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研究生:徐晁偉
研究生(外文):Jau-Wei Syu
論文名稱:HydA-mutated Chlorella sp. DT突變株之NADPH與H2生成及製備HydA-StrepII DT突變株
論文名稱(外文):NADPH reduction and H2 production of HydA-mutated-overexpressed Chlorella sp. DT mutants as well as generation of HydA-StrepII-overexpressed DT mutant
指導教授:簡麗鳳簡麗鳳引用關係
指導教授(外文):Lee-Feng Chien
口試委員:徐邦達朱修安
口試委員(外文):Ban-Dar HsuHsiu-An Chu
口試日期:2016-04-12
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:88
中文關鍵詞:綠藻氫氣葉綠體分離
外文關鍵詞:algaehydrogenNADPHChloroplast isolation
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綠藻光合作用系統在特定條件下,可利用太陽能,藉由產氫酶(HydA)產生氫氣(H2)。在過去的研究,得知HydA-overexpressed Chlorella sp. DT (DT)突變株及氧氣(O2)耐受性HydA-mutated-overexpressed DT突變株,相較於DT野生株(DT-WT),均可增加H2產量。然而,HydA蛋白表現量與H2產量不相符。為了瞭解DT突變株硫鐵蛋白-NADP+-氧化還原酶(FNR)是否與HydA競爭電子而導致上述情形,於是測量DT突變株葉綠體之NADPH還原與H2生成。
HydA-mutated-overexpressed DT突變株經過證實仍有hydA-mutated序列,且於5% O2環境時,可產生較多H2。HydA-mutated-overexpressed DT突變株葉綠體分離後,以NADP+處理並測量NADPH還原速率,發現HydA-mutated-overexpressed DT突變株NADPH還原速率約為DT-WT 0.3倍。測量HydA-mutated-overexpressed DT突變株葉綠體H2產量,結果顯示,未以NADP+處理之H2產量,比NADP+處理之H2產量,高0.3到0.4倍。HydA-mutated-overexpressed DT突變株之NADPH及H2產量百分比分別約為32%及68%。DT-WT之NADPH及H2產量百分比分別約為44%及56%。根據以上結果推測,FNR還原NADPH時,會限制HydA產生H2。
另外,將來可能進行蛋白純化,故而,以StrepII-tag標記HydA。將strepII-tag構築至載體pHyg3-HydA-StrepII,並以電穿孔方式轉殖於DT。藉由PCR,發現DT轉殖株含有hydA-strepII片段,確認載體已成功轉殖。以西方墨點法偵測,確認HydA-StrepII蛋白可表現於DT轉殖株。透過超音波,打破HydA-StrepII DT突變株細胞,並離心萃取總蛋白。利用Strep-Tactin親和性管柱,純化HydA-StrepII;然而,現階段HydA-StrepII純化尚不足量。


Green algae have a photosynthetic system that can produce H2 by hydrogenase (HydA, encoded by hydA) using sun light under certain condition. In the previous studies, an HydA-overexpressed mutant and some O2 tolerant HydA-mutated-overexpressed Chlorella sp. DT (DT) mutants were obtained to enhance the H2 production in this laboratory. However, the increased fold of HydA expression was not compatible to that of H2 production. In order to understand whether Ferredoxin-NADP+-oxidoreductase (FNR) competes electrons with HydA of DT mutants, NADPH reduction and H2 photoproduction of isolated chloroplasts of mutants were measured.
The mutated-hydA sequence and H2 production of O2 tolerant 3-year-old HydA-mutated-overexpressed DT mutants were confirmed. DT mutants still carried mutated-hydA gene and had O2 tolerance so that they could produce more H2 than DT-WT. The chloroplasts of DT mutants were isolated. With addition of NADP+ as FNR substrate, the NADPH reduction rates of isolated chloroplasts of one HydA-mutated-overexpressed DT mutants were 0.5 fold of DT-WT. Without addition of NADP+, the H2 photoproduction of isolated chloroplasts of DT mutants was about 0.3-0.4 fold higher than that with addition of NADP+ condition. The percentages of NADPH reduction and H2 production of one HydA-mutated-overexpressed DT mutants were 32% and 68%. The percentages of NADPH reduction and H2 production of DT-WT were 44% and 56%. The results suggested that the FNR-mediated NADPH reduction limited HydA-mediated H2 photoproduction in HydA-mutated-overexpressed DT mutants.
Moreover, in order to measure the activity of HydA for further investigation in the future, the wild type HydA protein was genetically attached a StrepII-tag. The gene of strepII-tag was constructed into plasmid pHyg3-HydA-StrepII, ligated to the 3'' end of hydA gene, and electrotransformed into DT. The observation of hydA-strepII fragment in DT transformants by PCR indicated that the plasmid was successfully transformed into DT. The detectable HydA-StrepII protein by western blot suggested that HydA-StrepII could be expressed in DT transformants. The HydA-StrepII DT mutant cells were broken by sonication and the total proteins were extracted by centrifugation. Furthermore, the HydA-StrepII was purified by Strep-Tactin affinity column. However, no detectable signal of HydA-StrepII was visualized. The results implied that there may be no enough HydA-StrepII extracted from HydA-StrepII DT mutant at present stage.


Abstract i
中文摘要 iii
Contents iv
List of tables and figures vii
Abbreviations x
1. Introduction
1.1 H2 photoproduction in green algae 1
1.2 HydA of green algae 1
1.3 Chlorella sp. DT gas channel mutants 2
1.4 NADPH reduction and H2 production in green algae 3
1.5 Purification of HydA 4
1.6 The purpose of this work 4

2. Materials and methods
2.1 Algal culture 6
2.2 Isolation of algal genomic DNA 6
2.3 polymerase chain reaction (PCR) 7
2.4 Isolation of algal DT total protein 7
2.5 Sodium dodecyl sulfate-polyacrylamide gel
electrophoreses (SDS-PAGE) analysis of algae
extracted total proteins 8
2.6 Western blotting analysis of HydA, StrepII, and PsbO
8
2.7 Standard curve of H2 concentration determined by GC 9
2.8 Measurement of in vitro H2 production by GC 10
2.9 Isolation of Chlorella DT chloroplasts 11
2.10 Measurement of chlorophyll fluorescence parameters
by fluorescence monitoring system 11
2.11 Measurement of in vitro NADPH reduction rate in
Chlorella chloroplasts by spectrophotometer 13
2.12 Measurement of in vitro H2 photoproduction in
Chlorella chloroplasts by GC 13
2.13 Site-Direct Mutagenesis 14
2.14 Construction of pHyg3-HydA-StrepII 15
2.15 E.coli heat shock transformation 15
2.16 Isolation of plasmids 15
2.17 Electroporation transformation of DT with plasmids
16
2.18 Purification of HydA-StrepII 17

3. Results
3.1 Confirmation of 3-year-old HydA-mutated-overexpressed
DT mutants
3.1.1 Culture of 3-year-old HydA-mutated-overexpressed DT
mutants on selected plate 18
3.1.2 Detection of b2TP-hydA gene fragment in 3-year-
old HydA-mutated-overexpressed DT mutants genomes
by PCR 18
3.1.3 Expression of HydA and PsbO proteins from 3-year-
old HydA-mutated-overexpressed DT mutants 18
3.1.4 In vitro H2 production of 3-year-old HydA-mutated-
overexpressed DT mutants 19
3.2 Photosynthetic properties of 3-year-old HydA-
mutated-overexpressed DT mutants
3.2.1 PSII activities of whole cells 20
3.2.2 PSII activities of isolated thylakoids 20
3.3 Properties of isolated chloroplasts of 3-year-old
HydA-mutated-overexpressed DT mutants
3.3.1 Isolation of chloroplasts from DT-WT, HydA-
overexpressed DT mutant, and 3-year-old HydA-
mutated-overexpressed DT mutants 20
3.3.2 PSII activities of isolated chloroplasts 21
3.3.3 In vitro NADPH reduction rate of isolated
chloroplasts 21
3.3.4 In vitro H2 photoproduction of isolated
chloroplasts 22
3.4 pHyg3-HydA-StrepII plasmid constructed and
electroporated into DT
3.4.1 Design of strepII-tag fragment 22
3.4.2 Point mutation of hydA stop codon of pHyg3-HydA
plasmid 23
3.4.3 Ponstruction of pHyg3-HydA-StrepII plasmid 23
3.4.4 Identification of hydA-strepII fragment by
sequencing 24
3.4.5 Electrotransformation of DT with pHyg3-HydA-StrepII
24
3.4.6 Existence of hydA-strepII fragment in the genomic
DNA of HydA-StrepII DT mutants 25
3.4.7 Expression of HydA, HydA-StrepII, and PsbO proteins
from HydA-StrepII DT mutants 25
3.4.8 Purification of HydA-StrepII from HydA-StrepII DT
mutants 26

4. Discussion
4.1 H2 production of 3-year-old HydA-mutated-
overexpressed DT mutants 27
4.1 PSII activity of isolated chloroplasts from DT 27
4.2 NADPH reduction and H2 production of isolated
chloroplast 28
4.3 Isolation of HydA from DT 28

5. Conclusions
5.1 Existence of HydA-mutated transgenes in 3-year-old
HydA-mutated-overexpressed DT mutants 30
5.2 Isolation of algae chloroplast 30
5.3 FNR mediated NADPH reduction limits HydA mediated the
H2 photoproduction 30
5.4 NADPH reduction in HydA-mutated-overexpressed DT
mutants 31
5.5 DT-BHS transformants carrying hydA-strepII gene and
overexpress HydA-StrepII fusion protein 31
5.6 Purification of HydA-StrepII 31

6. References 32

7. Appendices 83





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