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中国明对虾FBA基因克隆及其在白斑综合征病毒感染中的表达及功能分析
史晓丽1,2, 孟宪红1,2, 孔 杰1,2, 栾 生1,2, 罗 坤1,2, 曹宝祥1,2, 曹家旺1,2, 陈宝龙1,2
1.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 266071;2.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 266071
摘要:
醛缩酶(FBA)是糖酵解和糖异生中的关键酶,参与多种生物过程。本研究采用RACE技术,克隆获得中国明对虾(Fenneropenaeus chinensis)FBA基因(FcFBA)的全长cDNA序列,并对其进行生物信息学分析。结果显示,中国明对虾FcFBA基因的cDNA全长为2496 bp,其中,ORF长1098 bp,5´ UTR长79 bp,3´ UTR长1319 bp。完整的阅读框编码365个氨基酸,分子量为39.8 kDa,预测的理论等电点为6.6。同源性及系统进化分析表明,FcFBA与节肢动物的FBA聚为一类,与卤虫(Artemia franciscana)、家蚕(Bombyx mori)、沙漠蝗(Schistocerca gregaria)的相似度分别是86%、79%和78%。荧光定量PCR结果显示,FcFBA在肌肉中的相对表达量最高,肝胰腺中最低。WSSV感染后,该基因在鳃、肝胰腺和肌肉中呈现出不同的时空表达特点。dsRNA干扰24 h以后,抑制效率达到最大。与PBS对照组相比,FcFBA干扰组(dsRNA组)加快了对虾染病后的死亡速度。本研究表明,FcFBA基因可能参与了中国明对虾生物胁迫的应答反应。
关键词:  中国明对虾  FBA基因  基因克隆  组织表达  基因干扰
DOI:
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基金项目:
cDNA Cloning of the FBA Gene in Fenneropenaeus chinensis and Its Expression and Functional Analysis After WSSV Infection
SHI Xiaoli1,2, MENG Xianhong1,2, KONG Jie1,2, LUAN Sheng1,2, LUO Kun1,2, CAO Baoxiang1,2, CAO Jiawang1,2, CHEN Baolong1,2
1.Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;2.Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071
Abstract:
Fructose-1,6-bisphosphate aldolase (FBA) is a key glycolytic enzyme involved in both the glycolytic and gluconeogenic pathways. In the present study, fructose-1,6-bisphosphate aldolase from Chinese shrimp Fenneropenaeus chinensis (FcFBA) was cloned and sequenced. The full-length complementary DNA (cDNA) sequence of FcFBA is 2496 bp long, which contains a 79-bp 5¢-untranslated region (UTR), 1319-bp 3¢-UTR, and 1098-bp open reading frame, encoding a polypeptide of 365 amino acids with a molecular mass of 39.8 kDa and a theoretical isoelectric point of 6.6. Multiple sequence alignments showed the high similarity of FcFBA with the aldolases of other arthropods. Phylogenetic analysis showed that FcFBA of shrimp belonged to the same class as that of other arthropod aldolases. The transcript of FcFBA showed the highest expression in the muscle and lowest expression in the hepatopancreas, which indicates that FcFBA is similar to the muscle type (type A) aldolase that acts in the glycolytic pathway. The expression profile of FcFBA in the hepatopancreas, gills, and muscle was modulated when the shrimp were stimulated by white spot syndrome virus (WSSV), which resulted in differential expression of FcFBA. Further, RNA interference (RNAi) was used to analyze the role of FcFBA. After RNAi, the mortality of shrimp after WSSV infection was altered compared with that after phosphate-buffered saline injection. Within 24 h of RNAi, the expression level of FcFBA was significantly down regulated. These results show that FcFBA is inducible and might be involved in the immune response of shrimp.
Key words:  Fenneropenaeus chinensis  Fructose-1,6-bisphosphate aldolase gene  Cloning  Expression  RNA interference