文章摘要
三疣梭子蟹Apaf-1基因的克隆及其表达分析
Cloning and expression Analysis of Apaf-1 Gene in in swimming crab (Portunus trituberculatus)
投稿时间:2019-04-18  修订日期:2019-05-15
DOI:
中文关键词: 三疣梭子蟹  Apaf-1  细胞凋亡  低盐度胁迫  副溶血弧菌  WSSV
英文关键词: Portunus trituberculatus  Apaf-1  Cell apoptosis  Low salinity stress  Vibrio parahaemolyticus  WSSV
基金项目:国家自然科学基金面上项目(41876186,41576147)、泰山领军人才工程高效生态农业创新类计划项目(LJNY2015002)、国家虾蟹产业技术体系(CARS-48)
作者单位E-mail
王磊 上海海洋大学水产与生命学院 上海
青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室 青岛
农业部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 
1006966437@qq.com 
任宪云 青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室 青岛
农业部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 
 
宋柳 上海海洋大学水产与生命学院 上海  
吕建建 青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室 青岛
农业部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 
 
高保全 青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室 青岛
农业部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 
 
刘萍 青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室 青岛
农业部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 
liuping@ysfri.ac.cn 
孙东方 农业部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛  
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中文摘要:
      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1050bp,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表明, 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枝。组织表达分析表明,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。Apaf-1在Ⅱ期幼蟹经不同盐度胁迫后整体表达量均上调,3h达到最大值;Apaf-1在80日龄盐度20胁迫后鳃和肝胰腺组织中表现为先升高后降低的趋势,在盐度11时整体表达量下调(P<0.05),表明盐度的改变能影响该基因的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的表达量于48h到达峰值,为对照组的2.76倍(P<0.05),注射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72h到达峰值,为对照组的5.44倍(P<0.05),注射WSSV后在12h到达峰值,为对照组的5.89倍(P<0.05),整体呈上调表达。本研究为三疣梭子蟹凋亡途径的调控机制提供理论依据。
英文摘要:
      In this study, swimming crab (Portunus trituberculatus) apoptotic protein activator 1 (apoptotic protease activating factor-1, Apaf-1) gene of swimming crab (Portunus trituberculatus) was cloned by RACE technique. The full length of the gene was 2032 bp, in which ORF was 1050 bp, encoding 349 amino acids, predicted molecular weight was 39.13 kDa and theoretical isoelectric point was 7.67. The homology and phylogenetic analysis showed that the homology of Apaf-1 with Litopenaeus vannamei Apaf-1 was 51.27%, and it clustered with Penaeus vannamei. Tissue expression analysis showed that the relative expression level of Apaf-1 gene was highest in hepatopancreas, followed by muscle, heart, gill and eyestalk, and lowest in blood cells and epidermis. The expression of Apaf-1 in gill and hepatopancreas tissues of juvenile crabs at stage II was up-regulated after different salinity stress for 3 hours, and the expression of Apaf-1 in gill and hepatopancreas tissues first increased and then decreased after 20 days of salinity stress at 80 days of age, and decreased at 11 days of salinity (P < 0.05), suggesting that the change of salinity could affect the expression of Apaf-1 in gill and hepatopancreas tissues. The results showed that the expression of Apaf-1 reached its peak at 48 hours after injection of Vibrio parahaemolyticus, which was 2.76 times higher than that of the control group (P < 0.05), and increased at 12 hours after injection of WSSV, which was 1.25 times higher than that of the control group (P < 0.05); in hepatopancreas, the expression of Apaf-1 reached its peak at 72 hours after injection of Vibrio parahaemolyticus, which was 5.44 times higher than that of the control group (P < 0.05), and 12 hours after 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 theoretical basis for the regulation of apoptotic pathway in Portunus trituberculatus.
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