臺灣博碩士論文加值系統

景天酸代謝為三種二氧化碳固定途徑之一，其特性為光合速率較慢但呈現極高的水分利用。雖然先前研究指出表現甜椒 plant ferredoxin-like protein (PFLP) 蛋白可增強植物對細菌性病原的抵抗力。但是由於 PFLP 其蛋白質序列與光系統I (photosystem I)中的光合蛋白 ferredoxin-1 (Fd-1) 具有高度相似性，所以如在植物中過表達該基因，有可能促使轉殖植物其光合能力增加。為了證明 PFLP 可促進光合能力，我們將 pflp 基因片段轉殖進入蝴蝶蘭，並加以分析其光合指標。在電子傳遞鏈中，轉殖植物呈現較高的電子傳遞速率，且二氧化碳吸收速率及有機酸含量亦比野生型高。在酵素活性方面，進行貯存二氧化碳形成有機酸的酵素phosphoenolpyruvate carboxylase 以及釋放二氧化碳進入卡爾文循環的酵素NAD+-linked malic enzyme 兩者活性於轉殖株較高。此外可溶性醣類如：葡萄糖、果糖及蔗糖其含量於轉殖株中較高，澱粉粒亦大量累積於轉殖株中。以上結果顯示於轉殖株中過表達 pflp 基因將會刺激電子傳遞以增強醣類累積。總而言之 pflp 不僅可增加抗病亦可促進光合作用，於農業應用上為極有效用的基因。

Crassulacean acid metabolism (CAM) is one of three major models of carbon dioxide assimilation pathway with better water-use efficiency and slower photosynthetic efficiency in photosynthesis. Previous studies indicate that expressing gene of sweet pepper plant ferredoxin-like protein (PFLP) can enhance disease resistance to bacterial pathogens. The protein sequence of PFLP shows high homology to the ferredoxin-1(Fd-1) family that belongs to photosynthetic type Fd and involves in photosystem I. It is proposed that overexpressing pflp in transgenic plant may enhance photosynthetic efficiency. To investigate whether PFLP could improve photosynthetic efficiency, pflp transgenic Phalaenopsis were generated to analyze photosynthetic markers. Transgenic plant exhibited highly electron transport rate than that of wild type (WT). Compared with WT, transgenic plant showed higher CO2 assimilation rate and organic acids contents. Both of phosphoenolpyruvate carboxylase (PEPC) and NAD+-linked malic enzyme (NAD+-ME) activities also increased in transgenic plant. Finally, the photosynthesis products were examined between transgenic plant and WT. Soluble sugars contents such as glucose, fructose, and sucrose showed significantly increased in transgenic plant. The starch grains also highly accumulated in transgenic plant than that of WT. These above results indicate that overexpressing pflp in transgenic plant increases carbohydrates accumulation by the stimulation of electron transport flow. Altogether, we suggest pflp may be an extremely useful gene for agriculture that not only increases disease resistant but also enhances photosynthetic efficiency.

Contents
Page
Contents…………………..………………………..............…...........…I
List of tables…………………..………………………..............….…III
List of figures…………...…………….………........……………….…IV
Chinese abstract…………………………………………..........…….…01
English abstract……………………………….............................….….02
Chapter I Introduction…………………………..............………….…..04
Chapter II Materials and methods…………………...............…….…...07
2.1 Plant material, growth conditions and transformation…............07
2.2 Genomic PCR, reverse transcription PCR and GUS stain…......07
2.3 Analysis of photosynthetic gas exchange...................................08
2.4 Chlorophyll fluorescence measurements....................................08
2.5 Histological detection of starch grains........................................09
2.6 Metabolites analysis................................................................…09
2.7 Enzyme extraction and activity assays........................................10
Chapter III Results...................................................................................11
3.1 Generation and selection of Phalaenopsis transformants……....11
3.2 Overexpressing pflp enhances PETC in transgenic plants……11
3.3 Comparing the CO2 assimilation rate between transgenic
plants and WT.............................................................................12
3.4 Effects of increasing of CO2 assimilation on organic acid
contents in transgenic plants…...................................................13
3.5 The activities of PEPC and NAD+-ME......................................13
3.6 The impact of overexpressing pflp on carbohydrate
accumulation in transgenic plants…............................................14
Chapter IV Discussion……………………………....……………….16
Chapter V References…………………………..…….............………26
Chapter VI List of abbreviation..................................................………32
Chapter VII Supplement.…...………………………….............………33

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