臺灣博碩士論文加值系統

由於石化原料存量耗竭，將植物生質 (plant biomass) 經微生物轉換為可用能源或化學品的多元生物精煉技術，為一有力的永續資源發展方案。稻草在台灣目前為農業廢棄物，是台灣做為生物精煉工業發展時，最容易獲得的生質料源。由於稻草的木質纖維比例高，而纖維素在微生物的分解轉換效率比澱粉低，因此我們利用基因工程的策略，增加水稻葉片中澱粉的含量，以加速稻草生質在真菌轉換成葡萄糖的效率，以利微生物(如:酵母菌)發酵生成酒精或乳酸等較高價值的物質，以應用於生質能源或生物精煉的產業中。
植物以光合作用，利用太陽能裂解水分子，以還原二氧化碳形成碳水化合物，而植物主要的儲存性碳水化合物為澱粉，在植物葉片中的澱粉，由於在夜間會完全分解，而無法儲存累積，稱為暫存性澱粉(transitory starch)。在阿拉伯芥葉片中，主要參與暫存性澱粉降解的去磷酸化酵素AtSEX4，有三個同源性基因，分別為：AtSEX4 (Starch Excess Four)、AtLSF1 (Like SEX Four 1 )和AtLSF2 ( Like SEX Four 2 )，當這些基因突變時，阿拉伯芥葉片中的澱粉會逐漸累積，導致葉片高澱粉的性狀，但我們並不知道在水稻中是否存在相似的澱粉降解機制。
本研究利用基因轉殖的策略，以RNA干擾技術(RNA interference, RNAi) 配合持續大量表現的啟動子 (Ubiquitin promoter) 使主要水稻SEX4同源性基因(OsSEX4、OsLSF1與OsLSF2)表現量降低，希望能減緩水稻葉片中暫存性澱粉的降解速率，進而使澱粉逐日累積在水稻葉片中，形成高澱粉稻草，作為生質能源或生物精煉所需的優良料源。我們首先以水稻懸浮細胞系統觀察暫存性澱粉降解的情況，以缺糖處理0.5天誘導暫存性澱粉降解時，OsSEX4 RNAi轉殖水稻細胞比未轉殖野生型水稻細胞多出3倍的澱粉，OsLSF1 RNAi轉殖細胞則也多出3倍的澱粉。以此水稻懸浮細胞做為纖維素分解菌(黑黴和里氏木黴)的生質料源，初步發現以RNAi降低OsSEX4表現的水稻細胞生質可較快速被真菌降解轉換成較多的葡萄糖。OsSEX4 RNAi轉殖水稻植株的葉片較未轉殖野生型水稻葉片含3倍多的澱粉，RNAi水稻植株在外觀、穀粒數、粒重與發芽率上，皆與未轉殖野生型水稻無明顯差異。本研究利用基因工程減緩暫存性澱粉的降解速率，累積葉片澱粉，幫助微生物利用轉換此水稻細胞生質為葡萄糖。本計畫期望在不影響稻米產量下，以所改良的高澱粉稻草成為生質能源或生物精煉的優良料源。

Biorefinery, converting sustainable plant biomass to fuels or chemicals via microorganisms, is an attractive biotechnology to replace petroleum refinery. Since cellulosic biomass does not compete with human food, it is a good feedstock in biorefinery. In Taiwan, rice straw is an attractive lignocellulosic material, because it is an agricultural waste and easy to get. However, fungal conversion of rice straw into glucose or ethanol has low efficiency. Our goal is to make rice straw a better feedstock for cellulosic fermentation. We hypothesize that increasing soft carbohydrates, such as starch, in rice leaves via genetic engineering will facilitate microorganisms to break down the whole biomass. If degradation of transitory starch is interfered, there will be less exporting sugars to make cellulose in the engineered rice plants. The starch-rich rice leaves could be excellent feedstock to convert into glucose and facilitate yeast fermentation to high-value products, such as ethanol and lactic acid.
Transitory starch is made and accumulated in chloroplasts during the day, and then hydrolyzed during the following night in the plants. Starch Excess 4 (SEX4) locus of Arabidopsis encodes a phosphatase, and mutation in this gene results starch accumulation in leaves. There are three SEX4 homologous in Arabidopsis, SEX4, LSF1 (Like SEX Four-1) and LSF2. It is shown that SEX4 and LSF proteins play major roles in starch degradation in Arabidopsis; however, it is not clear whether similar regulation mechanism exists in cereals, such as rice.
We used RNA interference (RNAi) approach in combination with an constitutive promoter to reduce expression of SEX4 homologous genes in Oryza sativa, OsSEX4, OsLSF1 and OsLSF2. We anticipate that slowing down the starch degradation rate will lead to its accumulation in rice leaves day by day, and finally form starch-rich rice leaf or straw. We developed a quick evaluation system for starch levels in rice suspension cells. OsSEX4 and OsLSF1 RNAi cell lines showed 3.0-fold and 2.9-fold increase in starch level than wild type after 12 hours of sugar starvation. Our preliminary data showed that the biomass of OsSEX4 RNAi cell line was more easily digested than wild type by Trichoderma reesei and Aspergillus niger, and more fermentable glucose was converted. After generation of transgenic plants, we obtained RNAi lines with excess leaf starch contents. Compared to wild type, two-month-old T2 rice transgenic plants of OsSEX4 RNAi lines showed a 3-fold increase in leaf starch content. In addition, there is no penalty in starch accumulated OsSEX4 RNAi rice plants, including plant morphology, grain number, grain weight and seed germination. Thus, rice plants with leaf and straw containing excess transitory starch can be produced for efficient biofuel production without compromise in grain yield.

摘要 I
Abstract II
誌謝 III
第一章 前言 1
1-1 研究動機 1
1-2 研究目的 1
1-3 研究架構 2
第二章 文獻回顧 3
2-1能源危機 3
2-2生物精煉概念將生物質量轉換可用能源 3
2-3生質能源料源 4
2-4生質能源料源 – 水稻稻桿 6
2-5水解纖維素轉換植物生質為可發酵葡萄糖 6
2-5-1 纖維素 6
2-5-2 分解纖維素酵素 7
2-6植物葉片澱粉代謝 8
2-7 植物澱粉結構與種類 8
2-7-1 澱粉結構 8
2-7-2澱粉種類 9
2-8 短暫性澱粉為動態調控 10
2-9 短暫性澱粉降解路徑 10
2-10 磷酸化酵素SEX4同源性基因的角色 11
2-11 利用基因靜默策略降低水稻SEX4同源性基因的表現 15
2-11-1 RNA 干擾作用 15
2-12水稻 (Oryza sativa) 轉殖系統 16
2-13水稻懸浮細胞系統 17
2-14 RNA靜默基因水稻對生理影響 17
第三章 材料與方法 18
3-1實驗設備 18
3-2 實驗藥品 19
3-3 實驗材料 20
3-3-1 本研究所使用菌株與載體 20
3-3-2 本研究所轉殖質體抗生素 20
3-3-3 本實驗所使用之引子 20
3-3-4 資料庫序列找尋、比對和分析 20
3-3-4-1 資料庫找尋 20
3-3-4-2 分析軟體 21
3-4 水稻培養 21
3-4-1 水稻植株 21
3-4-2 水稻癒傷組織 21
3-5質體建構 21
3-5-1 引子設計 21
3-5-2 聚合酶連鎖反應 22
3-5-3 利用限制酵素檢測DNA 22
3-5-4 接合反應 22
3-5-5 線性載體DNA的去磷作用 23
3-5-6 洋菜膠體萃取DNA 23
3-6 重組質體之篩選 23
3-7細菌的轉殖作用 24
3-7-1製備勝任細胞 24
3-7-2熱休克法 24
3-7-3電穿孔法 24
3-8純化細菌質體DNA 25
3-9 菌種保存 25
3-10 菌體前培養 26
3-11 瓊脂凝膠電泳 26
3-12 DNA序列分析 26
3-13 農桿菌轉型 26
3-14 農桿菌的生化檢測 26
3-15 水稻癒傷組織誘導 26
3-16 水稻癒傷組織培養條件 27
3-17 水稻基因轉殖 27
3-18 水稻轉殖系分析方法 27
3-18-1 轉殖系GUS 分析 27
3-18-2 Genomic DNA的抽取與純化 28
3-18-3 Total RNA的純化 28
3-18-4 去除 RNA中DNA 29
3-18-5 合成 cDNA 29
3-19 水稻澱粉分析方法 29
3-19-1 澱粉標準品的配置 29
3-19-2葉片澱粉的萃取 29
3-19-3葉片澱粉的含量測定 30
3-19-4 水稻懸浮細胞澱粉的萃取 30
3-20 水稻葉片澱粉顆粒萃取 30
3-21 水稻懸浮細胞的建立 31
3-22 水稻懸浮細胞系含糖及缺糖處理 31
3-23 水稻懸浮細胞澱粉含量定性分析 31
3-24 發酵實驗 31
3-24-1 菌種保存 31
3-24-2 菌種培養 31
3-24-3 收集真菌細胞懸浮液 32
3-24-4 細胞計數 32
3-24-5 利用搖瓶發酵前培處理 32
3-24-6 利用搖瓶發酵主培處理 33
3-24-7 利用HPLC 分析葡萄糖含量 33
3-24-8 利用酵素方法分析葡萄糖定量法 33
第四章 結果 34
4-1 觀察水稻葉片在一天之中澱粉含量變化 34
4-2 尋找水稻OsSEX4同源性基 34
4-2-1相似水稻OsSEX4同源性基因胺基酸序列比對 34
4-2-2 水稻與阿拉伯芥中SEX同源基因序列相似度 34
4-2-3 透過軟體預測OsSEX4、OsLSF1和 OsLSF2在細胞內的位置 35
4-3 構築OsLSF1基因靜默之表達載體 35
4-3-1 利用 RT-PCR獲得OsLSF1專一性基因片段 35
4-3-2 OsLSF1基因片段載體接合確認之分析 36
4-3-3 RNAi spacer基因片段接合確認之分析 36
4-3-4 反向基因片段接合確認之分析 36
4-3-5 置換含有大量表現啟動子的載體確認分析 36
4-3-6 將pUOsLSFi與二元載體接合確認分析 37
4-4 確認為含建築載體農桿菌菌株分析 37
4-5 利用潮黴素篩選初步成功之轉殖癒傷組織 37
4-6 轉殖水稻癒傷組織之分析 37
4-6-1 利用GUS活性分析確認成功之轉殖癒傷組織 37
4-6-2 確認轉殖水稻癒傷組織細胞基因片段 38
4-6-3 利用RT-PCR分析確認成功之轉殖癒傷組織 38
4-7 OsSEX4 同源性基因RNAi 轉殖系水稻懸浮細胞分析 38
4-8 OsSEX4同型性基因 (homozygous gene) 的T3世代種子分析 39
4-8-1 OsSEX4同型性基因成熟種子誘導為癒傷組織之分析 39
4-9 以Trichoderma reesei和 Aspergillus niger將纖維素轉換為葡萄糖 39
4-9-1 模擬高澱粉質株情形分析 39
4-9-2 利用高澱粉懸浮細胞當作碳源分析 39
4-10 OsSEX4同源性轉殖系水稻懸浮細胞生長速率 40
4-11溫室種植高澱粉水稻轉殖株分析 40
4-11-1 GUS染色分析 40
4-11-2 利用RT-PCR分析葉片OsSEX4基因表現量分析 40
4-11-3 葉片澱粉含量分析 40
4-11-4 水稻葉片澱粉顆粒大小分析 41
4-11-5高澱粉轉殖株對植物生理影響 41
4-12 田間種植高澱粉水稻植株分析 41
4-12-1 GUS 染色 41
4-12-2箭葉澱粉含量分析 41
4-12-3 植株生長與種子外觀 42
4-12-4穗數與分櫱數情形 42
4-12-5種子產量影響 42
第五章 討論 43
5-1 選用水稻SEX4同源性基因做為抑制澱粉降解的酵素 43
5-2 OsSEX4同源性基因功能性區域與分別參與的功能 43
5-3 OsSEX4同源性基因在葉綠體中表現 43
5-4 選用RNAi技術降低基因表現量使澱粉累積 44
5-5 RNAi水稻轉殖細胞株的基因表現不同 44
5-6插入T-DNA的基因組分析 45
5-7 水稻SEX4與LSF1 功能確定 45
5-8 水稻葉片澱粉取樣 45
5-9不同時期葉片與萃取方式對澱粉含量的影響 46
5-10高澱粉水稻做為真菌碳源轉換葡萄糖 46
5-11 轉殖株葉片與種子澱粉顆粒大小 47
5-12分析轉殖水稻澱粉的支鏈長度 47
5-13葉片澱粉累積對種子產量與澱粉影響 47
5-14 轉殖水稻稻桿成分比例 47
5-15 稻桿的預處理方式不同 48
5-16 後續高澱粉的水稻葉片的應用 48
第六章 結論與未來展望 49
6-1 結論 49
6-2 未來展望 49
第七章 文獻參考 51
第八章 附錄 103
8-1 定序結果 103
8-2 組織培養配方 106
8-3 水稻葉片澱粉萃取液配方 113
8-4 發酵實驗培養基配方 113
8-5 載體構築 114

Arai-Sanoh, Y., Ida, M., Zhao, R., Yoshinaga, S., Takai, T., Ishimaru, T., Maeda, H., Nishitani, K., Terashima, Y., Gau, M., Kato, N., Matsuoka, M., and Kondo, M. (2011). Genotypic variations in non-structural carbohydrate and cell-wall components of the stem in rice, sorghum, and sugar vane. Biosci Biotechnol Biochem 75, 1104-1112.
Bahaji, A., Li, J., Ovecka, M., Ezquer, I., Munoz, F.J., Baroja-Fernandez, E., Romero, J.M., Almagro, G., Montero, M., Hidalgo, M., Sesma, M.T., and Pozueta-Romero, J. (2011). Arabidopsis thaliana mutants lacking ADP-glucose pyrophosphorylase accumulate starch and wild-type ADP-glucose content: further evidence for the occurrence of important sources, other than ADP-glucose pyrophosphorylase, of ADP-glucose linked to leaf starch biosynthesis. Plant Cell Physiol 52, 1162-1176.
Balat, H. (2010). Prospects of biofuels for a sustainable energy future: A critical assessment. Ener Educ Sci Tech-A 24, 85-111.
Baunsgaard, L., Lutken, H., Mikkelsen, R., Glaring, M.A., Pham, T.T., and Blennow, A. (2005). A novel isoform of glucan, water dikinase phosphorylates pre-phosphorylated alpha-glucans and is involved in starch degradation in Arabidopsis. Plant J 41, 595-605.
Beguin, P., and Aubert, J.P. (1994). The biological degradation of cellulose. FEMS Microbiol Rev 13, 25-58.
Bernhard Seiboth, C.I.a.V.S.-S. (2011). Trichoderma reesei: A Fungal Enzyme Producer for Cellulosic Biofuels. (Vienna University of Technology,Austria.).
Bhat, M.K., and Bhat, S. (1997). Cellulose degrading enzymes and their potential industrial applications. Biotechnology Advances 15, 583-620.
Binod, P., Sindhu, R., Singhania, R.R., Vikram, S., Devi, L., Nagalakshmi, S., Kurien, N., Sukumaran, R.K., and Pandey, A. (2010). Bioethanol production from rice straw: An overview. Bioresour Technol 101, 4767-4774.
Boisson, A.M., Gout, E., Bligny, R., and Rivasseau, C. (2012). A simple and efficient method for the long-term preservation of plant cell suspension cultures. Plant Methods 8, 4.
Brummell, D.A., Balint-Kurti, P.J., Harpster, M.H., Palys, J.M., Oeller, P.W., and Gutterson, N. (2003). Inverted repeat of a heterologous 3'-untranslated region for high-efficiency, high-throughput gene silencing. Plant J 33, 793-800.
Bushnell, L.D., and Haas, H.F. (1941). The Utilization of Certain Hydrocarbons by Microorganisms. J Bacteriol 41, 653-673.
Byrt, C.S., Grof, C.P., and Furbank, R.T. (2011). C4 plants as biofuel feedstocks: optimising biomass production and feedstock quality from a lignocellulosic perspective. J Integr Plant Biol 53, 120-135.
Chen, M.H., Liu, L.F., Chen, Y.R., Wu, H.K., and Yu, S.M. (1994). Expression of alpha-amylases, carbohydrate metabolism, and autophagy in cultured rice cells is coordinately regulated by sugar nutrient. Plant J 6, 625-636.
Claassen, P.A.M., van Lier, J.B., Contreras, A.M.L., van Niel, E.W.J., Sijtsma, L., Stams, A.J.M., de Vries, S.S., and Weusthuis, R.A. (1999). Utilisation of biomass for the supply of energy carriers. Appl Microbiol Biot 52, 741-755.
Comparot-Moss, S., Kotting, O., Stettler, M., Edner, C., Graf, A., Weise, S.E., Streb, S., Lue, W.L., MacLean, D., Mahlow, S., Ritte, G., Steup, M., Chen, J., Zeeman, S.C., and Smith, A.M. (2010). A putative phosphatase, LSF1, is required for normal starch turnover in Arabidopsis leaves. Plant Physiol 152, 685-697.
Delatte, T., Trevisan, M., Parker, M.L., and Zeeman, S.C. (2005). Arabidopsis mutants Atisa1 and Atisa2 have identical phenotypes and lack the same multimeric isoamylase, which influences the branch point distribution of amylopectin during starch synthesis. Plant J 41, 815-830.
Delatte, T., Umhang, M., Trevisan, M., Eicke, S., Thorneycroft, D., Smith, S.M., and Zeeman, S.C. (2006). Evidence for distinct mechanisms of starch granule breakdown in plants. Journal of Biological Chemistry 281, 12050-12059.
Dhillon, G.S., Oberoi, H.S., Kaur, S., Bansal, S., and Brar, S.K. (2011). Value-addition of agricultural wastes for augmented cellulase and xylanase production through solid-state tray fermentation employing mixed-culture of fungi. Ind Crop Prod 34, 1160-1167.
Fernando, S., Adhikari, S., Chandrapal, C., and Murali, N. (2006). Biorefineries: Current status, challenges, and future direction. Energ Fuel 20, 1727-1737.
FitzPatrick, M., Champagne, P., Cunningham, M.F., and Whitney, R.A. (2010). A biorefinery processing perspective: treatment of lignocellulosic materials for the production of value-added products. Bioresour Technol 101, 8915-8922.
Fulton, D.C., Stettler, M., Mettler, T., Vaughan, C.K., Li, J., Francisco, P., Gil, M., Reinhold, H., Eicke, S., Messerli, G., Dorken, G., Halliday, K., Smith, A.M., Smith, S.M., and Zeeman, S.C. (2008). Beta-AMYLASE4, a noncatalytic protein required for starch breakdown, acts upstream of three active beta-amylases in Arabidopsis chloroplasts. Plant Cell 20, 1040-1058.
Geddes, C.C., Nieves, I.U., and Ingram, L.O. (2011). Advances in ethanol production. Curr Opin Biotechnol 22, 312-319.
Goldemberg, J. (2008). The Brazilian biofuels industry. Biotechnol Biofuels 1, 6.
Graf, A., Schlereth, A., Stitt, M., and Smith, A.M. (2010). Circadian control of carbohydrate availability for growth in Arabidopsis plants at night. Proc Natl Acad Sci U S A 107, 9458-9463.
Gray, K.A., Zhao, L., and Emptage, M. (2006). Bioethanol. Curr Opin Chem Biol 10, 141-146.
Hejazi, M., Fettke, J., Kotting, O., Zeeman, S.C., and Steup, M. (2010). The Laforin-Like Dual-Specificity Phosphatase SEX4 from Arabidopsis Hydrolyzes Both C6-and C3-Phosphate Esters Introduced by Starch-Related Dikinases and Thereby Affects Phase Transition of alpha-Glucans. Plant Physiol 152, 711-722.
Hejazi, M., Fettke, J., Haebel, S., Edner, C., Paris, O., Frohberg, C., Steup, M., and Ritte, G. (2008). Glucan, water dikinase phosphorylates crystalline maltodextrins and thereby initiates solubilization. Plant J 55, 323-334.
Hiei, Y., Komari, T., and Kubo, T. (1997). Transformation of rice mediated by Agrobacterium tumefaciens. Plant Mol Biol 35, 205-218.
Jenkins, T., Bovi, A., and Edwards, R. (2011). Plants: biofactories for a sustainable future? Philos Transact A Math Phys Eng Sci 369, 1826-1839.
John, G., Shankar, B.V., Jadhav, S.R., and Vemula, P.K. (2010). Biorefinery: A Design Tool for Molecular Gelators. Langmuir 26, 17843-17851.
Jordan, D.B., Bowman, M.J., Braker, J.D., Dien, B.S., Hector, R.E., Lee, C.C., Mertens, J.A., and Wagschal, K. (2012). Plant cell walls to ethanol. Biochem J 442, 241-252.
Kamm, B., and Kamm, M. (2007). Biorefineries--multi product processes. Adv Biochem Eng Biotechnol 105, 175-204.
Kooi, C.W.V., Taylor, A.O., Pace, R.M., Meekins, D.A., Guo, H.F., Kim, Y., and Gentry, M.S. (2010). Structural basis for the glucan phosphatase activity of Starch Excess4. P Natl Acad Sci USA 107, 15379-15384.
Kotting, O., Santelia, D., Edner, C., Eicke, S., Marthaler, T., Gentry, M.S., Comparot-Moss, S., Chen, J., Smith, A.M., Steup, M., Ritte, G., and Zeeman, S.C. (2009). STARCH-EXCESS4 is a laforin-like Phosphoglucan phosphatase required for starch degradation in Arabidopsis thaliana. Plant Cell 21, 334-346.
Kusaba, M. (2004). RNA interference in crop plants. Curr Opin Biotechnol 15, 139-143.
Lam, M.K., and Lee, K.T. (2012). Microalgae biofuels: A critical review of issues, problems and the way forward. Biotechnol Adv 30, 673-690.
Li. (1979). Morphology and anatomy of grass family crops. . Shanghai Science and Technology Press,Shanghai,, 138–142.
Li, X., Qian, Q., Fu, Z., Wang, Y., Xiong, G., Zeng, D., Wang, X., Liu, X., Teng, S., Hiroshi, F., Yuan, M., Luo, D., Han, B., and Li, J. (2003). Control of tillering in rice. Nature 422, 618-621.
Lo, Y.C., Huang, L.F., Cheng, C.L., Chen, J., and Chang, J.S. (2011). Using a starch-rich mutant of Arabidopsis thaliana as feedstock for fermentative hydrogen production. Bioresour Technol 102, 8543-8546.
Lu, Y., Gehan, J.P., and Sharkey, T.D. (2005). Daylength and circadian effects on starch degradation and maltose metabolism. Plant Physiol 138, 2280-2291.
Maiorella, B. (1985). Ethanol fermentation. In: Comprehensive biotechnol. Young M (ed), Pergamon Press, Oxford. 3, 861-914.
Nishimura, A., Aichi, I., and Matsuoka, M. (2006). A protocol for Agrobacterium-mediated transformation in rice. Nat Protoc 1, 2796-2802.
Ohara, H. (2003). Biorefinery. Appl Microbiol Biot 62, 474-477.
Pan, X., Arato, C., Gilkes, N., Gregg, D., Mabee, W., Pye, K., Xiao, Z., Zhang, X., and Saddler, J. (2005). Biorefining of softwoods using ethanol organosolv pulping: preliminary evaluation of process streams for manufacture of fuel-grade ethanol and co-products. Biotechnol Bioeng 90, 473-481.
Park, J.Y., Arakane, M., Shiroma, R., Ike, M., and Tokuyasu, K. (2010). Culm in rice straw as a new source for sugar recovery via enzymatic saccharification. Biosci Biotechnol Biochem 74, 50-55.
Park, J.Y., Seyama, T., Shiroma, R., Ike, M., Srichuwong, S., Nagata, K., Arai-Sanoh, Y., Kondo, M., and Tokuyasu, K. (2009). Efficient recovery of glucose and fructose via enzymatic saccharification of rice straw with soft carbohydrates. Biosci Biotechnol Biochem 73, 1072-1077.
Pickett, D.A., D Bowles, T Bridgwater, P Jarvis, N Mortimer, M Poliakoff, J Woods (2008). Sustainable biofuels: prospects and challenges. Biological Sciences, 1-90.
Reddy, N., and Yang, Y. (2005). Biofibers from agricultural byproducts for industrial applications. Trends Biotechnol 23, 22-27.
Ritte, G., Heydenreich, M., Mahlow, S., Haebel, S., Kotting, O., and Steup, M. (2006). Phosphorylation of C6- and C3-positions of glucosyl residues in starch is catalysed by distinct dikinases. FEBS Lett 580, 4872-4876.
Rolland, F., Moore, B., and Sheen, J. (2002). Sugar sensing and signaling in plants. Plant Cell 14 Suppl, S185-205.
Saenger, W. (1984). The Structure of the Blue Starch-Iodine Complex.
Santelia, D., Kotting, O., Seung, D., Schubert, M., Thalmann, M., Bischof, S., Meekins, D.A., Lutz, A., Patron, N., Gentry, M.S., Allain, F.H., and Zeeman, S.C. (2011). The phosphoglucan phosphatase like sex Four2 dephosphorylates starch at the C3-position in Arabidopsis. Plant Cell 23, 4096-4111.
Sharkey, T.D., Laporte, M., Lu, Y., Weise, S., and Weber, A.P. (2004). Engineering plants for elevated CO(2): a relationship between starch degradation and sugar sensing. Plant Biol (Stuttg) 6, 280-288.
Smith, A.M. (2008a). Prospects for increasing starch and sucrose yields for bioethanol production. Plant J 54, 546-558.
Smith, A.M. (2008b). Prospects for increasing starch and sucrose yields for bioethanol production. Plant J 54, 546-558.
Smith, A.M., Zeeman, S.C., Thorneycroft, D., and Smith, S.M. (2003). Starch mobilization in leaves. J Exp Bot 54, 577-583.
Sokolov, L.N., Dominguez-Solis, J.R., Allary, A.L., Buchanan, B.B., and Luan, S. (2006). A redox-regulated chloroplast protein phosphatase binds to starch diurnally and functions in its accumulation. Proc Natl Acad Sci U S A 103, 9732-9737.
Stettler, M., Eicke, S., Mettler, T., Messerli, G., Hortensteiner, S., and Zeeman, S.C. (2009). Blocking the Metabolism of Starch Breakdown Products in Arabidopsis Leaves Triggers Chloroplast Degradation. Molecular Plant 2, 1233-1246.
Streb, S., Egli, B., Eicke, S., and Zeeman, S.C. (2009). The Debate on the Pathway of Starch Synthesis: A Closer Look at Low-Starch Mutants Lacking Plastidial Phosphoglucomutase Supports the Chloroplast-Localized Pathway. Plant Physiol 151, 1769-1772.
Takashi Matsumoto JW, H.K., Yuichi, Katayose, M.F., Nobukazu Namiki, Hiroshi Mizuno, Kimiko Yamamoto, Baltazar A. Antonio, Tomoya Baba, Katsumi Sakata,, Yoshiaki Nagamura, H.A., Koji Arikawa, Kohei Arita, Takahito Bito, Yoshino Chiden, Nahoko Fujitsuka, Rie Fukunaka, Masao, Hamada, C.H., Akiko Hayashi, Saori Hijishita, Mikiko Honda, Satomi Hosokawa, Yoko Ichikawa, Atsuko Idonuma, Masumi, Iijima, M.I., Maiko Ikeno, Kazue Ito, Sachie Ito, Tomoko Ito, Yuichi Ito, Yukiyo Ito, Aki Iwabuchi, Kozue Kamiya, Wataru, Karasawa, K.K., Satoshi Katagiri, Ari Kikuta, Harumi Kobayashi, Noriko Kobayashi, Kayo Machita, Tomoko Maehara,, Masatoshi Masukawa, T.M., Yoshiyuki Mukai, Hideki Nagasaki, Yuko Nagata, Shinji Naito, Marina Nakashima, Yuko, Nakama, Y.N., Mari Nakamura, Ayano Meguro, Manami Negishi, Isamu Ohta, Tomoya Ohta, Masako Okamoto, Nozomi, Ono, S.S., Miyuki Sakaguchi, Kumiko Sakai, Michie Shibata, Takanori Shimokawa, Jianyu Song, Yuka Takazaki, Kimihiro, Terasawa, M.T., Kumiko Tsuji, Shigenori Ueda, Kazunori Waki, Harumi Yamagata, Mayu Yamamoto, Shinichi Yamamoto,, and Hiroko Yamane, S.Y., Rie Yoshihara, Kazuko Yukawa, Huisun Zhong, Masahiro Yano, Takuji Sasaki. (2005). The map-based sequence of the rice genome. Nature 436, 793-800.
Trinder, P. (1969). Determination of blood glucose using 4-amino phenazone as oxygen acceptor. J Clin Pathol 22, 246.
Velten, J., and Schell, J. (1985). Selection-expression plasmid vectors for use in genetic transformation of higher plants. Nucleic Acids Res 13, 6981-6998.
Wang, B.B., and Xia, L.M. (2011). High efficient expression of cellobiase gene from Aspergillus niger in the cells of Trichoderma reesei. Bioresource Technol 102, 4568-4572.
Weise, S.E., Weber, A.P.M., and Sharkey, T.D. (2004). Maltose is the major form of carbon exported from the chloroplast at night. Planta 218, 474-482.
Weise, S.E., van Wijk, K.J., and Sharkey, T.D. (2011). The role of transitory starch in C-3, CAM, and C-4 metabolism and opportunities for engineering leaf starch accumulation. J Exp Bot 62, 3109-3118.
Weise, S.E., Aung, K., Jarou, Z.J., Mehrshahi, P., Li, Z., Hardy, A.C., Carr, D.J., and Sharkey, T.D. (2012a). Engineering starch accumulation by manipulation of phosphate metabolism of starch. Plant Biotechnol J 10, 545-554.
Weise, S.E., Aung, K., Jarou, Z.J., Mehrshahi, P., Li, Z.R., Hardy, A.C., Carr, D.J., and Sharkey, T.D. (2012b). Engineering starch accumulation by manipulation of phosphate metabolism of starch. Plant Biotechnology Journal 10, 545-554.
Werpy, T.P., G. (2010). Top value added chemical from biomass. Results of screening for potential candidates from sugars and syngas.Washington, DC: US Department of Energy.See http://www1.eere.energy.gov/biomass/pdfs/35523.pdf. 1.
Yan, J.Q., Zhu, J., He, C.X., Benmoussa, M., and Wu, P. (1998). Quantitative trait loci analysis for the developmental behavior of tiller number in rice (Oryza sativa L.). Theor Appl Genet 97, 267-274.
Yu, T.S., Kofler, H., Hausler, R.E., Hille, D., Flugge, U.I., Zeeman, S.C., Smith, A.M., Kossmann, J., Lloyd, J., Ritte, G., Steup, M., Lue, W.L., Chen, J., and Weber, A. (2001). The Arabidopsis sex1 mutant is defective in the R1 protein, a general regulator of starch degradation in plants, and not in the chloroplast hexose transporter. Plant Cell 13, 1907-1918.
Yu, T.S., Zeeman, S.C., Thorneycroft, D., Fulton, D.C., Dunstan, H., Lue, W.L., Hegemann, B., Tung, S.Y., Umemoto, T., Chapple, A., Tsai, D.L., Wang, S.M., Smith, A.M., Chen, J., and Smith, S.M. (2005). alpha-Amylase is not required for breakdown of transitory starch in Arabidopsis leaves. J Biol Chem 280, 9773-9779.
Zeeman, S.C., and Ap Rees, T. (1999). Changes in carbohydrate metabolism and assimilate export in starch-excess mutants of Arabidopsis. Plant Cell and Environment 22, 1445-1453.
Zeeman, S.C., Kossmann, J., and Smith, A.M. (2010). Starch: its metabolism, evolution, and biotechnological modification in plants. Annu Rev Plant Biol 61, 209-234.
林祐生與李文乾. (2009年一月). 生質酒精. 科學發展 44, 20-25.
纖維乙醇之技術與文獻探討. (2007). Version 12 分享․跨越門檻與障礙.