Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China.
Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China; Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian, 116023, China.
Fish Shellfish Immunol. 2019 Jun;89:207-216. doi: 10.1016/j.fsi.2019.03.065. Epub 2019 Mar 29.
Beclin-1, the mammalian ortholog of yeast Atg6, plays essential roles in the regulation of various processes, including autophagy, apoptosis, embryonic development and immune responses in vertebrates. However, the information about Beclin-1 in invertebrates especially in crustaceans is still very limited. In the present study, a novel Beclin-1 (designated as EsBeclin-1) was identified from Chinese mitten crab Eriocheir sinensis. The open reading frame of EsBeclin-1 cDNA was of 1,275 bp, encoding a typical APG6 domain. The deduced amino acid sequence of EsBeclin-1 shared high similarity ranging from 42.9% to 63.6% with the previously identified Beclins. In the phylogenetic tree, EsBeclin-1 was firstly clustered with Drosophila melanogaster Atg6 and then assigned into the branch of invertebrate Beclin-1. The mRNA transcripts of EsBeclin-1 were highly expressed in hepatopancreas, hemocytes and gill. After lipopolysaccharide (LPS) and Aeromonas hydrophila stimulations, the relative mRNA expression of EsBeclin-1 in hemocytes was significantly increased from 3 to 24 h with the peak level of 4.70-fold (p < 0.01) and 2.91-fold (p < 0.01) at 6 h, respectively. EsBeclin-1 protein was diffusely distributed in the cytoplasm of crab hemocytes under normal conditions, whereas it displayed predominantly punctuate distribution after LPS stimulation. After EsBeclin-1 was interfered with specific EsBeclin-1-dsRNA, the mRNA transcripts of some antimicrobial peptides, including EsALF2, EsLYZ, EsCrus and EsCrus2 in crab hemocytes were significantly decreased at 6 h post LPS stimulation. These results implicated that EsBeclin-1 played a role in regulating the antimicrobial peptides expressions in the immune responses of E. sinensis.
自噬相关蛋白 1（Beclin-1），酵母自噬相关蛋白 6（Atg6）的哺乳动物同源物，在各种过程的调节中发挥重要作用，包括自噬、凋亡、脊椎动物胚胎发育和免疫反应。然而，关于无脊椎动物，特别是甲壳类动物的 Beclin-1 的信息仍然非常有限。本研究从中华绒螯蟹（Eriocheir sinensis）中鉴定出一种新的 Beclin-1（命名为 EsBeclin-1）。EsBeclin-1 cDNA 的开放阅读框为 1275bp，编码一个典型的 APG6 结构域。EsBeclin-1 的推导氨基酸序列与已鉴定的 Beclin-1 具有高度相似性，相似性范围为 42.9%至 63.6%。在系统进化树中，EsBeclin-1 首先与黑腹果蝇（Drosophila melanogaster）的 Atg6 聚类，然后归入无脊椎动物 Beclin-1 分支。EsBeclin-1 的 mRNA 转录物在肝胰腺、血细胞和鳃中高度表达。在脂多糖（LPS）和嗜水气单胞菌刺激后，血细胞中 EsBeclin-1 的相对 mRNA 表达在 3 至 24 小时内显著增加，在 6 小时时分别达到 4.70 倍（p<0.01）和 2.91 倍（p<0.01）的峰值。在正常情况下，EsBeclin-1 蛋白在蟹血细胞的细胞质中呈弥散分布，而在 LPS 刺激后呈点状分布为主。用特异性 EsBeclin-1-dsRNA 干扰 EsBeclin-1 后，在 LPS 刺激后 6 小时，蟹血细胞中的一些抗菌肽，包括 EsALF2、EsLYZ、EsCrus 和 EsCrus2 的 mRNA 转录物明显减少。这些结果表明 EsBeclin-1 在中华绒螯蟹免疫反应中调节抗菌肽表达中发挥作用。
