赤眼鳟LGP2序列结构、组织表达及与MDA5互作特征

Sequence structure,tissue expression of LGP2,and its interaction with MDA5 in Squaliobarbus curriculus

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2940 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。

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 populations provide valuable resources for investigating the genetic mechanisms of the antiviral immunity.The laboratory of genetics and physiology 2(LGP2)is a member of RIG-Ⅰlike receptor family and is involved in recognition 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(2940 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 phosphorylation 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 Mylopharyngodon 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 disease resistant C.idella.