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研究生：	楊采文 Yang, Tsai-Wen
論文名稱：	全基因體篩選叢毛單孢菌誘發線蟲fat-7基因表達改變之因子 A Genome-wide Screen for the Bacterial Determinants Involved in Comamonas aquatica-induced fat-7 Alteration in C. elegans
指導教授：	陳昌熙 Chen, Chang-Shi
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學暨分子生物學研究所 Department of Biochemistry and Molecular Biology
論文出版年：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	45
中文關鍵詞：	秀麗隱桿線蟲、叢毛單孢菌、單不飽和脂肪酸去飽和酶
外文關鍵詞：	Caenorhabditis elegans, Comamonas aquatica, mono-unsaturated fatty acid desaturase
相關次數：	點閱：148 下載：0
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飲食深刻影響動物的新陳代謝，並與年齡相關的疾病有關。先前的研究發現，秀麗隱桿線蟲(Caenorhabditis elegans)在遇到不同的食物來源，如：大腸桿菌(Escherichia coli)、芽孢桿菌(Bacillus)和叢毛單胞菌(Comamonas)時，會調整其生理機能和脂肪儲存以調節壽命。然而，飲食與衰老之間的分子機制尚未闡明。在台灣大學吳益群教授實驗室發現，在餵食線蟲叢毛單胞菌菌株DA1877 後，其脂質含量有明顯的降低。其中，線蟲的單不飽和脂肪酸去飽和酶基因fat-7的表達被下調。我們好奇叢毛單胞菌是如何調控線蟲fat-7的表現，為此我們建立了一個基因篩選平台。為了系統性的鑑定參與在fat-7表達的叢毛單胞菌因子，我們使用綠螢光蛋白標記的fat-7基因轉殖線蟲，進行叢毛單胞菌的突變基因篩選。我們挑選線蟲fat-7的表達不會受到下調的突變株做為目標菌株。從21,897 個叢毛單胞菌Tn10跳躍子突變株中，利用液相初步篩選出294個可能參與在線蟲fat-7表達突變株，並在固相的二次篩選進一步證實有3個突變株fat-7的表達仍被上調。目前我們已鑑定出甲基丙二酸輔酶A變位酶基因和乙醯/丙醯輔酶A羧化酶基因的突變，會使線蟲fat-7表達不被下調，並且這二個基因都參與氨基酸和脂肪酸代謝。我們未來會將這二個基因在野生型叢毛單孢菌中進行突變，並在跳躍子突變株中回補這二個基因，再次確認我們的結果。概括上述，我們的結果顯示甲基丙二酸輔酶A變位酶基因和乙醯/丙醯輔酶A羧化酶基因會參與叢毛單胞菌誘導線蟲fat-7改變的細菌決定因素。

Diet profoundly affects metabolism and associates with age-related diseases in metazoans . Previous studies found that Caenorhabditis elegans, as a bacterivore, adjusts their physiology and fat storage to regulate lifespan when encounters distinct food sources, e.g., Escherichia coli¸ Bacillus, and Comamonas. However, the molecular mechanisms between diet and aging have not been elucidated. In Dr. Yi- Chun Wu's laboratory at NTU, they demonstrated that fat-7, a gene encodes mono-unsaturated fatty acid desaturase, was down-regulated in Comamonas aquatica DA1877-fed C. elegans. To identify the bacterial determinants involved in C. aquatica-mediated fat-7 expression systemically and unbiasedly, we established a high-throughput screening platform and conducted a C. aquatica genome-wide transposon mutant library screening using the GFP-labeled fat-7 transgenic C. elegans. We examined the fluorescence intensity for fat-7 expression and identified the DA1877 mutants that did not downregulate fat-7 expression compared to DA1877 wild-type. From a total of 21,897 Comamonas aquatica Tn10 transposon mutants, we found 294 candidates were potentially involved in the regulation of fat-7 in C. elegans from the primary screen. We further confirmed that 3 mutants still conferred fat-7::GFP upregulation in C. elegans by the secondary screen. We determined the transposon insertion gene through the arbitrary PCR method, and the mutations in the methylmalonyl-CoA mutase gene and the acetyl/propionyl-CoA carboxylase gene were identified. Of note, both of them participate in amino acids and fatty acids metabolism. We would generate deletion mutants and complement the gene in transposon mutants to reconfirm our results. Overall, our results suggested that the methylmalonyl-CoA mutase and acetyl/propionyl-CoA carboxylase are the two bacterial determinants involved in Comamonas aquatica-induced fat-7 alteration in C. elegans.

致謝 i
Abstract ii
中文摘要 iii
Table of Content iv
Introduction	1
Diet affects life-history traits in the organism 1
Fat Metabolism and Fat Content in Different Diets 1
Fatty Acid Desaturase-7 (fat-7)	2
fat-7 expression is a potential biomarker for screening the bacteria determinants that may participate in the regulation of C. elegans’  fat metabolism and lifespan 2
Material and Methods 3
Strain and Culture of Caenorhabditis elegans 3
Bacterial Strains and Plasmids	3
Construction of  C. aquatica Transposon Mutagenesis Library 3
Diet Shift Assay 4
Transposon Mutagenesis Library Screening Method	4
Real-Time Quantitative Reverse Transcription PCR(qRT-PCR) 5
Growth analysis of bacteria 6
C. elegans Lifespan Assay 6
Pore Forming Toxin Killing Assay 7
Arbitrary PCR for the Transposon Mutant Genes 7
Construction of Comamonas Deletion and Complementation mutants 8
Results 9
The expression of fat-7 was downregulated in DA1877-fed nematodes 9
Screening for bacterial determinants gene in Comamonas aquatica 10
The fat-7 expression was regulated during transcriptional or post-transcriptional processes 10
The DA1877 mutants were no growth defective and affected nematode’s lifespan 11
The fat homeostasis derived from the diet did not participate in the interaction of pore-forming toxin and their receptor 11
Identification of the transposon insertion gene confers the upregulation of fat-7 expression 12
Reconfirmation of the target gene by genetic complementation 13
Discussion 14
Reference 16
Figures	20
Figure 1. 20
Figure 2. 23
Figure 3. 25
Figure 4. 28
Figure 5. 29
Figure 6. 30
Figure 7. 31
Figure 8. 35
Table 37
Table 1. The nematode strains were used for this study 37
Table 2. The bacteria strains and plasmids were used for this study 37
Table 3. Primers of Arbitrary PCR 38
Table 4. The formula of Mini-Tn10 transposon mutagenesis library 38
Supplemental data 39
Figure S1. 39
Figure S2. 40
Figure S3. 41
Figure S4. 42
Figure S5. 43
Figure S6. 44
Figure S7. 45
                                    

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2026-09-03公開

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