The Activation of Superoxide Dismutase and Its Functional Mechanism in Crustacean

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Project title

Code/計畫編號

NSC100-2321-B019-010-MY3

Translated Name/計畫中文名

甲殼類超氧歧化酵素啟動與作用機制( I )

Project Coordinator/計畫主持人

Jiann-Chu Chen

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Aquaculture

Website

https://www.grb.gov.tw/search/planDetail?id=2396307

Year

2012

Start date/計畫起

01-08-2012

Expected Completion/計畫迄

31-07-2013

Bugetid/研究經費

1100千元

ResearchField/研究領域

生物技術(農)
漁業

白蝦與草蝦、斑節蝦為世界上重要的養殖對蝦（penaeid），根據FAO 2009年的統計，在世界上白蝦與草蝦2007年全年的養殖產量分別佔養殖魚蝦貝物種的第6位及第17位。對蝦在養殖過程常受到病原侵襲，當環境惡化、緊迫，衰弱宿主及病原聚集造交互影響下常導致疾病爆發。吞噬作用是對蝦重要的先天性免疫防禦，過程中會產生毒性高的超氧離子進行殺菌，藉由超氧歧化酵素將多餘的超氧離子解毒。甲殼類超氧歧化酵素已有包括選殖、特性、組織分佈、分子演化等的研究。我們發現對蝦僅具有兩種錳型超氧歧化酵素﹝cytosolic MnSOD (cytMnSOD)及mitochondrial MnSOD （mtMnSOD）﹞，尚未發現銅鋅型超氧歧化酵素（CuZnSOD，包括icCuZnSOD與ecCuZnSOD）。有關對蝦MnSOD所扮演超氧歧化的角色重要性與其在先天性免疫防禦研究缺乏。本計畫擬研對蝦兩型MnSOD（cytMnSOD與mtMnSOD）的啟動機制與功能性研究，第一年擬選殖對蝦兩型MnSOD promoter（cytMnSOD與mtMnSOD），進行序列比較與分析轉錄調控因子，建構帶有對蝦MnSOD promoter之表現載體。第二年擬研究對蝦兩型MnSOD基因在受到不同異物如lipopolysaccharide（LPS）、β-glucan（βG），受到Vibrio alginolyticus感染的基因表現，以及在受到環境（水溫、鹽度、pH、低溶氧）變化下的基因表現，進行核酸干擾（RNA interference）實驗，探討兩型MnSOD是否存在協同作用，探討超氧離子產生與降解超氧離子的MnSOD之表現性相關，評估以超氧離子生成與MnSOD活性作為先天性免疫防禦與自我保護的指標，並探討作為選種指標之適任性。第三年擬重組兩種MnSOD，進行重組蛋白表現、純化與復性以及抗體誘導，進行功能性研究，探討MnSOD與氧化壓力造成細胞壞死之關係。 White shrimp Litopenaeus vannamei, tiger shrimp Penaeus monodon, and Kuruma shrimp Marsupenaeus japonicus are the important penaeid shrimps currently being cultured worldwide. The production of white shrimp and tiger shrimp ranked 6th and 17th among cultured species of fish, cructacean and mollusk according to FAO report in 2009. It is known that deteriorated environment, weakening host and aggregation of pathogen cause an occurrence of disease. Phagocytosis is an important immune response for penaeid shrimp to against pathogen. During the phagocytosis, superoxide anion is generated to disinfect infected pathogen. The remaining superoxide is scavenged by superoxide dismutase (SOD). We have conducted molecular cloning, characterization, tissue distribution, and molecular evolution of SODs. In penaeid, there are two types of MnSODs (cytMnSOD and mtMnSOD). However, none is known on the activation of cytMnSOD and mtMnSOD and their functions in the innate immune system. This proposal is to study promoter cloning of MnSODs from white shrimp, its feature and transcripton factor binding motif, and construct a expression vector with shrimp MnSOD promoter during the first year. This proposal is to study the gene expressions of MnSODs (cytMnSOD and mtMnSOD) of white shrimp in different tissues, moulting stages, when shrimp have received LPS, β-glucan (βG), Vibrio alginolyticus and under environmental stressors like water temperature, salinity, pH, hypoxia and conduct gene knock-down during the second year. This proposal is to recombine MnSODs, purify the recombined rMnSODs, induce antibody and study the function of the protein, against cell apoptosis and its mechanism during post-phagocytosis during the third year.

Keyword(s)

超氧歧化酵素
對蝦
基因調控
基因表現
superoxide dismutase
penaeid
gene regulation
gene expression

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The Activation of Superoxide Dismutase and Its Functional Mechanism in Crustacean

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