| 摘要: |
| 本研究利用RACE方法克隆获得三疣梭子蟹(Portunus trituberculatus)凋亡蛋白酶激活因子-1 (Apoptotic protease activating factor-1, Apaf-1)基因，该基因cDNA全长为2032 bp，其中，ORF为1050 bp，编码349个氨基酸，预测分子量为39.13 kDa，理论等电点为7.67。同源性和系统发育分析显示，Apaf-1与凡纳滨对虾(Litopenaeus vannamei) Apaf-1的同源性最高，为51.27%，并与凡纳滨对虾聚为一支。组织表达分析显示，Apaf-1基因的相对表达水平在肝胰脏最高，其次是肌肉、心脏、鳃和眼柄，血细胞和表皮最低。不同盐度胁迫后，Apaf-1在Ⅱ期幼蟹体内表达水平在3 h达到最大值，此后，呈先下降再上升趋势；Apaf-1在80日龄盐度20胁迫后三疣梭子蟹的鳃中呈上升趋势，在肝胰腺中为先上升后下降趋势，表明盐度改变能影响该基因在鳃和肝胰腺组织中的表达。不同病原感染实验结果显示，在血细胞中，注射副溶血弧菌(Vibrio parahaemolyticus)后Apaf-1的表达于48 h到达峰值，为对照组的2.76倍(P<0.05)，注射白斑综合征病毒(WSSV)后，仅在12 h表达水平上调，为对照组的1.25倍(P<0.05)；在肝胰腺中，注射副溶血弧菌后在72 h到达峰值，为对照组的5.44倍(P<0.05)，注射WSSV后在12 h到达峰值，为对照组的5.89倍(P<0.05)，整体呈上调表达趋势。本研究为进一步理解三疣梭子蟹Apaf-1基因的生理功能提供了参考。 |
| 关键词: 三疣梭子蟹 Apaf-1 低盐度胁迫 副溶血弧菌 WSSV |
| DOI：10.19663/j.issn2095-9869.20190418001 |
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| Cloning and Expression Analysis of the Apaf-1 Gene in Portunus trituberculatus after Exposure to Low Salt and Pathogenic Stresses |
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WANG Lei1,2,3, REN Xianyun4,2,3, SONG Liu1,2,3, LÜ Jianjian4,2,3, GAO Baoquan4,2,3, LIU Ping4,2,3, SUN Dongfang2,3
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1.National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306;2.Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs;3.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;4.Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266071
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| Abstract: |
| In this study, the apoptotic protein activator 1 (apoptotic protease activating factor-1, Apaf-1) gene of the swimming crab (Portunus trituberculatus) was cloned using the RACE technique. The full length of the gene was 2032 bp, and the ORF was 1050 bp, encoding 349 amino acids, with a predicted molecular weight of 39.13 kDa, and a theoretical isoelectric point of 7.67. The homology and phylogenetic analysis showed that the homology of Apaf-1 with Litopenaeus vannamei Apaf-1 was 51.27%, and it was also clustered with L. vannamei. Tissue expression analysis showed that the relative expression level of the Apaf-1 gene was highest in the hepatopancreas, followed by the muscle, heart, gill, and eyestalk, and was lowest in the hemocytes and epidermis. After different salinity stresses, the expression level of Apaf-1 in the stage Ⅱ juvenile crab reached its maximum at 3 h, and then decreased and then increased. Apaf-1 showed an upward trend in the gills after salinity 20 stress after 80 d, and an upward and downward trend in the hepatopancreas, suggesting that the change of salinity could affect the expression of Apaf-1 in the gill and hepatopancreas. The results showed that the expression of Apaf-1 reached its peak at 48 h after injection of Vibrio parahaemolyticus, which was 2.76 times higher than that of the control group (P<0.05), and increased 12 h after the injection of white spot syndrome virus (WSSV), which was 1.25 times higher than that of the control group (P<0.05). In the hepatopancreas, the expression of Apaf-1 reached its peak at 72 h after the injection of Vibrio parahaemolyticus, which was 5.44 times higher than that of the control group (P<0.05), and 12 h after the injection of WSSV. The peak value was 5.89 times higher than that of the control group (P<0.05), and the expression was up-regulated as a whole. This study provides a reference for further understanding the physiological function of the Apaf-1 gene in Portunus trituberculatus. |
| Key words: Portunus trituberculatus Apaf-1 Low salinity stress Vibrio parahaemolyticus WSSV |
