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凡纳滨对虾ARMC8基因的克隆及表达
引用本文:黄金凤,杨奇慧,董晓慧,迟淑艳,刘泓宇,谭北平,章双. 凡纳滨对虾ARMC8基因的克隆及表达[J]. 水产学报, 2017, 41(2): 171-181
作者姓名:黄金凤  杨奇慧  董晓慧  迟淑艳  刘泓宇  谭北平  章双
作者单位:广东海洋大学,广东海洋大学水产学院,广东海洋大学水产学院,广东海洋大学水产学院,广东海洋大学水产学院,广东海洋大学水产学院;南海生物资源开发与利用协同创新中心,广东海洋大学水产学院
摘    要:为了研究含Armadillo重复蛋白8在凡纳滨对虾抗病感染过程中所起的免疫作用,本实验采用RACE-PCR技术首次克隆得到凡纳滨对虾ARMC8基因(LvARMC8,Gen Bank注册号:KX058562)的c DNA序列全长,并利用在线软件进行生物信息学分析;采用实时荧光定量PCR(Rt-PCR)技术对LvARMC8基因在凡纳滨对虾不同组织及感染白斑综合征病毒和副溶血弧菌过程中的表达变化特征进行分析。结果显示,LvARMC8基因全长为2917bp,其中开放阅读框长2046 bp,编码681个氨基酸,5′非编码区长49 bp,3′非编码区长822 bp。预测分析显示LvARMC8编码的蛋白质含有6个ARM结构域。同源性分析发现,LvARMC8基因与内华达古白蚁ARMC8基因的相似度最高,为71%。系统进化分析结果显示,LvARMC8和多种无脊椎动物ARMC8聚为一支,其中与昆虫类动物赤拟谷盗、致倦库蚊和柑橘凤蝶的亲缘关系最近。Rt-PCR分析发现,LvARMC8基因在凡纳滨对虾多个组织中均能检测出,在表皮中表达量最高,眼柄中表达量最低。WSSV感染后12 h,LvARMC8基因在凡纳滨对虾血液中的表达量显著降低,但48 h后显著升高,72 h达到最高水平。除副溶血弧菌感染后24 h外的时间点,LvARMC8基因在凡纳滨对虾血液中的表达量均显著升高。研究表明,LvARMC8基因可能参与凡纳滨对虾抗病免疫应答途径。

关 键 词:凡纳滨对虾  ARMC8基因  基因克隆  组织表达  WSSV感染  弧菌
收稿时间:2016-04-23
修稿时间:2016-09-21

Cloning and expression profile analysis of ARMC8 gene from Litopenaeus vannamei
HUANG Jinfeng,YANG Qihui,DONG Xiaohui,CHI Shuyan,LIU Hongyu,TAN Beiping and ZHANG Shuang. Cloning and expression profile analysis of ARMC8 gene from Litopenaeus vannamei[J]. Journal of Fisheries of China, 2017, 41(2): 171-181
Authors:HUANG Jinfeng  YANG Qihui  DONG Xiaohui  CHI Shuyan  LIU Hongyu  TAN Beiping  ZHANG Shuang
Affiliation:College of Fisheries,Guangdong Ocean University,College of Fisheries,Guangdong Ocean University,College of Fisheries,Guangdong Ocean University,College of Fisheries,Guangdong Ocean University,College of Fisheries,Guangdong Ocean University;China;South China Sea Resource Exploitation and Protection Collaborative Innovation Center SCS-REPIC;China,College of Fisheries,Guangdong Ocean University
Abstract:The purpose is to study the potential role of Litopenaeus vannamei armadillo repeat-containing protein 8 (ARMC8) in the immune response triggered by the virus and bacterial pathogens. Full-length cDNA sequence of ARMC8 gene from L. vannamei (named LvARMC8, Genbank Accession Number: KX058562) was first cloned using RACE method. The full-length cDNA sequence of LvARMC8 was 2917 bp, which contains a 50 bp 5''UTR, 822 bp 3''UTR and 2046 bp open reading frame (ORF) that encoded 681 amino acid residues. SMART analysis results showed that LvARMC8 contains six armadillo repeat (ARM) domains. Multiple alignment analysis shows that LvARMC8 shared 71% amino acid identity with Zootermopsis nevadensis, which is highest. Phylogenetic analysis showed that LvARMC8 was clustered together of invertebrates group and most closely related to Tribolium castaneum ARMC8, Culex quinquefasciatus ARMC8 and Papilio xuthus ARMC8. RT-PCR analysis showed that LvARMC8 was constitutively expressed in all the examined tissues with the highest expression in epithelium and lowest expression in eyestalk. Upon WSSV challenge, the expression of LvARMC8 was significantly down-regulated at 4 hpi but significantly up-regulated starting at 48 hpi, reaching the peak at 72 hpi. By V. parahaemolyticus challenging, the level of LvARMC8 expression were markedly increased in all the detected time points except 24 hpi. The result suggests that LvARMC8 might take part in the innate immune response of L. vannamei triggered by pathogens.
Keywords:Litopenaeus vannamei   Armadillo repeat-containing protein 8   gene cloning   tissue expression   WSSV infection   Vibrio parahaemolyticus
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