減少水稻SEX4同源基因的表現以利生物精鍊之應用

Accumulation of tansitory starch by reducing gene expression of SEX4 homologs in Oryza sativa for biorefinery application

吳青蓉(Ching-Rong Wu)

指導教授 ： 黃麗芬

元智大學/工程學院/生物科技與工程研究所/碩士(2012年)

摘要

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。

關鍵字

短暫性澱粉 ； 水稻 ； 阿拉伯芥 ； SEX4 ； LSF1 與生質燃料

並列摘要

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.

並列關鍵字

LSF1 and biofuel ； Transitory starch ； Oryza sativa ； Arabidopsis ； SEX4

參考文獻

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.