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大黄鱼cGAS-like基因变型1isofrom X1的克隆鉴定及在感染刺激隐核虫后cGAS的表达变化
郑利兵1, 洪越群2, 洪宇建2, 孙恺辉2, 洪宇聪2
1.厦门大学;2.越群海洋生物研究开发有限公司
摘要:
大黄鱼(Larimichthys crocea)作为重要的海水经济鱼类之一,是我国海水网箱养殖单一品种产量最大的物种。近年来,由刺激隐核虫(Cryptocaryon irritans)感染引起的海水白点病对大黄鱼养殖业造成了巨大的威胁。刺激隐核虫的感染不仅有寄生引起的对宿主的伤害,同时还伴随有病原菌的二次继发性感染。cGAS 是近几年来新发现的先天免疫中一个新型的信号分子。本文基于大黄鱼基因组信息,用 cDNA 末端快速扩增(RACE)技术在鳃组织中克隆鉴定了cGAS cDNA 全长序列:其全长包含一个 231 bp 的5''端-非翻译区(5''-UTR),、207 bp 的3''-UTR 和 1488 bp 的开放阅读框(ORF),命名为 Lc-cGAS-like X1;。该基因编码一个由 495 个氨基酸残基组成的蛋白质多肽,预测的蛋白质分子量(Mw)大小为56.57 KDa,理论等电点(pI)为9.45;序列分析表明其编码的蛋白无信号肽,第115~431位氨基酸序列为 Mab-21功能域,第470~492 位氨基酸序列为跨膜结构域;。基因组全长 3153 bp,包含 8 个外显子和 7 个内含子,所有内含子的剪切特点都符合GT-AG规则。同源比对结果显示:Lc-cGAS-like X1与鱼类cGAS的相似性大于66%,与高等脊椎动物的相似性较低。系统进化分析表明Lc-cGAS-like X1 与鱼类 cGAS 聚成一支。荧光定量PCR(qRT-PCR)检测显示Lc-cGAS基因(两种变型两种isoform)在大黄鱼 9 种组织中均有表达,其中在鳃中的表达量最高;刺激隐核虫感染后,大黄鱼免疫器官头肾中Lc-cGAS mRNA 分别在刺激隐核虫幼虫感染阶段和滋养体脱落阶段出现了极显著上调(P<0.01);解毒器官肝脏中 Lc-cGAS mRNA 分别在滋养体脱落阶段和细菌感染阶段出现极显著上调变化(P<0.01)。本研究结果暗示大黄鱼cGAS基因参与了大黄鱼感染刺激隐核虫的免疫防御过程,为进一步了解鱼类cGAS的多样化功能提供参考。
关键词:  大黄鱼  刺激隐核虫  cGAS  基因鉴定  基因表达
DOI:
分类号:S917.4
基金项目:广东越群海洋水产动物苗种繁育及饲料研发技术体系院士工作站(2014B090905008)
Molecular identification of cyclic GMP-AMP synthase-like (cGAS) variant isoform X1 of Larimichthys crocea and their expression change after challenged by Cryptocaryon irritans
zhenglibing1, hong yue qun2, hong yu jian2, sun kai hui2, hong yu cong2
1.Xiamen University;2.Yuequn Ocean Biological Research Development CO., LTD
Abstract:
Larimichthys crocea, one of the important marine economic fishes, composed the largest yield for a single species of Chinese marine net-cage farming. The marine white spot disease caused by Cryptocaryon irritans is a huge threat to large yellow croaker farming industry. The parasitic process could not only lead to tissue damage, but also cause secondary bacterial infection. Cyclic GMP-AMP synthase-like (cGAS) was found to combine dsDNA free in the cytoplasm to induce I type IFN or inflammatory cytokines in the recent years. In the present study, cGAS-like variant X1 from L.crocea was obtained by rapid amplification of cDNA ends (RACE) method based on the partial genome information in this study. The results showed that the full-length cDNA of Lc-cGAS-like X1 was 1925 bp, containing a 5′ untranslated region (UTR) of 231 bp, 3′-UTR of 207 bp, and an open reading frame (ORF) of 1488 bp which encoded a peptide with 495 amino acids. The full length genome was 3153 bp, containing eight exons and seven introns. And all the exon-intron boundaries were in accordance with classical GT-AG rule (GT/intron/AG). The predicted molecular weight (Mw) of encoding protein was 56.57 KDa and the theoretical isoelectric point (pI) was 9.45. There was no a signal peptide in the N-terminal, and three potential glycosylation sites were predicted in the deduced amino acids. There are trans-membrane region and Mab-21 domain. The results of multiple alignment showed that Lc-cGAS-like X1 shared the highest identity of 99 %with the Lc-cGAS-like X2, the identity of Lc-cGAS-like X1 shared with fish was higher than 66%, and high vertebrates was about 38-40 %. Phylogenetic analysis showed the cGAS cluster was mainly in accordance with the traditional classification. qRT-PCR detected the transcripts of Lc-cGAS (two variants) distributing in all nine examined tissues of L. crocea, and it was the most abundant in gill. After challenged by C.irritans, Lc-cGAS mRNA in the head kidney appeared highly significant up-regulation (P<0.01) during the infective and parasitic stages; while Lc-cGAS mRNA in the liver was highly significant up-regulation (P<0.01) during the trophonts released stage and the secondary bacterium infective stage. In all, the data showed Lc-cGAS was involved in the immune response to C.irritans.
Key words:  Larimichthys crocea  Cryptocaryon irritans  cGAS  gene cloning  gene expression
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