臺灣博碩士論文加值系統

植物賀爾蒙乙烯調控植物花朵及果實的成熟與凋落、種子萌發及免疫防禦機制。在阿拉伯芥中 ACS 是生合成乙烯的關鍵酵素，可以將 AdoMet 催化成乙烯前驅物 ACC 而一旦乙烯生成之後便會與乙烯接收器 ETR1 結合來開啟乙烯訊息，傳導路徑。為了進一步了解文心蘭中 ACS 及 ETR1 所扮演的角色，我們選殖出文心蘭的 OnACS12 及 OnETR1，經胺基酸比對後與阿拉伯芥相似度達 55%及 71%。
將此兩基因分別異位表達於阿拉伯芥中進行功能性分析，結果顯示，35S::OnACS12 轉基因植物透過三種方式抑制開花時間，分別抑制開花基因 LFY表現、正調控 DELLAs 蛋白(GAI, RGL1 及 RGL2)以及負調控吉貝素 GA 的生合成基因(CPS 及 GA3ox1)；OnACS12 同時藉由促進 DELLAs 蛋白的累積來抑制JA(茉莉酸)生合成基因 DAD1 的表現，使 OnACS12 植物中與花藥內壁生成有關的基因 MYB26, NST1 和 NST2 被負調控，進而導致花藥內壁無法形成。另外，大量表現 OnETR1-C65Y 顯性抑制突變(dominant negative mutation)後的植株性狀與 etr1-1 相似，皆對乙烯不敏感及延遲花朵老化凋謝。在大量表現 OnETR1-C65Y之阿拉伯芥中，乙烯訊息傳導路徑基因 EDF1-4 和 ERF1 以及 JA 訊息傳遞路徑基因 MYC2 和 WRKY33 表現量受到抑制，藉由抑制 MYC2 和 WRKY33 的表現使MYB26, NST1, NST2 和 MYB85 被負調控。因此我們提出假設，因為大量表達OnACS12 及 OnETR1-C65Y 影響 JA 生合成以及 JA 的訊息傳遞 使花藥內壁生成，有關的基因 MYB26, NST1, NST2 和 MYB85 受到抑制進而影響花藥的正常發育。

Plant hormone ethylene regulates organ senescence and abscission, fruit ripening,seed germination and plant defense system. In Arabidopsis, the key enzyme of
ethylene biosynthesis is ACS which catalyzes AdoMet to ACC, the precursor of ethylene. After synthesis, ethylene binds to its receptors such as ETHYLENE RECEPTOR 1 (ETR1) to switch on the ethylene signal transduction. To understand the function of ACS and ETR1 in Oncidium Gower Ramsey, OnACS12 and OnETR1 were isolated and analyzed in Arabidopsis. 35S::OnACS12 caused late flowering phenotype due to the suppression of LFY, up-regulation of DELLAs (GAI, RGL1 及 RGL2) and repression of the GA biosynthesis genes (CPS and GA3ox1). Furthermore,OnACS12 promoted accumulation of DELLAs to repress DAD1 to cause anther
endothecium thickening related genes MYB26, NST1 and NST2 down-regulated in OnACS12 plants. An OnETR1 dominant-negative mutation at site 65 Cys to Tyr(OnETR1-C65Y) was generated. A similar etr1 mutant has been demonstrated to cause ethylene insensitivity in Arabidopsis. Ectopic expression of OnETR1-C65Y delayed the flower senescence which is similar to the etr1-1 Arabidopsis. The expression levels of downstream genes of ethylene signaling such as EDF1-4 and ERF1 and JA signaling such as MYC2 and WRKY33 were down-regulated in OnETR1-C65Y
Arabidopsis. Repression of MYC2 and WRKY33 caused the down-regulation of MYB26, NST1, NST2 and MYB85. We proposed a model to describe that ectopic expression of OnACS12 and OnETR1-C65Y negatively regulated JA synthesis and JA signaling transduction to suppress anthers secondary cell wall thickening relative genes MYB26, NST1, NST2 and MYB85 to control the normal anther development.

Abstract ................................................................................................. i
摘要………………........................................................................................... i
Abstract …………................................................................................................ ii
1. Introduction .......................................................................................................... 1
2. Materials and methods ........................................................................................... 3
3. Results .................................................................................................................... 12
3.1.1 Isolation and construction of OnACS12 from O. Gower Ramsey...............12
3.1.2 Ethylene biosynthesis increased in the ectopic expression OnACS12........13
3.1.3 Ectopic expression OnACS12 delayed flowering time via repression of
the flowering gene LFY........................................................................................13
3.1.4 Late-flowering of OnACS12 Arabidopsis was regulated by
gibberellin-DELLA signaling pathway..........................................................14
3.1.5 OnACS12 caused male sterility due to producing indehiscent anthers.....15
3.1.6 Ectopic expression OnACS12 caused indehiscent anthers via secondary
cell wall thickening failure ..................................................................................15
3.1.7 Ectopic expression OnACS12 down-regulated MYB26, NST1and NST2
expression that associated with anther dehiscence ..............................................16
3.2.1 Isolation of OnETR1and construction of OnETR1-C65Y cDNA from O.
Gower Ramsey.....................................................................................................17
3.2.2 OnETR1-C65Y delayed flower senescence by suppressing the ethylene
signal transduction. ..............................................................................................18
3.2.3 OnETR1-C65Y delayed flower senescence after detaching analysis and
ethylene treatment. ..............................................................................................19
3.2.4 Ectopic expression of OnETR1-C65Y lacks the triple response
phenotype.............................................................................................................19
3.2.5 35S::OnETR1-C65Y delayed senescence after ACC and JA treatment ....20
3.2.6 35S::OnETR1-C65Y delayed senescence by suppressing WRKY33 ...........20
3.2.7 35S::OnETR1-C65Y caused male sterility due to producing indehiscent
anthers...............................................................................................................21
3.2.8 Ectopic expression OnETR1-C65Y caused indehiscent anthers via
secondary cell wall thickening failure..................................................................22
3.2.9 Ectopic expression OnETR1-C65Y down-regulated MYB26, NST1, NST2
and MYB85 that associated with anther dehiscence.............................................23
4. Discussion................................................................................................................24
5. Reference..................................................................................................................31

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