摘要
为了研究自噬相关基因ATG10(Autophagy related gene 10)在鱼类免疫应答中的功能,研究克隆了大黄鱼(Larimichthys crocea)ATG10基因(LcATG10)的cDNA序列,其开放阅读框全长747个核苷酸,编码248个氨基酸的蛋白,包含1个Autophagy_act_C结构域。系统进化分析显示,LcATG10和其他硬骨鱼类ATG10聚成一支,与金头鲷和石斑鱼ATG10亲缘关系最近。LcATG10在所检测的正常大黄鱼各组织中都有表达。在病毒类似物poly(I:C)刺激后,大黄鱼脾脏和头肾组织中LcATG10的表达水平显著上调,分别在12h和6h达到峰值(9.4和5.9倍)。LcATG10在大黄鱼头肾细胞系(LYCK)、原代巨噬细胞、淋巴细胞和粒细胞中也均有表达,在原代粒细胞中的表达量相对较高;在poly(I:C)刺激后,大黄鱼原代头肾粒细胞和LYCK细胞中LcATG10的表达水平显著上调。过表达LcATG10的鲤上皮瘤(Epithelioma papulosum cyprinid,EPC)细胞受鲤春病毒血症病毒(Spring viremia of carp virus,SVCV)感染48h后,细胞病变效应(Cytopathic effects,CPEs)明显低于对照组;细胞培养上清中SVCV病毒滴度为103.55 TCID50/mL,显著少于对照组;SVCV标志基因SVCV-G、SVCV-M和SVCV-P的表达水平也显著低于对照组,分别是对照组的0.022、0.015和0.022倍。这些研究结果表明LcATG10在大黄鱼抗病毒免疫应答中发挥作用,为深入研究自噬在大黄鱼抗病毒免疫中的分子机制奠定了基础。
Large yellow croaker(Larimichthys crocea)is an important economic marine fish species in China.In recent years,the frequent outbreaks of infectious diseases caused by bacteria or viruses have led to great economic losses to the large yellow croaker aquaculture.Thus,the understanding of immune mechanisms of large yellow croaker will contribute to prevent and control these diseases.Autophagy is an essential and widely conserved cellular degradation process that mediates the elimination of defective proteins and organelles.Autophagy is regulated by various autophagy-related genes(ATG),of which the ATG10 gene has been demonstrated to play a vital role in the immune response.To study the function of fish ATG10 in immune response,the ATG10 gene(LcATG10)was cloned from large yellow croaker(Larimichthys crocea).The open reading frame(ORF)of LcATG10 cDNA is 747 nucleotides long,encoding a protein of 248 amino acids with an Autophagy_act_C domain.Multiple amino acid sequence alignment showed that LcATG10 shared a high homology with ATG10 molecules in other known bony fishes.Phylogenetic analysis revealed that LcATG10 and other fish ATG10 molecules were clustered into a major clade,separated from the clade formed by the amphibian,avian,and mammalian ATG10s.Real-time qPCR results showed that the LcATG10 were widely expressed in tested tissues and immune-related cells of large yellow croaker.Poly(I:C)induced the mRNA levels of LcATG10 in the head kidney and spleen tissues with 5.9-and 9.4-fold,respectively,and it also increased LcATG10 in primary head kidney granulocytes and head kidney cell line(LYCK)cells of large yellow croaker.LcATG10-overexpression in epithelioma papulosum cyprinid(EPC)cells decreased the cytopathic effects(CPEs)caused by spring viremia of carp virus(SVCV)infection and reduced the viral titer of SVCV in the culture supernatant to 103.55 TCID50/mL.LcATG10-overexpression significantly down-regulated the expression levels of three viral genes(SVCV-G,SVCV-M,and SVCV-P)to 0.022-,0.015-and 0.022-folds of those in control group,respectively.These results indicate that LcATG10 played an important role in the antiviral immune response of large yellow croaker,which provides a basis for in-depth study of the molecular mechanism of autophagy in the antiviral immunity of large yellow croaker.
作者
魏祖运
卢利霞
任秋磊
何天良
陈新华
WEI Zu-Yun;LU Li-Xia;REN Qiu-Lei;HE Tian-Liang;CHEN Xin-Hua(Key Laboratory of Marine Biotechnology of Fujian Province,Institute of Oceanology,Fujian Agriculture and Forestry University,Fuzhou 350002,China)
出处
《水生生物学报》
CAS
CSCD
北大核心
2022年第4期521-528,共8页
Acta Hydrobiologica Sinica
基金
现代农业产业技术体系(CARS-47)资助。
