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研究生：	吳宛霖 Wu, Wan-Lin
論文名稱：	探討蝴蝶蘭原球體發育時期蔗糖對乙醛酸循環代謝路徑之調控 Study of glyoxylate cycle regulation by sucrose during protocorm growth in Phalaenopsis aphrodite
指導教授：	蔡文杰 Tsai, Wen-Chieh
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 熱帶植物科學研究所 Institute of Tropical Plant Sciences
論文出版年：	2014
畢業學年度：	102
語文別：	英文
論文頁數：	67
中文關鍵詞：	台灣白花蝴蝶蘭、萌發、蔗糖、乙醛酸循環
外文關鍵詞：	Phalaenopsis aphrodite, germination, sucrose, glyoxylate cycle
相關次數：	點閱：147 下載：0
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乙醛酸循環(glyoxylate cycle)為油類種子萌發的特有代謝路徑，在油脂的代謝中扮演了不可或缺的角色。研究顯示，在種子萌發過程中，乙醛酸循環關鍵酵素isocitrate lyase (ICL)及malate synthase (MLS)的酵素活性及基因表現受到外加碳水化合物所抑制。蝴蝶蘭為單子葉植物，但其構造卻和其他單子葉植物的澱粉類種子不同。成熟的蘭花種子中具有一個未分化的胚，不具胚乳組織，且在未分化的胚中含有大量的油脂組織。由於蘭花種子所儲存的能量極少，自然萌發時須真菌共生提供足夠的能量。在試管培養播種時，碳水化合物的添加扮演重要的角色，使蘭花種子正常生長發育。為了解碳水化合物對於蝴蝶蘭種子萌發及原球體發育時油脂運轉情形的影響，本研究首先在OrchidBase中取得PaICL和PaMLS兩個基因的全長，探討蝴蝶蘭種子在組織培養過程中，蔗糖對台灣白花蝴蝶蘭種子在萌發後不同時期中乙醛酸循環的影響。結果顯示，在萌發後原球體發育過程中，蔗糖的添加使PaICL及PaMLS的基因表現量下降，其中PaMLS的基因表現在蔗糖的處理後顯著降低。為了進一步找出可能調控PaMLS轉錄的轉錄因子，本研究選用在1/2MS及1%蔗糖培養基中，培養4天及7天原球體進行次世代定序，並分析兩時期基因體中表現量具有差異的基因。此外，利用plantPAN資料庫預測PaMLS 2-kb的啟動子序列上可能的轉錄因子結合位點。綜合原球體差異表現基因以及PaMLS啟動子序列分析之結果，篩選出一個正調控轉錄因子基因(PaHB5)及七個負調控轉錄因子基因(PaANT, PaMADS2, PaMYB4, PaPIF3, PaRAV1-1, PaWRKY18, PaWRKY71)。利用雙螢光素酶活性分析，發現PaHB5對於malate synthase的啟動子具有2.73倍啟動的能力。此研究的結果首次揭開了乙醛酸循環在分子機制上的調控機制。

The glyoxylate cycle plays a central role in converting storage oil to soluble carbohydrate in oilseeds to support growth during germination. Enzyme activity or transcript accumulation of key enzymes of the glyoxylate cycle, isocitrate lyase (ICL)/ malate synthase (MLS), are downregulated by carbon catabolite repression during seed germination. Without endosperm and cotyledon, orchid seeds was found large amount of lipid reserved in the immature embryo, and carbohydrates play a vital role to support protocorm development during tissue culture. To investigate the metabolism of orchid seed during germination, I explored how the sucrose regulates the glyoxylate cycle during the Phalaenopsis aphrodite protocorm development. We identified ICL and MLS from OrchidBase, and named as PaICL and PaMLS. Expression analysis showed that sucrose depressd the expression of both PaICL and PaMLS, and the transcripts level of PaMLS was extremely downregulated by sucrose treatment. For identification of putative genes encoded transcription factor which regulate expression of malate synthase, digital expression analysis comparing transcriptomes derived from day 4 and day 7 protocorms cultured on 1/2 MS medium and sucrose was performed. In addition, 2-kb upstream sequence of malate synthase gene was retrieved from OrchidBase for analyzing regulatory motif by plantPAN. Combining transcriptomic and regulatory motif analysis, one putative positive (PaHB5) and seven negative transcription factor genes (PaANT, PaMADS2, PaMYB4, PaPIF3, PaRAV1-1, PaWRKY18, PaWRKY71) are identified. Dual luciferase assay was adopted to analyze binding ability of PaHB5 to the promoter of malate synthase gene. Results show that PaHB5 could increase expression of malate synthase gene for 2.73 fold. This is the first study reveal that the molecular mechanism underlying glyoxylate cycle.

中文摘要………………………………………………………………………i
Abstract ……………………………………………………………………iii
致謝 ………………………………………………………………………………v
Table of Content ………………………………………………vi
List of Tables ..…………...……………………………x
List of Figures ...…………………………………………xi
List of Appendix Figures ...…………………xiii

1.Introduction
1.1 Orchid seeds
1.1. 1The feature of orchid seed morphology ………………………………1
1.1.2 Germination of orchid seed …………………………………………..……...2
1.2 Metabolism during flowering-plant seed germination
1.2.1 Storage products in flowering-plant seeds ……………………4
1.2.2 Metabolism of endospermic seed during germination 4
1.2.3 Metabolism of oil seed during germination ….………………5
1.3 Glyoxylate cycle in plant ………………………………………………………….....6
1.4 Regulation of glyoxylate cycle ……………………………………………………..7

2. Specific aim …………………………………………………………………………....9

3. Material and Method
3.1 Plant materials and growth conditions …………….……………10
3.2 Histological analysis ………………………………..………………………………..10
3.3 Transmission electron microscopy ……………………………………………10
3.4 RNA preparation …………………………………………………….…………….......11
3.5 RT-PCR and real-time quantitative PCR ……………………………11
3.6 5’ Rapid amplification of PaICL cDNA ends (RACE).12
3.7 3’ Rapid amplification of PaMLS cDNA ends (RACE)…13
3.8 Sequence alignments and phylogenetic analysis …….14
3.9 Comparison of transcriptomic profiling by RNA sequencing
3.9.1 Sequencing the protocorm transcriptomes by next generation sequencing …………………………15
3.9.2 Transcriptomic comparison …………………………………………………15
3.10 Promoter analysis of PaMLS
3.10.1 Promoter analysis of PaMLS …………………………………………………16
3.10.2 Transcription factor analysis …………………………………………16
3.11 Transient expression experiments and dual luciferase reporter assay
3.11.1 Genomic DNA extraction ……………………………………………………....17
3.11.2 Promoter-reporter plasmid construction…………………….17
3.11.3 PaHB5 plasmid construction ………………………………………………..18
3.11.4 Transient transformation by using particle bombardment ………...18
3.11.5 Dual luciferase assy …………………………………………………………....19

4. Result
4.1 Distribution of storage products in Phalaenopsis aphrodite mature seeds ……….20
4.2 TEM of a section through a P. aphrodite 0 DAI protocorm ……………………….20
4.3 Effects of sucrose on protocorm development in P. ahrodite ……………………...21
4.4 Identification of PaICL and PaMLS in P. aphrodite …………………………….....21
4.5 Phylogenetic analysis of PaICL and PaMLS …………………………………….....22
4.6 Temporal expression of PaICL and PaMLS during protocorm development ……22
4.7 Comparison of sucrose-affected transcript profiles between 4 DAI and 7 DAI protocorms …………………………………………………23
4.8 Function assessment of developing stage-dominant unigenes …………………….24
4.9 Characterization of developing stage-dominant unigenes by KEGG patyays ......25
4.10 Digital comparison of expression level of genes involved in oil mobilization pathway …………………………………………………………………..………….....26
4.11 Analysis of PaMLS promoter ………………………………………………..…...27
4.12 Temporal expression of putative transcription factors during protocorm development ………………………………………………………………………...….28
4.13 Activation ability of PaHB5 on PaMLS promoter ……………………….……..28

5. Discussion
5.1 Different effects of sucrose on PaICL and PaMLS expression ..…………………30
5.2 Transcriptional regulation of sucrose on PaMLS ………………………………...31

6. Reference………………………………………………………………..……………34

List of Tables
Table 1. List of primers used in this study ……………………..38
Table 2. Number of unigenes mapped in KEGG pathways...40
Table 3. Predicted unigenes that may bind to PaMLS promoter …………………..44
Table 4. Putative transcription factor of PaMLS ………………………45
Table 5. Predicted ATHB5 binding sites on 2000 bp upstream sequence of PaMLS ………………………………………………………………………………….46

List of Figures
Figure 1. Distribution of storage products in Phalaenopsis aphrodite mature seeds …………………………………………………………………………………….47
Figure 2. Transmission electron micrograph of a section through a Phalaenopsis aphrodite 0 DAI protocorm ……………………………………………………………48
Figure 3. Phenotypes of different developmental protocorm stage in Phalaenopsis aphrodite ………………………………………………………………………………..49
Figure 4. Effect of sucrose on expression of PaICL and PaMLS during Phalaenopsis aphrodite protocorm development …………………………………………………….50
Figure 5. Phylogenetic analysis of ICL by neighbor-joining method ……………….51
Figure 6. Phylogenetic analysis of MLS by neighbor-joining method ………………52
Figure 7. Venn diagram of Phalaenopsis aphrodite protocorm transcriptomes ……53
Figure 8. Representation of gene ontology assignments for 4 DAI and 7 DAI dominant genes derived from Phalaenopsis aphrodite protocorm transcriptomes …………..54
Figure 9. Digital comparison of expression level of glyoxylate cycle genes …….....56
Figure 10. Digital comparison of expression level of TCA cycle genes ………..…..57
Figure 11. Digital comparison of expression level of glycolysis/ gluconeogenesis genes ……………………………………………………………………………………58
Figure 12. The predicted transcription factor function of unigenes derived from Phalaenopsis aphrodite protocorm transcriptomes ………………………………….60
Figure 13. RT-PCR analysis of putative transcription factor genes during Phalaenopsis aphrodite protocorm development ……………………………………61
Figure 14. Real-time quantitative RT-PCR analysis of putative transcription factor genes during Phalaenopsis aphrodite protocorm development ………………………62
Figure 15. Activation ability of PaHB5 on the PaMLS promoter ……………………64

List of Appendixs Figures
Appendixs Fig 1. Oil mobilization pathway in oil seeds ……………………………65
Appendixs Fig 2. pJD301 map ……………………………………………………….66
Appendixs Fig 3. pBI221 map ………………………………………………………..67



                                    

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