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赤眼鳟LGP2序列结构、组织表达及与MDA5互作特征

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为探究赤眼鳟遗传学和生理学实验室蛋白 2(laboratory of genetics and physiology 2，LGP2)的功能特征及抗草鱼呼肠孤病毒(grass carp reovirus，GCRV)育种参考潜力，实验克隆获得了 2 940 bp的赤眼鳟lgp2(Sclgp2)全长cDNA和 721 bp的 5′端上游序列。Sclgp2 cDNA编码 680 个氨基酸，包含DEXDc(DExD/H-box helicase domain)、HELICc(helicase superfamily C-terminal domain)和CTD(C-terminal regulatory domain)结构域;其 5′端上游序列含有MafB(muscle aponeurosis fibromatosis B)和IRF3(interferon regulatory factor 3)等转录因子结合位点。不同物种LGP2 的功能结构域、磷酸化修饰位点数具有相似性，同时也存在结构域排布位置及序列的差异。赤眼鳟和草鱼lgp2 cDNA序列比较初步发现 2 个位于RNA结合功能区的GCRV抗性关联位点。系统进化分析显示，赤眼鳟LGP2 先与草鱼、鲫和青鱼聚在一起，再与鲤科鱼类等聚为一大支。荧光定量表达分析显示，赤眼鳟脾脏中sclgp2 表达水平显著高于其他组织，肌肉、心脏中表达量次之，而肠中表达量最低。GCRV感染后，肝脏中ifn1 表达水平在 24～72 h显著下降，其他组织sclgp2 和ifn1 表达水平未有显著变化。相关性分析结果显示，赤眼鳟肌肉sclgp2 与ifn1 表达水平呈极显著正相关(0。999)。酵母双杂交互作检测发现，赤眼鳟LGP2 与MDA5 存在弱相互作用，而其DEXDc(1～201 aa)、HELICc(390～476 aa)以及CTD(553～668 aa)结构域与MDA5 无互作。该研究成功获得了sclgp2 全长cDNA及 5′端上游序列，明确了其序列结构、免疫表达及与 MDA5 的互作特征，为赤眼鳟 LGP2 免疫功能属性研究奠定了基础，并为草鱼抗GCRV育种提供了参考。
Sequence structure,tissue expression of LGP2,and its interaction with MDA5 in Squaliobarbus curriculus
Grass carp(Ctenopharyngodon idella)are susceptible to grass carp reovirus(GCRV)and exhibit severe hemorrhage when infected.In contrast,Squaliobarbus curriculus(Sc)are resistant to GCRV.We have generated C.idella/S.curriculus hybrid fish which are relatively more resistant to GCRV than C.idella.These fish popula-tions provide valuable resources for investigating the genetic mechanisms of the antiviral immunity.The laborat-ory of genetics and physiology 2(LGP2)is a member of RIG-Ⅰlike receptor family and is involved in recogni-tion of viral nuclear acid at the first line of defense.Understanding its functions in antiviral immunity will have a significant impact on the molecular breeding of fish with resistant traits.In this study,the full-length cDNA(2 940 bp)and the 5′upstream sequence(721 bp)of the S.curriculus lgp2(Sclgp2)gene were sequenced.The 5'upstream region contains several putative transcription factor binding sites for MafB(muscle aponeurosis fibromatosis B)and IRF3(interferon regulatory factor 3).The Sclgp2 encodes a protein of 680 amino acids which consists of conserved DEXD/H(DExD/H-box helicase domain),HELICc(helicase superfamily C-terminal domain)and CTD(C-terminal regulatory domain).The functional domains and the number of predicted phos-phorylation sites are similar to that in other fish species,with differences in domain arrangement and amino acid sequences.Furthermore,by comparing the sequences of C.idella and S.curriculus LGP2 proteins,two GCRV sites associated with GCRV resistance in the RNA binding region were identified.In the phylogenetic tree,ScLGP2 was clustered in a clade containing the homologs from C.idella,Carassius auratus and Mylopharyn-godon piceus,forming an expanded clade with the homologs from other cyprinid fish.Quantitative PCR analysis revealed that the mRNA expression level of Sclgp2 was the highest in the spleen,followed by muscle and heart,whereas the lowest was detected in the intestine.The expression levels of Sclgp2 and ifn1 remained unchanged in most tissues except that the ifn1 expression was significantly down-regulated in the liver at 24-72 h after GCRV infection.Interestingly,the mRNA expression levels of Sclgp2 in the muscle positively correlated with that of ifn1(0.999,P<0.01).ScLGP2 weakly interacted with ScMDA5,yet DEXDc(1-201 aa),HELICc(390-476 aa)and CTD(553-668 aa)were not engaged in such interaction.Taken together,the present study provides basis for studying the functions of ScLGP2 and exploring the Sclgp2 gene as a potential marker for genetic breeding of dis-ease resistant C.idella.

Squaliobarbus curriculuslaboratory of genetics and physiology 2sequence structureexpression characteristicprotein interactionGCRV resistance

李耀国、廖依静、王静安、肖调义

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湖南农业大学,湖南省特色水产资源利用工程技术研究中心,湖南长沙 410128

赤眼鳟 生理学实验室蛋白2(LGP2) 序列结构 表达特征 蛋白互作 GCRV抗性

国家自然科学基金国家自然科学基金国家自然科学基金湖南省教育厅科研项目中央引导地方科技发展专项

3217296631802288U20A206319B2652022ZYC085

2024

10.11964/jfc.20211013107

水产学报
中国水产学会

水产学报

CSTPCD北大核心
影响因子：1.148
ISSN：1000-0615
年,卷(期)：2024.48(1)
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