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研究生:陳世殷
研究生(外文):Shih-YinChen
論文名稱:水稻葉綠體基因轉殖的研究
論文名稱(外文):The study of rice chloroplast transformation
指導教授:張清俊張清俊引用關係
指導教授(外文):Ching-Chun Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:109
中文關鍵詞:水稻葉綠體轉殖電穿孔PEG轉殖
外文關鍵詞:Oryza sativaplastid transformationelectroporationPEG-mediated transformation
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植物葉綠體的基因轉殖,最普遍使用的技術是基因槍的轟擊法,一旦將轉殖載體送至葉綠體中,便能藉由同源重組的方式將外源基因嵌入葉綠體基因體中,再經過一系列篩選與再生培養方能得到葉綠體基因轉殖植株。雖然轟擊法具有高效率但成本較高,也曾成功應用於水稻的葉綠體基因轉殖,但尚未獲得葉綠體同質體的植株,且轉殖效率仍低。因此,本研究嘗試發展其它轉殖方法,首先嘗試利用電穿孔法,將具有綠色螢光蛋白基因的葉綠體表現載體送至水稻懸浮細胞的葉綠體之中,雖然觀察到表現GFP的細胞,但高壓電擊導致細胞嚴重損傷,最終無法獲得葉綠體轉殖植株。此外,亦嘗試利用PEG轉殖法將葉綠體表現載體送至水稻葉肉原生質體的葉綠體中,並利用R9 peptide與PAMAM dendrimer來提升轉殖效率,也觀察到綠色或黃綠色螢光表現於原生質體的葉綠體之中,但受限於PEG法的再生效率,仍未得到轉殖的植株。最後,藉由表現與純化葉綠體transit peptide與R9 peptide的融合蛋白,利用transit pepetide的指引能力與R9 peptide的DNA結合能力,嘗試提升PEG葉綠體轉殖法的效率。
Biolistic bombardment is the most common used method for plant plastid transformation. Once expression vectors were delivered into plastid, the transgene could be integrated into plastid genome via homologous recombination. Subsequently under a series of selection and regeneration process, the transplastomic plant could be obtained. Generally, the efficiency of plastid transformation by bombardment is higher than other methods, but it is costly. Although the rice plastid transformation has been demonstrated by bombardment, the homoplasmic transplastomic plant was not obtained and the efficiency is still low. In this study, several approaches were tested to improve the efficiency and reduce the cost. First, the delivery of chloroplast expression vectors carrying gfp reporter gene into plastids of rice suspension cells by electroporation was tested. Although the GFP expressing cells were transiently observed under fluorescence microscope, the transplastomic plantlet could not obtain, probably because the cells were seriously damaged after high voltage of electroporation. In addition, the delivery of chloroplast expression vector into plastids of rice mesophyll protoplasts by either PEG alone or in combination with R9 peptides or PAMAM dendrimers was also tested. The addition of R9 peptides or PAMAM dendrimers with PEG failed to increase the transformation efficiency. The GFP was transiently observed in the plastids of a few protoplasts after PEG-mediated transformation; however, subsequently the GFP expressing cells were lost after subjecting to regeneration process. Finally, the recombinant fusion proteins that the transit peptide of nuclear-encoded chloroplast RNA polymerase was fused with R9 peptide were used as a DNA carrier. It was hypothesized that the DNA-protein complex could be recognized and imported into chloroplast through translocon complex. The preliminary results suggested that it is potentially able to enhance the plastid transformation by PEG-mediated method.
Abstract in Chinese 3
Abstract in English 4
Acknowledgement 6
1. Introduction 9
1.1. Plastid, the unique organelle in plant 9
1.2. Developments of plastid transformation 10
1.3. Advantages of plastid transformation 13
1.4. Application of plastid transformation 14
1.4.1. Agronomic traits 14
1.4.2. Phytoremediation 17
1.4.3. Biopharmaceuticals 17
1.4.4. Metabolic pathway engineering 18
1.4.5. Biomaterials 19
1.4.6. Rubisco engineering 19
1.4.7. Basic research 21
1.5. Advance of cell-penetrating peptides in plant 23
2. Aim 26
3. Materials and Methods 27
3.1. Expression vector 27
3.1.1. Nuclear transient expression vector 27
3.1.2. Plastid transformation vector 27
3.2. Plant materials and culture conditions 27
3.2.1. Rice callus induction 27
3.2.2. Establishment of rice suspension cell culture 28
3.2.3. Maintenance of tobacco BY-2 cell suspension culture 28
3.3. Plastid transformation of rice cells by electroporation 29
3.3.1. Plastid transformation by electroporation 29
3.3.2. Test of cell viability 30
3.4. Plastid transformation of rice cells by PEG treatment 30
3.5. PEG-mediated protoplast transformation 31
3.5.1. Plant growth 31
3.5.2. Protoplast isolation 32
3.5.3. Small-scale PEG-mediated transformation 33
3.5.4. Large-scale PEG-mediated transformation 33
3.6. CPP-mediated transformation 34
3.6.1. Plasmid construction 34
3.6.2. Purification of His-tag proteins 36
3.6.3. Transient expression by R9-mediated method 37
3.6.4. Transient expression by CPP-mediated method 37
4. Results 39
4.1. Electroporation method 39
4.2. PEG-mediated method 41
4.3. R9 peptide & PAMAM dendrimer method 42
4.4. Protoplast transformation 43
4.5. CPP-mediated method 44
5. Discussion 47
6. Bibliography 53
7. Tables 62
8. Figures 79
9. Appendix 104

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