野生型灰楊及PtiCSLA1增量轉殖株之細胞壁特性分析

Abstract

由於石油資源日漸枯竭，因此開發再生的替代能源一直是重要的研究課題。生質乙醇(Bioethanol)為目前重要的再生能源之一，可藉由發酵木質纖維中的單糖來製成。唯目前工業發酵的製程尚以利用六碳糖(Hexose)為最有效率的方法，因此利用基因調控來提高木質纖維中六碳糖的含量將有助於能源作物的開發與利用。甘露聚醣(Mannan)為細胞壁多醣之一，是裸子植物細胞壁中含量最多的非纖維素多醣(含量約20%)，同時也是被子植物細胞壁非纖維素多醣的次要組成分之一(含量2 - 5%)，其單糖的六碳結構非常適合作為生質乙醇生產的材料。本研究首先以Mannan之專一性抗體LM21對野生型灰楊(Populus trichocarpa)不同節間之莖部組織進行免疫標定，以觀察Mannan於植物生長過程中的累積情形。結果顯示Mannan在植物發育初期，最先堆積於初生木質部（Primary xylem），並隨著植株的次級生長而逐漸累積於木質部及韌皮纖維（Phloem fiber）的次級細胞壁中。另一方面，LM21的訊號也會受到果膠質(Pectin)及乙醯基(Acetyl group)的遮蔽，但不受木質化(Lignification)及木聚醣(Xylan)堆積的影響。本研究另以轉基因方式利用35S啟動子(Promoter)正向調控灰楊Mannan生合成基因PtiCSLA1(Cellulose synthase-like A1)表現，並以即時定量聚合酶鏈鎖反應(Quantitative real time polymerase chain reaction, qRT-PCR)分析基因表現量，以酵素連結免疫吸附試驗(Enzyme-linked immunosorbent assay, ELISA)分析酵素活性，再以醣類分析及免疫標定技術觀察野生型及轉基因型植株細胞壁多醣的組成及分布。醣類分析的結果顯示，利用單一35S啟動子正向調控PtiCSLA1基因表現的植株，其Mannan含量最高可達到野生型植株的1.5倍。同時免疫標定的結果顯示轉殖株的Mannan含量確實增加，證實灰楊PtiCSLA1基因參與Mannan的生合成。

Due to fossil fuel reserve will be completely depleted, it has been an important issue to seek any alternative and renewable source for fuels. Bioethanol is regarded as the most potential alternative for fossil fuel substitutes. Bioethanol can obtain from fermentation of sugars in lignocelluloses. For current industrial-based fermentation, hexoses are the preferred substrates for ethanol production. Mannan polysaccharides are the most abundant non-cellulosic cell wall polysaccharides in gymnosperm (~20%), however they are minor non-cellulosic polysaccharides in angiosperm (2 - 5%). Mannose is one of hexose members and suitable for bioethanol production. In this study, mannan specific antibody LM21 was used to investigate mannan deposition in wild-type black cottonwood (Populus trichocarpa) stems from different internodes. Results of immunolabeling reveal that mannan deposits in primary xylem in early developmental stages, and then accumulates in secondary walls of xylem cells and phloem fibers during wood formation. On the other hand, LM21 signals can be masked by pectin and acetyl group, but are less affected by lignification and xylan deposition. To investigate the function of PtiCSLA1 (Cellulose synthase-like A1), 35S promoter was used to overexpress PtiCSLA1 in Populus trichocarpa. Gene expression were analyzed by qRT-PCR (Quantitative real time polymerase chain reaction), and enzyme activities were analyzed by enzyme-linked immunosorbent assay (ELISA), and wood compositions were analyzed through carbohydrate analysis and immunolabeling. Results of carbohydrate analysis revealed that mannan contents of PtiCSLA1 transgenic lines driven by single 35S promoter were higher than wild-type plants, and the highest content to be 1.5 fold of that in wild-type plants. Results of immunolabeling by mannan specific antibody also revealed that LM21 signals in transgenic lines were stronger than that of wild-type plants, which provides a strong evidence to support PtiCSLA1 gene involving in mannan biosynthesis.