麻竹筍生長之細胞壁特性及基因表現分析

Abstract

麻竹（Dendrocalamus latiflorus）為臺灣常見竹種之中，分布最廣且適應力最佳之竹種。麻竹具有生長快速及竹種高大等優勢，為良好之木質纖維材料應用來源。本試驗以四周齡大之麻竹筍為材料，依生長部位之型態區分為頂部、中部及基部，分析其不同生長階段之細胞壁組成特性、代謝成分差異及基因表現，期能藉由上述分析，瞭解麻竹的生長特性，以利未來進一步推廣利用。 由組織切片之免疫標定及染色觀察結果得知，麻竹筍於頂部初生長階段，導管即開始有明顯的分化，此時之細胞壁結構以初級細胞壁為主，尚未有明顯的木質素及纖維素的累積。麻竹筍基部可觀察到完整的維管束組織，且木質素及纖維素大量累積於維管束組織。維管束的發育會先由原生木質部開始，並伴隨著導管的生成；接著為韌皮部的成熟，最後才為木質部導管及週邊組織的發育及纖維鞘的累積。細胞壁成分分析結果顯示，麻竹筍木質素含量約占6 – 9%，且於麻竹筍生長初期，酸可溶木質素含量會高於酸不溶木質素。麻竹筍細胞壁醣類組成主要為葡萄糖及木糖，並在生長過程有明顯的累積，葡萄糖的含量由頂部至基部分別為34.5、43.8及47.9%；木糖的含量則分別為14.8、27.0及29.2%。 代謝成分分析結果顯示，葡萄糖、呋喃木糖、果糖及蔗糖為極性層主要代謝成分。蔗糖於麻竹筍頂部有較高代謝，而葡萄糖、呋喃木糖及果糖此3種糖類於基部代謝較為旺盛。脂質層則以Pentadecanoic acid及8,11-Octadecadienoic acid為主要代謝成分，且此2種成分於麻竹筍頂部代謝較為旺盛。 基因差異表現結果顯示，於麻竹筍頂部主要為細胞活動及產生能量相關之基因，進一步以墨點分析及即時定量聚合酶鏈鎖反應結果可知，麻竹筍頂部之myo-Inositol phosphate synthase、Auxin response factor、Signal recognition particle 14 kDa protein及Glutathione S-transferase的表現量較高；麻竹筍基部表現量較高的基因則與光合作用香觀，分別為Chloroplast oxygen-evolving enhancer protein 及Succinate dehydrogenase。 本研究結合了物理、化學性質、代謝及基因表現分析，瞭解單株麻竹筍於不同生長階段之特性。由試驗結果可知，麻竹筍頂部之維管束發育尚未完全，此時細胞壁結構以初級細胞壁為主，基因表現及代謝以細胞分化及能量產生較為相關；麻竹筍基部具有成形之維管束，此時細胞壁已有明顯之二次生長，基因表現及代謝則以醣類生成及光合作用為主。

Ma bamboo (Dendrocalamus latiflous) is a common bamboo specie that is the most distributed and well adapted in Taiwan. Having the advantages of rapid growth and relatively large bulk, it provides a great source of ligno-cellulosic material. One four-week-old ma bamboo shoot was used for this experiment and was divided into top, middle and base portions. Each portion was analyzed according to different growth phases for differences in cell wall properties, metabolites and gene expression. A better understanding of the growth of ma bamboo shoot provides opportunities for further utilization in the future. From immunolocalization and staining results, the vessels in the top portion of ma bamboo shoot began to show apparent cell division in primary growth phases. No clear accumulation of cellulose and lignin were observed in this primary growth phase. However, complete vascular bundles were observed in the base portion along with vast accumulation of cellulose and lignin. Initiation of vascular bundles starts in the region of the protoxylem accompanied by the formation of vessels, and follows the maturation of the phloem. Finally, the fiber sheath and surrounding tissue develop around the xylem vessel. Cell wall component analysis shows that lignin takes up 6 - 9% of the cell wall and that during early stages of growth, the contents of acid soluble lignin are higher than that of acid insoluble lignin. Glucose and xylose are the main sugar components of ma bamboo shoot cell wall and show significant deposition during growth. Glucose contents from top to base portion are 34.5%, 43.8%, and 47.9% respectively; while xylose contents from top to base portion are 14.8%, 27.0%, and 29.2% respectively. Metabolite profiling shows that glucose, xylofuranose, fructose, and sucrose are the major compounds of the polar phase. Of these compounds, the metabolism of sucrose in the top portion of ma bamboo shoot is greater than that of the base portion, whereas the metabolism of glucose, xylofuranose, and fructose is greater in the base portion. In the lipid phase, pentadecanoic acid and 8, 11-octadecadienoic acid are the main compounds and they both present higher metabolism in the top portion. According to gene differential expression results, the genes expressed in the top portion of ma bamboo shoot were mostly found to be related to cell activity and energy generation. Using dot blot and qRT-PCR methods, the main genes were recognized as myo-inositol phosphate synthase, auxin response factor, signal recognition particle 14 kDa protein, and glutathione S-transferase. In the base portion, the most expressive genes were chloroplast oxygen-evolving enhancer protein and succinate dehydrogenase which deals with photosynthesis. Current research is an integrated analysis of physical, chemical, metabolic, and gene expressional properties, which allows us to understand the growth characteristics of ma bamboo shoot. In the top portion of the bamboo shoot, vascular bundles are immature. Functions of gene expression and metabolic processes involve cell differentiation and energy generation. On the other hand, the base portion contains fully developed vascular bundles, gene expression is related to sugar metabolism and photosynthesis, and cell wall exhibits noticeable secondary growth.