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An Attempt To Reestablishing The Lost Function Of Ascorbic Acid Biosynthesis In Zebrafish (Danio rerio), By Constructing A Transgenic Zebrafish With A Functional L-Guluno- -Lactone Oxidase (gulo) Isolated From Cloudy Catshark (Schliorhinus torazame).

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Abstract
The vitamin C is one of the essential micronutrients in living organisms. Vitamin C, also known as ascorbic acid, is a water-soluble low molecular weight carbohydrate which acts as a strong antioxidant. It involves a vast array of biological processes in the body such as collagen synthesis, gene regulation, epigenetic regulation, hormonal regulation, in metabolic energy synthesis, and immunity. Both plants and animal kingdoms synthesize vitamin C in their body except for few exceptions. In animal kingdom including human, anthropoid primates, guinea pigs, some bats, some passerine birds, and teleost fish have lost the ascorbic acid biosynthesis ability throughout the evolution. This functional loss is caused by a genetic lost in those animal species. The animals mentioned above have lost the gulo (L-gulono- -lactone oxidase) gene that codes for the L-gulono lactone oxidase enzyme which catalyzes the final enzymatic reaction of the animal ascorbic acid synthesis pathway. Up to date scientists were unable to elucidate a proper solid reason for the fluctuated gulo loss throughout the evolution, there are several hypotheses around the science community, but those hypotheses are clashing each other. So, up to now the gulo loss remain as a paradox in science. With the modern development of molecular biology and transgenic studies, we tend to address this problem from a new approach. We attempt to revoke the ascorbic acid biosynthesis in teleost fish using one of the most popular vertebrate model Danio rerio (zebrafish). In search of gulo gene, we found an ascorbic acid synthesizing fish species which was available in fish markets in Korea. The cloudy catshark (Scyliorhinus torazame) gulo, which was designated as Sgulo, was isolated from the catshark kidney and used to revoke the ascorbic acid biosynthesis in zebrafish. In the process, we constructed a Tol2 based expression clone which was included a b-actin promoter, Sgulo and mcherry reporterTg(bactin2:Sgulo-mcherry). We used the multi-site gateway cloning technique in constructing the expression clone. The constructed expression clone was microinjected to zebrafish embryos and selected the F0 positive embryos by the red fluorescent, which was given by the mcherry.
Though we used b-actin promoter F0 generation gave a mosaic expression of the fluorescent construct. This mosaic expression was due to the different construct integration time at the embryo development. Afterward, we checked for the germline transmission of F0 generation and obtained the F1 generation by mating the F0 positive male with wild-type female. The positive F1 was used to make the F2 generation. After making the transgenic F1 zebrafish line, we confirmed the Sgulo genomic insertion and the Sgulo mRNA expression in the transgenic fish.
Furthermore, we checked the physiological and developmental differences, growth differences, endogenous ascorbic acid levels, and Sgulo enzyme activity in transgenic and wild-type zebrafish. The physiological and development properties between the transgenic and wild-type zebrafish did not show any difference between the two groups, but the growth and the weight of the fish, endogenous ascorbic acid levels and Sgulo enzyme activity showed a significant difference in Tg with compared to Wt group. With these results, we could confirm that we were able to create a transgenic zebrafish which consist of a Sgulo gene in its genome, which expresses ubiquitously and able to re-establish the ascorbic acid biosynthesis. As future aspects, further, analysis can be done using the developed transgenic model. High precision and high-tech analysis like HPLC could confirm our results at a high precision level and could clear the roads to use the Sgulo transgenic model as a regenerative, toxicological, immune, behavioral, therapeutic and cancer research model.
Author(s)
Nimod Dilushan Janson
Issued Date
2019
Awarded Date
2019. 8
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/common/orgView/000000009018
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
Lee, Je Hee
Table Of Contents
Summary i
Acknowledgment . iii
List of Figures vi
List of Tables vii
Introduction . 1
1.1. Ascorbic acid . 1
1.2. Biological importance of vitamin C . 2
1.3. Ascorbic acid (vitamin C) biosynthesis . 4
1.4. The loss of vitamin C biosynthesis 6
1.4.1. The genetics and the loss of GULO throughout vertebrate evolution 6
1.4.2. Vitamin C deficiency 7
1.5. Previous attempts on restoring vitamin C biosynthesis in scurvy prone animals 7
1.6. Zebrafish (Danio rerio) and Cloudy catshark (Scyliorhinus torazame) 8
1.7. Objectives and the summary of the project 9
2. Materials and Methods 11
2.1. Zebrafish husbandry. 11
2.2. Isolation of Sgulo from cloudy catshark 11
2.2.1. In-silico analysis of Sgulo . 11
2.2.2. Dissection of cloudy catshark to obtain kidney. . 12
2.2.3. Total RNA extraction. . 12
2.2.4. The cDNA synthesis. 12
2.3. Constructing the Expression construct. 13
2.3.1. Primer designing . 13
2.3.2. Gateway cloning vectors . 14
2.3.3. Amplifying attB-PCR products. 14
2.3.4. Constructing entry clones using the BP recombination reaction. . 14
2.3.5. Constructing expression clones using the LR recombination reaction . 15
2.4. Synthesizing the capped transposase mRNA . 15
2.5. Construction of transgenic zebrafish . 16
2.5.1. Microinjection . 16
2.5.2. Selection of transgenic F0 embryos 16
2.5.3. Raising the F0 transgenic zebrafish 17
2.5.4. Making the F1 transgenic zebrafish line . 18
2.5.4. Making the F2 transgenic zebrafish line . 19
2.5.5. Confirmation of transgenic fish by genotyping 19
2.6. Development and physiological assessment between transgenic and control fish groups 19
2.7. Analysis of growth rate between transgenic and control zebrafish . 20
2.8. The Sgulo expression in transgenic zebrafish 20
2.9. Gene expression analysis . 21
2.10. GULO assay 21
2.11. Statistical analysis 22
3. Results and discussion 23
3.1. In-silico analysis 23
3.2. Gulonolactonase expression . 28
3.3. Construct expression clone 29
3.4. Transgenic zebrafish 30
3.4.1. F0 generation 30
3.4.2. F1 generation 33
3.5. Genotyping and expression confirmation 34
3.6. Development and physiological assessment 35
3.6.1. The physiological difference . 36
3.7. Growth analysis . 38
3.8. Endogenous ascorbic acid 39
3.9. Differential gene expression 41
3.10. Sgulo enzyme activity. . 44
4. Conclusion 46
References . 47
Degree
Master
Publisher
제주대학교 대학원
Citation
Nimod Dilushan Janson. (2019). An Attempt To Reestablishing The Lost Function Of Ascorbic Acid Biosynthesis In Zebrafish (Danio rerio), By Constructing A Transgenic Zebrafish With A Functional L-Guluno- -Lactone Oxidase (gulo) Isolated From Cloudy Catshark (Schliorhinus torazame).
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General Graduate School > Marine Life Sciences
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