Innate immunity is the primary defense of crustaceans against pathogens. Crustins, as antimicrobial peptides, are important to crustacean innate immunity. In this study, two kinds of Gly-rich crustin genes were cloned from Macrobrachium nipponense and were referred to as Mn-Gly-Cru1 and Mn-Gly-Cru2. These crustins belong to type II crustins with typical type II crustin structures. The full-length cDNA of Mn-Gly-Cru1 is 677 bp and contains a 576 bp open reading frame (ORF) encoding 191 amino acids. The full-length cDNA of Mn-Gly-Cru2 is 727 bp, with 573 bp ORF encoding 190 amino acids. The constructed phylogenetic tree indicated that Mn-Gly-Cru1 and Mn-Gly-Cru2 belong to the type IIa subfamily. RT-PCR analysis showed that Mn-Gly-Cru1 and Mn-Gly-Cru2 are widely distributed in various tissues. qRT-PCR results indicated that Mn-Gly-Cru1 is mainly expressed in the gills, whereas Mn-Gly-Cru2 is expressed at the highest level in hemocytes. The transcripts of Mn-Gly-Cru1 and Mn-Gly-Cru2 respond to bacterial or white spot syndrome virus (WSSV) stimuli. After injection of 48 h dsMnRelish, the expression of MnRelish, Mn-Gly-Cru1, and Mn-Gly-Cru2 were all inhibited. After WSSV, Vibrio parahaemolyticus, or Staphylococcus aureus challenge, MnRelish, Mn-Gly-Cru1, and Mn-Gly-Cru2 were all upregulated. However, the expression levels of MnRelish, Mn-Gly-Cru1, and Mn-Gly-Cru2 at 6 h bacteria or 36 h WSSV challenge were downregulated in Relish-silenced prawns when compared with the control (bacteria or WSSV challenge only, bacteria or WSSV challenge plus dsGFP injection). Results suggest that Mn-Gly-Cru1 and Mn-Gly-Cru2 play essential roles in M. nipponense innate immunity against bacteria or WSSV, and the expression levels of both genes are regulated by Relish transcriptional factor.

中文翻译：

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东方河虾日本沼虾两个II型Crustin基因的分子克隆和表达分析。

天然免疫是甲壳类对病原体的主要防御作用。甲壳素作为抗菌肽，对于甲壳类动物的先天免疫至关重要。在这项研究中，从日本沼虾（Macrobrachium nipponense）中克隆了两种富含Gly的克鲁斯汀基因，分别称为Mn-Gly-Cru1和Mn-Gly-Cru2。这些cru质属于具有典型II型cru质结构的II型cru质。Mn-Gly-Cru1的全长cDNA为677 bp，包含一个576 bp的开放阅读框（ORF），编码191个氨基酸。Mn-Gly-Cru2的全长cDNA为727 bp，ORF为573 bp，编码190个氨基酸。所构建的系统发育树表明Mn-Gly-Cru1和Mn-Gly-Cru2属于IIa型亚科。RT-PCR分析表明，Mn-Gly-Cru1和Mn-Gly-Cru2广泛分布在各种组织中。qRT-PCR结果表明，Mn-Gly-Cru1主要在the中表达，而Mn-Gly-Cru2在血细胞中以最高水平表达。Mn-Gly-Cru1和Mn-Gly-Cru2的转录本可响应细菌或白斑综合症病毒（WSSV）刺激。注射dsMnRelish 48 h后，MnRelish，Mn-Gly-Cru1和Mn-Gly-Cru2的表达均被抑制。WSSV，副溶血性弧菌或金黄色葡萄球菌攻击后，MnRelish，Mn-Gly-Cru1和Mn-Gly-Cru2均被上调。然而，与对照（仅细菌或WSSV攻击，细菌或WSSV挑战加dsGFP注入）。结果表明Mn-Gly-Cru1和Mn-Gly-Cru2在M中起重要作用。