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研究生:李昱錡
研究生(外文):Yu-Chi Li
論文名稱:IbpreproHypSys在甘藷中的訊息傳遞與調控
論文名稱(外文):Signal Transduction and Regulation of IbpreproHypSys in Sweet Potato
指導教授:鄭石通鄭石通引用關係
口試委員:葉國楨靳宗洛陳玉琪黃皓瑄張英峯
口試日期:2017-03-03
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:植物科學研究所
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:2017
畢業學年度:105
語文別:英文
論文頁數:70
中文關鍵詞:IbpreproHypSysipomoelinIbHypSys訊息傳遞系統性作用茉莉酸過氧化氫斜紋夜盜蛾木質素
外文關鍵詞:IbpreproHypSysipomoelinIbHypSyssignal transductionsystemic effectjasmonatehydrogen peroxideSpodoptera lituralignin
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Hydroxyproline-rich glycopeptides (HypSys)是一種小片段的訊號傳遞胜肽,包含了18到20個胺基酸。當甘藷(Ipomoea batatas cv. Tainung 57)受到傷害或IbHypSys處理後,透過茉莉酸(jasmonic acid)和過氧化氫(H2O2),IbHypSys的前驅物IbpreproHypSys基因會被誘導表現。透過基因轉殖甘藷大量表現 (OE) 和抑制表現 (RNAi) IbpreproHypSys來驗證IbpreproHypSys在生物體內的功能。在OE基改甘藷中進行傷害處理,傷害誘導基因Ipomoelin (IPO)在原位葉和系統葉上的基因表現量大於野生種和RNAi基改甘藷。除此之外,嫁接實驗顯示,野生種甘藷當砧木的情況下對接穗轉殖甘藷葉片進行傷害處理,接穗為OE基改甘藷較RNAi基改甘藷能在砧木野生種甘藷中誘導較高的IPO基因表現。然而,當基改甘藷為砧木野生種為接穗時,對野生種甘藷葉片進行傷害處理,在OE基改甘藷砧木中的IPO也是表現最高的,這代表了大量表現IbpreproHypSys轉殖甘藷不但能加強傷害的訊息傳送,也能加強傷害的訊息接收。分析參與在phenylpropanoid pathway的基因表現指出,人工合成的IbHypSys可以誘導木質素的生合成。並且IbpreproHypSys的基因表現可以抑制斜紋夜盜蛾幼蟲的生長。因此,傷害處理能夠誘導IbpreproHypSys基因表現,IbpreproHypSys基因能夠轉譯出IbpreproHypSys蛋白並且被剪切成IbHypSys,其中IbHypSys能夠促進IbpreproHypSys和IPO基因的表現,並且增強木質素的生合成,這些結果都有助於植物對抗昆蟲的侵襲。
Hydroxyproline-rich glycopeptides (HypSys) are small signaling peptides containing 18–20 amino acids. The expression of IbpreproHypSys, encoding the precursor of IbHypSys, was induced in sweet potato (Ipomoea batatas cv. Tainung 57) through wounding and IbHypSys treatments by using jasmonate and H2O2. Transgenic sweet potatoes overexpressing (OE) and silencing (RNAi) IbpreproHypSys were created. The expression of the wound-inducible gene ipomoelin (IPO) in the local and systemic leaves of OE plants was stronger than the expression in wild-type (WT) and RNAi plants after wounding. Furthermore, grafting experiments indicated that IPO expression was considerably higher in WT stocks receiving wounding signals from OE than from RNAi scions. However, wounding WT scions highly induced IPO expression in OE stocks. These results indicated that IbpreproHypSys expression contributed toward sending and receiving the systemic signals that induced IPO expression. Analyzing the genes involved in the phenylpropanoid pathway demonstrated that lignin biosynthesis was activated after synthetic IbHypSys treatment. IbpreproHypSys expression in sweet potato suppressed Spodoptera litura growth. In conclusion, wounding induced the expression of IbpreproHypSys, whose protein product was processed into IbHypSys. IbHypSys stimulated IbpreproHypSys and IPO expression and enhanced lignin biosynthesis, thus protecting plants from insects.
誌謝 II
摘要 III
Abstract V
Acronym table VII
Contents VIII
Introduction 1
Materials and Methods 6
Plant materials and treatments 6
Construction of transgenic plants 7
Gene expression assays 8
H2O2 content 8
Lignin content assay 9
Insect bioassay 10
Construction and purification of partial IbpreproHypSys in the expression plasmid vector (pGEX6P-1) by E.coli BL21. 10
The effect of protease inhibitors in IbpreproHypSys procession by sweet potato extracts. 11
Results 13
Isolation and expression of IbpreproHypSys 13
Effects of IbHypSys on H2O2 and jasmonate accumulation 13
IbHypSys IV induced IPO expression 15
Expression of IbpreproHypSys in transgenic plants 16
IPO expression in local and systemic leaves upon wounding 16
Requirement of IbpreproHypSys for producing systemic wounding signals 18
Effect of IbHypSys on lignin biosynthesis 19
Effects of IbpreproHypSys expression of plants on their resistance to S. litura 20
IbpreproHypSys processing 21
Inhibition of proteinase inhibitor in IbpreproHypSys procession 22
Processing efficiency of IbHypSysIV region by plant extract 23
Discussion 24
Expression of IbpreproHypSys 24
Signal transduction of IbHypSys 25
Effects of IbpreproHypSys on local and systemic wounding responses 26
Induction of the phenylpropanoid pathway and herbivore resistance 28
IbpreproHypSys processing by plant extract 31
Conclusion 33
Figures and figure legends 34
Figure 1. Similarity of IbpreproHypSys from sweet potatoes Georgia Jet and Tainung 57 (TN 57). 34
Figure 2. IbpreproHypSys expression after wounding. 35
Figure 3. Effects of the synthetic IbHypSys IV on H2O2 accumulation, IbAOS and Ib-13-LOX expression, and IbpreproHypSys expression. 36
Figure 4. H2O2 generation after the synthetic IbHypSys IV treatment. 38
Figure 5. IPO expression in WT after the synthetic IbHypSys IV treatment. 39
Figure 6. IbpreproHypSys expression in the transgenic sweet potatoes overexpressing and silencing IbpreproHypSys. 40
Figure 7. Local and systemic wounding responses in WT and transgenic plants. 41
Figure 8. DAB staining of H2O2 in the leaves of WT, OE, and RNAi plants. 43
Figure 9. The appearance of a grafted sweet potato. 44
Figure 10. Grafting experiments. 45
Figure 11. Effects of the synthetic IbHypSys IV or wounding on IbPAL2-8, IbHCT, and IbCHS expression and lignin contents. 48
Figure 12. Bioassays with Spodoptera litura larvae. Second instar larvae were fed with the leaves of WT, OE, or RNAi plants. 49
Figure 13. IbpreproHypSys process by plant extracts. 50
Figure 14. Inhibition by proteinase inhibitors in IbpreproHypSys process. 51
Figure 15. The effect of IbHypSysIV region in processing efficiency. 52
Figure 16. Functions of IbHypSys in sweet potato upon wounding. 53
Table 1. Primers for this study 54
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