利用斑馬魚暴露氯化鉛的影響評估及應用CRISPR-Cas9工具調解斑馬魚肌肉氧結合myoglobin基因

Evaluation of exposing lead chloride in zebrafish and CRISPR-Cas9 knockout tool of muscular oxygen-binding myoglobin gene in zebrafish

裴氏玉孝(THI NGOC HIEU BUI)

指導教授 ： 蕭崇德

中原大學/理學院/化學研究所/博士(2020年)

https://doi.org/10.6840/cycu202000434

摘要

論文是四項研究的全部內容。 第一項研究包括在改進的T型迷宮規程中，以評估成年斑馬魚記憶能力的學習情況。 T型迷宮裝置有多種類型，規程，在先天研究中得到了廣泛的應用。 雖然以前的研究都是基於食品獎勵協議，但程序的效率要求長期的培訓過程。 在這裡，我們旨在增強具有寬臂深度的設備，進行色彩調節以及通過電擊調節（1-2 V，0.3 – 0.5 mA）改進協議。 三種條件添加的組合導致在三天的持續時間內縮短方案。 我們證明了成年性別，記憶障礙 （ZnCl2處理）和肥胖表型突變體（lepa KO）在斑馬魚中的空間記憶性能。
第二項研究集中於低濃度（50 ppb）成年斑馬魚暴露於慢性毒性氯化鉛的影響。用氯化鉛處理一個月後進行行為和生化測試。我們發現攻擊性和短期記憶力測試顯著降低。生化分析的結果表明，大腦中5-羥色胺，褪黑激素表達的皮質醇活性顯著增加而顯著降低。最終，長期暴露於氯化鉛的影響可能導致生態毒性，並可能降低其使用壽命。
第三,四項研究是檢查斑馬魚中細胞內氧轉移基因的功能。根據我們的轉錄組學篩選，斑馬魚在常見進化枝中與正常的血紅蛋白和肌紅蛋白重疊群數對齊。在冰魚中缺乏血紅蛋白和肌紅蛋白表達的比較中，我們應用CRISPR / Cas9（聚類的規則間隔的短回文重複序列）/相關蛋白9）工具進行基因組編輯的突變株。目的探討肌紅蛋白基因在改變斑馬魚行為活動中的作用。

關鍵字

慢性中毒 ； 內存容量 ； 學習能力 ； CRISPR / Cas9(常間回文重複序列叢集關聯蛋白) ； 斑馬魚

並列摘要

The dissertation is fully inclusive contents of four studies. The first study was included in advancement in modified T-maze protocol to evaluate learning in mnemonic capacity of adult zebrafish. T-maze apparatuses are various types, protocols and extensively utilized in congenital research. While previous studies have based on food reward protocol, the efficiency of procedure is required a long training process. Here, we aimed to the enhancement of equipment with spacious arm depth, color conditioning and the improvement of protocol with electric shock conditioning (1-2 V, 0.3 – 0.5 mA). The combination of three conditioning additions resulted in shorten protocol in three-day proceeding duration. We demonstrated the spatial memory performance of adult gender, memory impairment (ZnCl2 treated) and obesity phenotype mutant (lepa KO) effect in zebrafish. The second study was focused on the effect of chronic toxicity lead chloride exposed in adult zebrafish at low concentration (50 ppb). Behavioral and biochemical tests were performed after treating with lead chloride for one month. We found significantly decreased of aggressiveness and short-term memory test. The results of biochemical assays showed the activities of cortisol increased significantly and decreased dramatically of serotonin, melatonin expressions in the brain. In conclusive, the effect of chronic lead chloride exposure cause possible ecotoxicity and may reduce their life-span fittingness. The third and fourth studies was examination of the function of intracellular oxygen transfer genes in zebrafish. According to our metatranscriptomics screening, zebrafish aligned in common clades with the normal hemoglobin and myoglobin contig numbers. In comparison of the lacking hemoglobin and myoglobin expression in ice fish, we established a mutant line with a genome editing by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat)/ associated protein 9) tool. To investigate the role of myoglobin gene in altering the behavior activities in zebrafish.

並列關鍵字

chronic toxicity ； memory capacity ； learning capacity ； CRISPR/Cas9 ； zebrafish

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