为了探索表皮酚氧化酶原激活因子 (prophenoloxiase activating factors，PPAFs) 在甲壳动物免疫反应中的功能，基于前期转录组数据，本实验利用PCR和RACE技术从日本沼虾表皮克隆并鉴定了一个新的PPAF基因，命名为MnPPAF1 (GenBank登录号为OP784577)，利用生物信息学、实时荧光定量PCR (qPCR) 和RNA干扰(RNAi)等方法，研究了该基因序列特征、时空表达模式、嗜水气单胞菌攻毒后表皮MnPPAF1的转录水平、酚氧化酶 (phenoloxidase，PO) 活性以及日本沼虾死亡率的变化。结果显示，MnPPAF1 cDNA全长1 718 bp，编码460个氨基酸，具有夹子结构域丝氨酸蛋白酶 (clip-domain serine proteinases，Clip-SPs) 和胰蛋白酶样丝氨酸蛋白酶 (trypsin-like serine proteases，Tryp-SPs) 结构域。MnPPAF1在腹背部的表皮、血细胞、鳃、胃、心脏、肝胰腺等多种组织均有表达；表皮MnPPAF1的表达与蜕皮周期有关，与蜕皮间期 (C期) 相比，蜕皮前晚期 (D₄期) MnPPAF1表达量增加857%。嗜水气单胞菌攻毒后，表皮MnPPAF1表达水平在6 h达到了峰值，较对照组增加227%，而表皮PO活性在12 h达到峰值，同比增加24%。在腹背部第2节注射3 μg dsRNA溶液，每12 小时注射1次，共注射3次，最后1次注射后12 h，干扰效率最高，表皮MnPPAF1表达同比下降71.79%，至24 h，表皮PO活性降低72.31%。在干扰效率最高的时间点攻毒，在120 h，攻毒+干扰组虾的累计死亡率比攻毒+未干扰组增加26%。结果表明，MnPPAF1是表皮重要的免疫因子，参与调节酚氧化酶原系统的激活，该基因表达下调能显著增加嗜水气单胞菌感染虾的死亡率。本研究为阐明表皮在甲壳动物免疫系统中的作用积累了资料。

Prophenoloxidase activation is a crucial innate immune response in crustaceans against pathogenic microorganisms infections, with prophenoloxidase-activating factors (PPAFs) being pivotal in this activation. Despite their importance, crustacean epidermal PPAFs have been seldom studied. To elucidate the role of epidermal PPAFs in crustaceans immunity, a novel PPAF gene, MnPPAF1 (GenBank accession: OP784577), was cloned and characterized from the epidermis of Macrobrachium nipponense using PCR and RACE techniques, based on prior transcriptome data. Bioinformatics, qPCR and RNAi methods were employed to analyze the gene sequence, spatial and temporal expression patterns, transcription levels of epidermal MnPPAF1, phenoloxidase (PO) activity, and shrimps mortality following Aeromonas hydrophila challenge. The complete MnPPAF1 cDNA sequence is 1 718 bp, encoding a 460-amino acid protein with clip-domain serine proteinase (Clip-SP) and trypsin-like serine protease (Tryp-SP) domains. MnPPAF1 expression was detechted in various tissues, including ventral and dorsal epidermis, hemocytes, gills, stomach, heart, hepatopancreas, with epidermal expression correlating to the molting cycle. Expression levels in the later premolt stage (D₄ stage) were 857% higher than in the intermolt stage (C stage). After A. hydrophila challenge, epidermal MnPPAF1 expression peaked at 6 h, increasing by 227% over the control group, while epidermal PO activity peaked at 12 h, increasing by 24%. 3 μg of dsRNA solution was injected into the second segment of the abdomen and back every 12 h, 3 injections in total. RNAi has the highest interference efficiency 12 h after the last injection, where the expression of epidermal MnPPAF1 decreased by 71.79%, compared to the control group, and after 24 h, the activity of epidermal PO decreased by 72.31%. Challenge at the time point with the highest interference efficiency and within 120 h, the cumulative mortality of shrimp in the challenged + RNAi group increased by 26% compared with that in the challenged + non-RNAi group. These findings indicate that MnPPAF1, an important immune factor in the epidermis, was involved in regulating the activation of the prophenoloxidase system. The down-regulation of this gene expression can significantly increase the mortality of shrimp infected by A. hydrophila. This study contributes to a better understanding of the epidermis's role in the crustaceans immune system.

Q 786; S 917.4

河南省自然科学基金 (182300410033)