在綠竹生長過程中具差異性表現基因之檢定

Abstract

竹為生長速度最快之陸生植物。為了解綠竹於快速生長時期之基因調控方式，本研究使用差異性表現分析法 (differential display)，從八種不同生長時期綠竹之 cDNA 樣品中，篩選出306條具差異性表現之DNA 片段，並一一加以定序。經過 blast 比對後，將結果以 Gene ontology (GO) 資料庫進行分類比較。利用 DNA Microarray 分析，確認差異性表現分析法之結果，並透過逐步叢集分析 (k-means clustering analysis），將 306 條 DNA 片段區分為六個分群。在推測具有轉錄調節功能之 ESTs 中，挑選出一條BohLOL1。序列分析顯示其包含三個具有 DNA 結合功能的 zinc finger 保守區域，與阿拉伯芥 LSD1 及 LOL1 (LSD-One-Like 1) 具同源性。Reverse northern 分析顯示 BohLOL1 在綠竹快速生長時表現量上升。Real-time RT-PCR 分析顯示無菌竹苗中之 BohLOL1，會因為 auxins 及 cytokinins 之作用、病菌感染、2,6-dichloroisonicotinic acid (salicylic acid 之類似物) 及hydrogen peroxide 之添加而提高表現量。細胞定位與 Protein-DNA 交互作用分析結果指出，BohLOL1 可能在細胞核中，藉由與目標基因結合之方式，發揮其在綠竹快速生長與遭受逆境時之作用。本實驗提出並證實BohLOL1.與綠竹生長之關聯及其結合 DNA 之特性，對於後續針對綠竹快速生長時期基因調控之研究方向，極具參考價值。

Growth of woody bamboo is generally rapid and faster than other woody plants. To investigate what genes are involved in controlling the growth of bamboo, differential display RT-PCR was performed to analyze the gene expression profiles of bamboo (Bambusa oldhamii). 306 cDNAs were isolated and sequenced. The results of BLAST analysis were classified with Gene Ontology (GO) database. DNA microarray was carried out to confirm the results of the differential display. Real-time RT-PCR was applied to exam the results of microarray. Among the 306 ESTs, A cDNA, BohLOL1, encoding a protein containing three zf-LSD1 (zinc finger- Lesions Simulating Disease resistance 1) domains was choose for futher study. A phylogenetic analysis revealed that BohLOL1 is a homolog of Arabidopsis LSD1 and LOL1 (LSD-One-Like 1). The BohLOL1 gene was upregulated in shoots with higher rates of culm elongation. The expression level of this gene in multiple shoots of bamboo, was also upregulated by auxins, cytokinins, pathogen infection, 2,6-dichloroisonicotinic acid (a functional analog of salicylic acid) and hydrogen peroxide. The results suggest that BohLOL1 participates in bamboo growth and in the response to biotic stress. The DNA-binding assays and subcellular localization studies demonstrated that BohLOL1 is a nuclear protein.and affects the expression of target genes through protein-DNA interactions The growth-dependent up-regulation of BohLOL1 expression, which uniquely occurs in growing bamboo, might be one of the critical factors that contribute to the rapid growth of this remarkable plant.