臺灣博碩士論文加值系統

根據先前研究結果顯示ethephon誘導葉片老化及老化相關基因SPCP1的表現受reduced glutathione, EGTA及cycloheximide抑制(Chen et al., 2009)，此結果顯示calcium influx、reactive oxygen species (ROS)及新合成(de novo)蛋白質會影響ethephon的作用。因此根據此研究結果本研究利用扣減雜交和RACE-PCR的方法從ethephon處理6及24小時的混合樣品中共選殖了五個全長的cDNAs具有可能的功能為calmodulin (SPCAM)、catalase (SPCATA)、anionic peroxidase (SPPA)、ACC oxidase (SPACO) 和DSS1-like protein (SPDSS1)。SPCAM其ORF包含450個核甘酸，產生149個胺基酸，蛋白質結構具有四個明顯EF-hand motifs，與阿拉伯芥分離的calmodulins比較其胺基酸相似度從48%至100%，其中與CaM7相似度最高。SPCATA其ORF包含1479個核甘酸，產生492個胺基酸；與不同物種的catalases胺基酸相似度從71.2%至80.9%，其中與水筆仔的catalase相似度最高。SPPA其ORF包含1068個核甘酸，產生355個胺基酸；與甘藷其他已發表的同功酵素比較其胺基酸相似度從28.7%至97.5%，其中與甘藷的anionic peroxidase SWPA4相似度最高。SPACO其ORF包含930個核甘酸，產生309個胺基酸；與不同物種ACC oxidase比較其胺基酸相似度從62.3%至81.5%，其中與菸草的ACC oxidase相似度最高。SPDSS1其ORF包含228個核甘酸，產生75個胺基酸；與不同物種DSS1比較其胺基酸相似度從25.2%至62.3%，其中與玉米的DSS1相似度最高。在自然老化過程葉片的葉綠素含量及光合作用效率會顯著減少，然而選殖基因的表現量則有顯著增加；DAB染色結果顯示H2O2的量於全部黃化的葉片(S3)比其它葉片發育階段有顯著增加；Evan blue染色結果亦顯示全部黃化的葉片(S3)細胞死亡較年青的葉片(S0)為多。另外利用ethephon誘導葉片老化過程也有相類似的結果。ethephon處理的葉片其葉綠素含量及光合作用效率也比對照組顯著減少，然而選殖基因的表現量則比對照組顯著增加；DAB染色結果顯示H2O2的量於ethephon處理三天的葉片比其對照組有顯著增加；Evan blue染色結果亦顯示ethephon處理三天的葉片細胞死亡較其對照組亦有顯著增加。根據這些實驗的結果結論SPCAM、SPCATA、SPPA、SPACO和SPDSS1在自然葉片老化及ethephon誘導葉片老化過程中其基因表現量會顯著增加；其功能是否與ethephon誘導葉片老化過程calcium influx、ROS增加或清除、及後續的訊息傳導等有關將會討論。

According to our previous results, ethephon-induced sweet potato leaf senescence and senescence-associated gene SPCP1 expression was affected by reduced glutathione, EGTA, and cycloheximide (Chen et al., 2009). These data suggest that calcium influx, reactive oxygen species (ROS) and de novo synthesized proteins can affect ethephon-mediated effects. Therefore, PCR-selective substractive hybridization and RACE-PCR methods were used to clone 5 full-length cDNAs encoded putative calmodulin (SPCAM), catalase (SPCATA), anionic peroxidase (SPPA), ACC oxidase (SPACO), and DSS1-like protein (SPDSS1) from mixed samples of ethephon-treated leaves for 6 and 24 hours. The ORF of SPCAM contains 450 nucleotides and encodes 149 amino acids. There are 4 putative EF-motifs in the deduced protein structure. SPCAM exhibited amino acid sequence identity with isolated Arabidopsis calmodulins from 48% to 100%, and was completely the same as CaM7 calmodulin. The ORF of SPCATA contains 1479 nucleotides and encodes 492 amino acids. SPCAM exhibited high amino acid sequence identity with other plant catalases from 71.2% to 80.9%, and had the highest identity with mangrove catalase. The ORF of SPPA contains 1068 nucleotides and encodes 355 amino acids. SPPA exhibited amino acid sequence identity with other published sweet potato peroxidase isoforms from 28.7% to 97.5%, and had the highest identity with anionic peroxidase SWPA4. The ORF of SPACO contains 930 nucleotides and encodes 309 amino acids. SPACO exhibited high amino acid sequence identity with other plant ACC oxidases from 62.3% to 81.5%, and had the highest identity with tobacco ACC oxidase. The ORF of SPDSS1 contains 228 nucleotides and encodes 75 amino acids. SPDSS1 exhibited amino acid sequence identity with other DSS1 from 25.2% to 62.3%, and had the highest identity with maize DSS1. The chlorophyll contents and Fv/Fm values were significantly reduced, however, the isolated gene expression was remarkably enhanced in natural senescent leaves. DAB staining showed that H2O2 amount was remarkably elevated at S3 senescent leaves compared to leaves of the other developmental stages. Evan blue staining also demonstrated that S3 senescent leaf had more cell death compared to S0 young leaves. In addition ethephon-induced leaf senescence exhibited similar results. The chlorophyll contents and Fv/Fm values were significantly reduced, however, the isolated gene expression was remarkably enhanced in ethephon-treated leaves compared to dark control. DAB staining showed that H2O2 amount was remarkably elevated at 72 hours in ethephon-treated leaves compared to dark control. Evan blue staining also demonstrated that ethephon-treated leaf for 72 hours had more cell death compared to dark control. Based on these data we conclude that SPCAM, SPCATA, SPPA, SPACO and SPDSS1 gene expression were significantly increased in natural and ethephon-induced senescent leaves. The possible functions of these isolated genes in association with events in ethephon-induced leaf senescence, including calcium influx, ROS elevation or scavenge, and following signaling will be discussed.

目錄
頁次
目錄…………………………… …………… ………… I
內容目次…………………………………… ………… II
圖目錄………………………………………………… IV
縮寫表………………………………………………… VI
中文摘要……………………………………………… VII
英文摘要……………………………………………… IX

壹、 緒論…………… …………………………………………1
I. 老化的定義與過程……………………………………… 1
II. 影響葉片老化的因子…………………………………… 2
III. Ethylene 的合成及信息傳導途徑……………………… 6
IV. 與Ca2+-binding，H2O2 清除，乙烯合成及其他相關基因的
研究………………………………… …………………… 8
貳、材料與方法…………………… ………… ………………13
I. 實驗材料……………… ……………………………………13
II. 實驗方法……………………………………………………13
A. 葉片處理…………………………………………………13
B. RNA extraction………………………… ……… …… …15
C. PCR select cDNA subtraction … … … … ………………19
D. 差異性cDNA 選殖以及親緣樹分析……………………29
E. RACE PCR………………… ………………… …… ……33
F. RT-PCR……………………………………………………38
G. 色素測定…………………………………………………42
H. 光合作用測定……………………………… ……………43
I. DAB 染色及Evan blue ……………………………………43
参、結果…………………………………………………………45
I. 差異性表現之cDNA 片段選殖………… …… … ………45
II. 選殖全長cDNA 之核甘酸序列、胺基酸序列及親緣樹之分
析…………………………… …………… …………… …45
III. 自然老化的甘藷葉片會增加選殖基因的表現… … ……50
IV. Ethephon 誘導甘藷葉片老化及增加選殖基因的表現 …52
肆、討論…………………………………………………… ……55
參考文獻……………………………………………………… …60

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