agomiR-56和antagomiR-56干扰novel miR-56表达,筛选出agomiR-56和antagomiR-56的最佳注射剂量,并以实时荧光定量PCR检测罗氏沼虾免疫相关基因的表达变化。【结果】感染副溶血弧菌后,罗氏沼虾鳃组织中的novel miR-56相对表达量随感染时间的推移呈逐渐上升趋势,至感染后72 h,novel miR-56相对表达量约是感染前(0 h)的6.18倍。TLR1是novel miR-56的靶基因,二者的结合位点位于TLR1基因3'非编码区(3'-UTR),且novel miR-56过表达会抑制TLR1基因的表达。于罗氏沼虾围心腔注射0.6、1.0和1.5μg/g的agomiR-56均能显著抑制TLR1基因表达(P<0.05,下同),且以1.5μg/g agomiR-56的抑制效果最优;罗氏沼虾其他免疫相关基因(Spz、TLR2、TLR3、MyD88、IMD、Crustin1、Wntless、ALF1和proPO)在注射agomiR-56后的相对表达量也显著降低。antagomiR-56注射试验结果则恰好相反,注射不同剂量的antagomiR-56对罗氏沼虾TLR1基因有显著促进效果,且随着注射剂量的升高,对罗氏沼虾TLR1基因表达的促进效果呈先上升后下降的变化趋势,以1.0μg/g为antgomiR-56体外注射的最适剂量;罗氏沼虾其他免疫相关基因在注射antgomiR-56后的相对表达量均显著上升。【结论】TLR1是novel miR-56的靶基因,其结合位点位于TLR1基因3'-UTR。novel miR-56通过靶向抑制TLR1基因表达而参与Toll信号通路调控,具有抑制罗氏沼虾免疫相关基因表达的功能。
【Objective】To analyze the regulatory role of novel miR-56 on immune-related genes in Macrobrachium rosenbergii,which could provide theoretical basis for further exploring the involvement of miRNAs in the immune regula‐tion mechanism in M.rosenbergii.【Method】The expression changes of novel miR-56 in the gill tissues of Macrobra‐chium rosenbergii after Vibrio parahaemolyticus infection were detected by real-time fluorescence quantitative PCR.The potential target gene TLR1 of novel miR-56 was predicted and screened by RNAhybrid,and dual-luciferase reporter gene assay verified the targeting relationship between novel miR-56 and target gene TLR1.The expression of novel miR-56 was interfered by in vitro injection of agomiR-56 and antagomiR-56 at different concentrations(0.6,1.0 and 1.5μg/g),the optimal injection concentration of agomiR-56 and antagomiR-56 was screened,and changes in the expression of immune-related genes of Macrobrachium rosenbergii were detected by rel-time fluorescence quantitative PCR.【Result】After infec‐tion with Vibrio parahaemolyticus,the relative expression level of novel miR-56 in the gill tissues of Macrobrachium rosenbergii showed a gradual upward trend with the progression of infection.At 72 h post-infection,the relative expres‐sion level of novel miR-56 was approximately as 6.18 times as that before infection(0 h).TLR1 was the target gene of novel miR-56,and the binding site between them was located in the 3'untranslated region(3'-UTR)of TLR1 gene.More‐over,the overexpression of novel miR-56 could inhibit the expression of TLR1 gene.Intracardiac injection of agomiR-56 at doses of 0.6,1.0 and 1.5μg/g into Macrobrachium rosenbergii significantly inhibited the expression of TLR1 gene(P<0.05,the same below),with the optimal inhibitory effect observed at 1.5μg/g agomiR-56.The relative expression levels of other immune-related genes in Macrobrachium rosenbergii(including Spz,TLR2,TLR3,MyD88,IMD,Crustin1,Wntless,ALF1 and proPO)also significantly decreased after agomiR-56 injection.In contrast,the results of the an‐tagomiR-56 injection experiment showed the opposite trend.Injection of different doses of antagomiR-56 significantly pro‐moted the expression of TLR1 gene in Macrobrachium rosenbergii.With the increase of injection dose,the promoting ef‐fect on TLR1 gene in Macrobrachium rosenbergii expression first increased and then decreased,and 1.0μg/g was the opti‐mal in vitro injection dose of antagomiR-56.The relative expression levels of other immune-related genes in in Macrobra‐chium rosenbergii significantly increased after antagomiR-56 injection.【Conclusion】TLR1 is a target gene of novel miR-56.The binding site is located in the 3'-UTR of TLR1 gene.Novel miR-56 regulates Toll signaling pathway by targeting and inhibiting TLR1 gene,and has the function of inhibiting the expression of immune-related genes in Macrobrachium rosenbergii.
作者
徐齐
戴习林
付元帅
李云
XU Qi;DAI Xi-lin;FU Yuan-shuai;LI Yun(National Demonstration Center for Experimental Fisheries Science Education(Shanghai Ocean University),Shanghai 201306,China;Shanghai Collaborative Innovation Center for Cultivating Elite Breeds and Green-culture of Aquaculture Animals(Shanghai Ocean University),Shanghai 201306,China)
出处
《南方农业学报》
北大核心
2025年第6期1802-1812,共11页
Journal of Southern Agriculture
基金
国家自然科学基金项目(41506159)
上海市科技兴农重点攻关项目(2021-02-08-00-12-F00748)。
