Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
Fish Shellfish Immunol. 2018 Jun;77:419-428. doi: 10.1016/j.fsi.2018.03.059. Epub 2018 Mar 31.
Astakine is a cytokine-like factor containing a prokineticin domain, which directly participates in hematopoiesis and blood cell differentiation. In the present study, a novel Astakine gene was identified from Chinese mitten crab Eriocheir sinensis (designated as EsAst). The full-length cDNA of EsAst was of 1163 bp, consisting of a 5' untranslated region (UTR) of 120 bp, a 3' UTR of 656 bp, and an open reading frame (ORF) of 387 bp encoding a polypeptide of 128 amino acids. There were a signal peptide and a prokineticin domain with nine conserved cysteine residues in the deduced amino acid sequence of EsAst. EsAst shared higher similarity with Astakines from Penaeus monodon and Pacifastacus leniusculus, and it was closely clustered with the Astakine from shrimp P. monodon in the phylogenetic tree. The EsAst mRNA transcript was higher expressed in hemocytes and hepatopancreas. The relative expression level of EsAst in hemocytes was continuously increased from 1.5 to 48 h after Vibro anguillarum challenge compared that in the untreated control group. After Pichia pastoris GS115 challenge, the relative expression level of EsAst in hemocytes was also up-regulated. After rEsAst injection, ROS levels in HPT cells were also increased at 12 and 24 h, and the total hemocyte counts were also significantly increased at 6, 9, 12, and 24 h post rEsAst injection. The interference of EsAst expression with dsRNA injection could delay the recovery of hemocytes production post A. hydrophila stimulation. When mitochondrial complexes I was knock down by dsRNA, ROS levels were decreased and THCs were also decreased. Recovery of hemocyte production inducing by A. hydrophila stimulation and rEsAst injection were delayed with dsEsbc1 injection. When ROS levels were increased after RNAi of Lon protease, THCs were also increased. The expression levels of five genes (EsJNK, EsSTAT, EsPI3K, EsAKT1, EsP70S6K) involved in SAPK-JNK and mTOR signaling pathways were up-regulated at 12 and 24 h in rEsAst group and EsLon dsRNA group compared with that in EGFP dsRNA group, and were similar to the trend of ROS levels. These results collectively suggested that EsAst should be a novel Astakine to promote the production of hemocytes in a ROS-dependent way in E. sinensis.
雅科比素是一种细胞因子样因子，含有前动力蛋白结构域，直接参与造血和血细胞分化。本研究从中华绒螯蟹（命名为 EsAst）中鉴定出一种新型雅科比素基因。EsAst 的全长 cDNA 为 1163bp，包含 5'非翻译区（UTR）120bp、3'UTR 656bp 和开放阅读框（ORF）387bp，编码 128 个氨基酸的多肽。在 EsAst 的推导氨基酸序列中，存在一个信号肽和一个具有 9 个保守半胱氨酸残基的前动力蛋白结构域。EsAst 与斑节对虾和克氏原螯虾的雅科比素具有更高的相似性，并且在系统发育树中与对虾的雅科比素紧密聚类。EsAst mRNA 转录物在血细胞和肝胰腺中表达较高。与未处理对照组相比，在鳗弧菌刺激后 1.5 至 48 小时，血细胞中 EsAst 的相对表达水平持续增加。在巴氏毕赤酵母 GS115 刺激后，血细胞中 EsAst 的相对表达水平也上调。rEsAst 注射后，HPT 细胞中的 ROS 水平在 12 和 24 小时也升高，rEsAst 注射后 6、9、12 和 24 小时总血细胞计数也显著升高。dsRNA 注射干扰 EsAst 表达可延迟 A. hydrophila 刺激后血细胞生成的恢复。当线粒体复合物 I 被 dsRNA 敲低时，ROS 水平降低，THCs 也降低。dsEsbc1 注射可延迟由 A. hydrophila 刺激和 rEsAst 注射诱导的血细胞生成恢复。当 Lon 蛋白酶的 RNAi 增加 ROS 水平时，THCs 也增加。在 rEsAst 组和 EsLon dsRNA 组中，与 EGFP dsRNA 组相比，参与 SAPK-JNK 和 mTOR 信号通路的五个基因（EsJNK、EsSTAT、EsPI3K、EsAKT1、EsP70S6K）的表达水平在 12 和 24 小时均上调，与 ROS 水平的趋势相似。这些结果共同表明，EsAst 应该是一种新型的雅科比素，能够以 ROS 依赖的方式促进中华绒螯蟹血细胞的生成。
