仿刺参SARM基因的克隆、表达和功能研究

Characterization,Expression and Function of SARM in Sea Cucumber Apostichopus japonicus

采用RACE方法克隆了仿刺参TLR信号通路MyD88非依赖途径接头分子SARM,命名为AjSARM。AjSARM基因的序列全长3874 bp,开放阅读框为2412 bp,编码803个氨基酸。通过SMART软件对AjSARM蛋白进行蛋白结构预测,发现AjSARM蛋白由2个SAM结构域和1个C端TIR结构域组成。采用实时荧光定量PCR的方法检测AjSARM基因在仿刺参发育不同时期的表达量,发现在小耳幼虫期开始高表达,神经发育也是从这一时期开始的,表明AjSARM蛋白在仿刺参的发育过程中发挥了重要作用。仿刺参体腔细胞经过4种病原菌(灿烂弧菌、假交替单胞菌、希瓦氏菌和蜡样芽孢杆菌)刺激后,在灿烂弧菌、假交替单胞菌和希瓦氏菌刺激组,AjSARM基因在4 h和24 h的表达均受到抑制,然而在蜡样芽孢杆菌组,24 h表达上调。除了希瓦氏菌组外,其他3个组的AjSARM基因在96 h表达量达到最高。AjSARM在应对不同病原菌刺激后不同时间点所表现出的动态表达模式表明,它参与了仿刺参体腔细胞免疫应答的过程。通过对AjSARM的结构以及在不同发育阶段和不同病原菌刺激后的表达模式进行分析,探索仿刺参MyD88非依赖途径接头分子SARM的功能,可以为棘皮动物的发育和免疫系统进化研究提供参考。

SARM,an adaptor of MyD88 independent signal pathway,was cloned in sea cucumber Apostichopus japonicus,named AjSARM by RACE method.The full length of AjSARM was 3874 bp with an ORF of 2412 bp encoding 803 amino acids.Structure prediction by SMART indicated that AjSARM contained two sterile motifs(SAM)and a C-terminal TIR domain.Quantitative real-time PCR was selected to examine AjSARM expression at different developmental stages of sea cucumber.AjSARM was rapidly up-regulated from auricularia at nervous development stage.It was indicated that AjSARM played an important role in the process of development.After four pathogens stimulus,the expression of AjSARM was inhibited at 4 h and 24 h in the groups of Vibrio splendidus,Pseudoalteromonas nigrifacien and Shewanella baltica chanlenged,but upregulated at 24 h in Bacillus cereus group.Except in S.baltica group,AjSARM reached top expression level all in other three groups at 96 h.The dynamic expression patterns of AjSARM at different times in response to different pathogens proved that it participated in the immune reaction in sea cucumber coelomocytes.The structure and expression analyses of AjSARM help to explore its functions as an adaptor and will lay foundations for the evolution study of immune system in echinoderm and disease prevention in sea cucumber.