香港牡蛎(Crassostrea hongkongensis)新型凝集素ChPerlucin的基因克隆与功能研究  

Molecular cloning and functional studies of ChPerlucin in Crassostrea hongkongensis

作　　者：赵泽慧 张爱娇 杨雨澄 毛帆[1] 肖述[1] 李军[1] 张扬[1] 向志明[1] 喻子牛[1] ZHAO Zehui;ZHANG Aijiao;YANG Yucheng;MAO Fan;XIAO Shu;LI Jun;ZHANG Yang;XIANG Zhiming;YU Ziniu(CAS Key Laboratory of Tropical Marine Bio-resources and Ecology,South China Sea Institute of Oceanology,Chinese Academy of Science,Guangzhou 510301,China;University of Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[1]中国科学院热带海洋生物资源与生态重点实验室(中国科学院南海海洋研究所),广东广州510301 [2]中国科学院大学,北京100049

出　　处：《热带海洋学报》2022年第1期42-51,共10页Journal of Tropical Oceanography

基　　金：广东省自然科学基金项目(2021A1515010541)。

摘　　要：C-型凝集素(C-type lectin)是一类能识别并结合多糖的免疫蛋白质超家族,本文首次克隆了一种新型香港牡蛎C-型凝集素基因——甘露聚糖结合凝集素,并获得了该基因的全长序列,命名为ChPerlucin。结果显示,ChPerlucin基因cDNA全长577bp,包括21bp的5′末端非翻译区(untranslated region,UTR)、73bp的3′UTR,以及483bp的开放阅读框(open reading frame,ORF),编码160个氨基酸,ChPerlucin蛋白理论分子量为18kD,等电点为5.95。ChPerlucin蛋白含有保守的C-型凝集素样结构域(C-type lectin or carbohydrate-recognition domain,CLECT);邻接法(Neighbor-Joining,NJ)进化树分析表明,ChPerlucin与其他贝类的Perlucin聚为一支,说明该基因是软体动物Perlucin家族的新成员。qRT-PCR结果显示,ChPerlucin在香港牡蛎所检测组织和各个胚胎发育时期中均有表达;病原菌刺激后,ChPerlucin表达量显著升高;利用毕赤酵母真核成功表达了ChPerlucin重组蛋白,发现ChPerlucin重组蛋白具有抑菌效果。以上结果表明ChPerlucin在香港牡蛎的先天性免疫过程中发挥了重要的作用。The C-type lectin superfamily is a class of immune proteins that can recognize and bind to polysaccharides.In this study,a new type C-type lectin gene of Crassostrea hongkongensis was cloned for the first time,and the full-length sequence of the gene was obtained.The results show that the full length of ChPerlucin gene cDNA is 577 bp,including 5′-untranslated region(UTR)in 21 bp,3′-UTR in 73 bp,and an open reading frame(ORF)in 483 bp open reading that code 160 amino-acid polypeptide.The predicted molecular mass is 18kD,and the isoelectric point is 5.95.Amino acid sequence alignment showed that the Perlucin gene of Crassostrea hongkongensis contains a conserved carbohydrate recognition domain(CLECT).Neighbor-Joining(NJ)evolutionary tree analysis showed that ChPerlucin clustered with Perlucin from other shellfish,indicating that this gene is a new member of the Perlucin family of mollusks.The results of qRT-PCR showed that ChPerlucin was broadly expressed in various tissues and during different stages of the oyster’s embryonic and larval development;the expression of ChPerlucin increased significantly after bacteria stimulation;the recombinant protein ChPerlucin was successfully expressed using Pichia pastoris eukaryotic;and we find that ChPerlucin recombinant protein has antibacterial effect.The above results indicate that ChPerlucin plays an important role in the innate immunity of Crassostrea hongkongensis.

关 键 词：香港牡蛎 先天性免疫 C-型凝集素 ChPerlucin 

分 类 号：S944.4[农业科学—水产养殖] Q939.91[农业科学—水产科学]