摘要:幹細胞調控植物細胞發育 (葉與花)及幹細胞在基因轉殖技術上的應用,成了現代生物學裡熱門的話題。了解植物幹細胞在發育演化上所扮演的角色更是此議題中最重要的成份。一般而言植物的幹細胞存在頂端分生組織中,負責建構整個植物的骨架。然而,一種頗受歡迎的景觀植物,苦苣苔科植物的成員可以在葉子上持續維持幹細胞的活性,甚至在部分苦苣苔科成員的葉子 (或子葉)可以不停地生長。這種特異的現象 (子葉不等大)已經吸引許多植物學家的目光。然而子葉不等大的發育機制在近四十年內依然無解,我們若能解開子葉不等大機制的謎團,未來可以在組織培養等生物技術上獲得應用,更可以在分子育種上有更大的貢獻。
目前我們發現分生組織相關基因KNOX1能在子葉上異位表現,以及植物激素細胞分裂素可以調控KNOX1基因的表現,這項突破性的發現提供了有力的證據:植物細胞可以利用生理及環境因子的交互作用調控分生組織活性基因的表現,並重新活化分生組織的活性。我們認為細胞分裂素與分生組織活性基因可以在葉部上共同作用,而分生組織活性基因可以經由一連串訊息傳遞刺激更多植物細胞分裂。此外,KNOX1基因的啟動子區域可能具有調控該基因在葉子上表現的能力,這些啟動子區域可能為苦苣苔科植物特異生長模式的關鍵。
為了解苦苣苔科葉部分生組織形成的機制及其演化上扮演的角色,本研究擬分析細胞分裂素的生合成路徑與分生組織活性是否存在交互作用,實驗首先分析KNOX1基因的上游啟動子內的順式作用元素,我們將苦苣苔科KNOX1基因啟動子與報導基因結合轉殖到阿拉伯芥上,以此檢測該分生組織活性基因表現的區域以及細胞分裂素處理是否能增加植物分生組織的活性。之後,更進一步利用去氧核糖核酸-蛋白質結合檢測法分析預期的順式作用元素及阿拉伯芥的IPT蛋白是否存在交互作用,細胞分裂素可能藉此機制或蛋白質與蛋白質的交互作用調控分生組織活性基因。此外藉由分析分生組織活性基因的親緣關係,以冀了解苦苣苔科不同生長型式的植物,其不同葉型幹細胞形成模式與基因序列間的關係。
最後我們期望這些結果可以對植物幹細胞的分子遺傳機制有更深一層的了解及貢獻。
Abstract: Understanding plant stem cell development and evolution is the most important issue in modern biology as stems cells control organ development (leaf / flower), and stem cells can be tissue cultured with gene transformation biotechnology. Plant stem cells are usually located in shoot apices and involved in shoot apical meristem in creating plant architecture. However, species of the popular house plant family Gesneriaceae (African Violet, Cape Primrose) can have continuously active stem cells / meristem in leaves. Continuous meristematic activity in their cotyledons for example causes a phenomenon unique in plants, anisocotyly. It amazes many botanists but its developmental and genetic mechanisms remain unresolved. By unraveling the induction of meristem activity, we can apply this knowledge not only to modern biotechnology applications such as meristem tissue culture but this may also help techniques of molecular breeding.
Our preliminary data from RNA in-situ hybridization showed that shoot apical meristem genes, such as KNOX1 and STM are ectopically expressed in cotyledons and leaf primordial in Gesneriaceae anisocotylous species (genus Streptocarpus). In addition, we found that the plant hormone cytokinin may enhance anisocotyly. Our interpretation is that cytokinin may regulate the meristem gene or vice versa, and resulted in prolonged cotyledon meristematic activity with the enhanced cell division activity.
To test this hypothesis, we like to analyze how the cytokinin biosynthesis interacts with meristem genes activity. We propose that cytokinin act on cis regulatory region of KNOX1 to control its ectopically expression and localization in macrocotyledon /leaves. To prove that, a promoter analysis will be conducted by a reporter assay via transformation of Streptocarpus KNOX1 promoters into Arabidopsis to test whether the promoter itself regulate the localization of meristematic gene or not. Furthermore we would like to clarify how cytokinin regulate KNOX1, either by binding its promoter, or through protein-protein interactions. As it was known the biosynthetic enzymes of cytokinin have important roles in the cell division signal transduction (meristematic activity), we decide to use the cytokinin synthesizing Isopentenyltransferase (IPT) enzymes derived from Arabidopsis thaliana as experimental material, and analyze whether the KNOX1 and IPT interaction is observed via the promoter - protein or protein - protein interactions through a DNA-protein binding assay, and yeast two hybrid assay.
Different KNOX1 / STM gene sequences and their unique expression pattern may result in different types of meristematic activities in leaves, which seems to correlate with various leaf morphology in Gesneriaceae species, such as equal or unequal cotyledons, isophyllous or anisophyllous foliage leaves. A phylogenetic analysis of all KNOX1 / STM genes copies from representative Gesneriaceae species will be related to their cotyledon meristem activity including their expression patterns, in an attempt to find out the phenotypic effect of different KNOX1 / STM. These data will contribute to a better understanding of the molecular genetic control in meristem / stem cell maintenance in diversity of plant species.
Keywords: Anisocotyly, anisophylly, macrocotyledon, meristem, KNOX1 gene, promoter analysis, cytokinin, plant hormone, stem cell maintenance