日本沼虾表皮Serpin基因克隆及免疫功能的初步研究

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摘要:

丝氨酸蛋白酶级联反应介导的黑化是甲壳动物重要的免疫反应,丝氨酸蛋白酶抑制剂(serine proteinase inhibitor,Serpin)是黑化反应的重要调节因子,而甲壳动物表皮Serpin的有关研究较少.为了探索表皮Serpin在日本沼虾(Macrobrachium nipponense)免疫反应中的功能,基于前期转录组数据,利用PCR、RACE和生物信息学从日本沼虾表皮克隆并鉴定了1个新的Serpin基因,命名为MnSerpin,利用RT-qPCR和RNAi等方法,研究了该基因时空表达模式、嗜水气单胞菌(Aeromonas hydrophila)攻毒后表皮MnSerpin的转录水平以及日本沼虾死亡率的变化.结果显示,MnSerpin cDNA全长2181 bp,编码419个氨基酸,具有Serpin结构域.MnSerpin在腹背部表皮、鳃、血细胞、胃、心、肝胰腺等多种组织均有表达;表皮MnSerpin的表达与蜕皮周期有关,与蜕皮间期(C期)相比,在蜕皮后早期(A期)最高,增加6.06倍(P<0.01).嗜水气单胞菌攻毒后,表皮MnSerpin的相对表达量在6 h达到了峰值,比对照组增加3.77倍(P<0.01).在腹背部第二节的关节膜内注射3μg dsRNA溶液,每12 h注射1次,共注射3次,最后一次注射后12 h,干扰效率最高,表皮MnSerpin的相对表达量同比下降58%(P<0.01).在干扰效率最高的时间点攻毒,120 h内,干扰组虾的累计死亡率比未干扰组增加16%(P<0.01).结果表明,日本沼虾表皮MnSerpin是重要的免疫因子,MnSerpin的表达存在组织以及蜕皮周期不同阶段的差异,该基因表达下调能显著增加嗜水气单胞菌感染虾的死亡率.

Melanization mediated by serine protease cascade reaction is an important immune response of crustaceans in which serine proteinase inhibitor(serpin)are important regulators.However,crustacean epidermal serpin is rarely studied.In order to explore the function of epidermal serpin in the immune response of Macrobrachium nipponense,a new serpin gene,named MnSerpin,was firstly cloned and identified from epidermis using PCR and RACE based on previous transcriptome data.Bioinformatics,RT-qPCR and RNAi methods were used to study the gene sequence characteristics,spatial and temporal expression pattern,transcription level of epidermal MnSerpin and mortality of shrimps after Aeromonas hydrophila challenge.The results showed that the full length of MnSerpin cDNA was 2181 bp,encoding 419 amino acids,which formed a serpin domain.MnSerpin was expressed in abdominal epidermis,gill,hemocyte,stomach,heart and hepatopancreas.The expression level of MnSerpin in epidermis was related to the molting cycle.Compared with the intermolt stage(C stage),it was the highest in the early postmolt stage(A stage)and increased by 6.06 times(P<0.01).After A.hydrophila challenge,the relative expression of MnSerpin in epidermis reached the peak after 6 h,and increased by 3.77 times compared with the control group(P<0.01).3μg of dsRNA solution was injected into the abdominal second segment articular membrane every 12 h,3 injections in total.Compared to control group,RNAi had the highest interference efficiency 12 h after the last injection,where the relative expression of epidermal MnSerpin decreased by 58%(P<0.01).Challenge at the time point with the highest interference efficiency and within 120 h,the cumulative mortality of shrimps increased by 16%compared with the non-RNAi-challenged group(P<0.01).The results showed that MnSerpin from the epidermis of M.nipponense was an important immune factor,its expression was different in tissues and stages of molting cycle,and its down-regulation could significantly increase the mortality of shrimp infected with A.hydrophila.

作者:

宁黔冀彭彦新岳凯迪李亦君

Ning Qianji;Peng Yanxin;Yue Kaidi;Li Yijun(College of Life Sciences,Henan Normal University,Xinxiang 453007,China)

机构地区:

betway官方app 生命科学学院

引用本文:

《betway官方app 学报（自然科学版）》 CAS 2024年第2期123-129,I0006,I0007,共9页

Journal of Henan Normal University(Natural Science Edition)

基金:

国家自然科学基金(30940008) 河南省自然科学基金(182300410033).

关键词:

日本沼虾(Macrobrachium nipponense) 表皮 MnSerpin RNAI