| 摘要: |
| 中华乌塘鳢(Bostrychus sinensis)属于广盐广温、低氧耐受性强、药用价值高的鱼类,为我国东南沿海重要养殖对象。谷胱甘肽过氧化物酶(glutathione peroxidase,GPx)是生物体内重要的抗氧化酶,能够清除过氧化氢防止机体生物膜氧化受损,研究中华乌塘鳢GPx基因对其养殖及海洋生态研究具有重要意义。利用分子克隆技术获得了中华乌塘鳢GPx7和GPx8(分别命名为BsGPx7和BsGPx8)的cDNA序列。BsGPx7编码186个氨基酸,含有2个半胱氨酸(Cys56和Cys85)催化位点;BsGPx8编码210个氨基酸,含有1个半胱氨酸(Cys109)催化位点。利用RT-PCR方法研究了BsGPx7、BsGPx8基因在中华乌塘鳢组织中的分布表达,以及不同浓度镉离子(7.5,15,30 mg/L)胁迫下BsGPx7、BsGPx8基因的表达量变化。结果显示,BsGPx7和BsGPx8在组织中呈泛在性表达,其中肝脏的表达量最高,其次是鳃和皮肤。不同浓度的镉离子胁迫下,BsGPx7基因在鳃和皮肤中的表达量均显著上调(P<0.05),BsGPx8基因在肝脏、鳃和皮肤中的表达量均显著上调(P<0.05)。成功构建了原核表达载体,表达并纯化获得了BsGPx7重组蛋白(rBsGPx7)。对rBsGPx7在不同温度和pH条件下的酶活性进行了测定,表明rBsGPx7在35℃和pH 8条件下酶活性最高。综上提示,BsGPx7与BsGPx8参与了机体对抗镉离子诱导的氧化应激的过程,可能在抗氧化防御中起一定作用。 |
| 关键词: 谷胱甘肽过氧化物酶(GPx) 中华乌塘鳢(Bostrychus sinensis) 镉离子 基因表达 酶活性 |
| DOI：10.11693/hyhz20211100268 |
| 分类号:S917.4 |
| 基金项目:国家自然科学基金项目,42106101号。 |
附件 |
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| MOLECULAR CLONING, EXPRESSION AND ENZYMATIC ACTIVITITY ANALYSES OF GPx7 AND GPx8 FROM CHINESE BLACK SLEEPER (BOSTRYCHUS SINENSIS) |
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DING Lang, XIA Li-Ping, SHEN Bin, ZHANG Jian-She
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National Engineering Research Center for Marine Aquaculture, Zhejiang Ocean University, Zhoushan 316022, China
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| Abstract: |
| Glutathione peroxidase (GPx) is an important antioxidant enzyme that can remove hydrogen peroxide to protect the biofilm. We sequenced and characterized the GPx7 and GPx8 from Chinese black sleeper (Bostrychus sinensis). The BsGPx7 encoding 186 amino acids and contains two catalytic residues (Cys56, Cys85), and BsGPx8 encoding 210 amino acids and contains one catalytic residue (Cys109). The expressions of BsGPx7 and BsGPx8 in organs of Chinese black sleeper were analyzed in RT-PCR (reverse transcription polymerase chain reaction), and those under different concentrations (7.5, 15, 30 mg/L) of Cd2+stimulation were also studied. Results show that the BsGPx7 was found ubiquitously and highly expressed in 10 examined organs of Chinese black sleeper, with predominant expression in liver, followed by gill and skin. The expression of BsGPx7 was significantly upregulated in gill and skin under different concentrations of Cd2+(P<0.05). The expression of BsGPx8 was also significantly upregulated in liver, gill, and skin (P<0.05). The recombinant BsGPx7 (rBsGPx) protein was obtained by the construction of prokaryotic expression vector and purification, and the effects of temperature or pH on the activity of rBsGPx7 were determined. The rBsGPx7 protein had poor thermal stability and narrow range of pH resistance. The purified rBsGPx7 had optimal activity at 35 °C and pH 8. In conclusion, BsGPx7 and BsGPx8 are involved in antioxidant defense. |
| Key words: glutathione peroxidase (GPx) Chinese black sleeper (Bostrychus sinensis) Cd2+ gene expression enzymatic activity |
