Studying the Desaturase Functions in the Cold Acclimation of Fish

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Project title

Code/計畫編號

NSC85-2321-B019-021-A15

Translated Name/計畫中文名

環境適應---去飽和脂肪酸酵素在抗寒機制的研究

Project Coordinator/計畫主持人

Shye-Jye Tang

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=227717

Year

1996

Start date/計畫起

01-08-1995

Expected Completion/計畫迄

01-07-1996

Bugetid/研究經費

653千元

ResearchField/研究領域

生物科學

研究報告已顯示,生物體為能生存在較低溫的環境中,其體組成的不飽和脂肪酸含量會增加,以維持細胞膜的流動性。而脂質的不飽和程度除了可直接由外界攝取外,亦受到去飽和脂肪？(Desaturase)活性的影響。在廣溫性魚類的研究報告亦指出,魚體對低溫的耐受性增加,提高膜不飽和脂質含量為主要原因之一,且分析其Desaturase活性,亦發現到受低溫誘導後Desaturase活性會增加。植物與藻類已有多種的Desaturase被純化定序。哺乳動物的老鼠(Rat)及小老鼠(Mouse)的Desaturase也有被定序。但卻無任何一種魚類的Desaturase被純化定序。為能直接的探討魚的Desaturase表現情形,與體組成脂質成分與環境溫度的關係。首先希望能將魚類的Desaturase選殖定序。採用斑馬魚(Zebrafish)當實驗材料來源,選殖Desaturase基因。根據老鼠、小老鼠、酵母菌(Yeast) stearyl-CoA desaturase DNA序列,設計出Degenerative primers。將斑馬魚的第一股cDNA以反轉錄作用合成後,當成模板(Template)以Degenerative primers進行PCR(polymerase chain reaction)。並構築在選殖用載體(pGEM-T vector),進行DNA定序後,與Genebank進行比對分析,所選殖的DNA序列無一與Desaturase基因有較高的相似性。另外再以老鼠的Desaturase DNA當探針對選殖出來的菌株進行Colony hybridization。同時,亦進行斑馬魚cDNA基因庫(cDNA library of zebrafish)的篩選。研究報告已顯示,生物體為能生存在較低溫的環境中,其體組成的不飽和脂肪酸含量會增加,以維持細胞膜的流動性。而脂質的不飽和程度除了可直接由外界攝取外,亦受到去飽和脂肪？(Desaturase)活性的影響。在廣溫性魚類的研究報告亦指出,魚體對低溫的耐受性增加,提高膜不飽和脂質含量為主要原因之一,且分析其Desaturase活性,亦發現到受低溫誘導後Desaturase活性會增加。植物與藻類已有多種的Desaturase被純化定序。哺乳動物的老鼠(Rat)及小老鼠(Mouse)的Desaturase也有被定序。但卻無任何一種魚類的Desaturase被純化定序。為能直接的探討魚的Desaturase表現情形,與體組成脂質成分與環境溫度的關係。首先希望能將魚類的Desaturase選殖定序。採用斑馬魚(Zebrafish)當實驗材料來源,選殖Desaturase基因。根據老鼠、小老鼠、酵母菌(Yeast) stearyl-CoA desaturase DNA序列,設計出Degenerative primers。將斑馬魚的第一股cDNA以反轉錄作用合成後,當成模板(Template)以Degenerative primers進行PCR(polymerase chain reaction)。並構築在選殖用載體(pGEM-T vector),進行DNA定序後,與Genebank進行比對分析,所選殖的DNA序列無一與Desaturase基因有較高的相似性。另外再以老鼠的Desaturase DNA當探針對選殖出來的菌株進行Colony hybridization。同時,亦進行斑馬魚cDNA基因庫(cDNA library of zebrafish)的篩選。 To live in cold environment, organisms should increase the unsaturativity of lipid composition to keep membrane fluidity. Unsaturativity can be not only regulated directly by lipid intake, but also affected by desaturase. Studies about thermally-acclimating fishes indicated that increased unsaturativity of membrane lipid composition is one of the major reason of increased tolerance against cold environment. Activity analysis of desaturase also revealed that increased activity can be induced by low temperature. Many desaturases had been purified and sequenced in algae, plants, and mammalians including rats and mouses, but no one in fishes. In order to investigate the relation of desaturase expression between lipid composition and environmental temperature in fish, gene screenings and sequencings are the first things to be done. Zebrafish is used as experimental source to select desaturase gene, and reverse transcribed cDNA are used as templates to proceed PCR. Degenerative primer sequences are designed from stearoyl-CoA desaturases of rat, mouse, and yeast. Resulting dsDNAs are cloned into pGEM-T vector for selections and sequencing. No homologies are obtained when sequences are compared with desaturases sequence from Genebank. Furthermore, rat's desaturase DNA is used as probe to select those PCR---colonies (colony hybridization). At the same time, cDNA library screening of zebrafish is under way.

Keyword(s)

冷馴化
去飽和脂肪酸酵素
適應
脂質組成
組織結構馴化
Cold acclimation
Desaturase
Adaptation
Lipid composition
Ultrastructuralacclimation

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Studying the Desaturase Functions in the Cold Acclimation of Fish

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