| 摘要: | MYB家族是一群能夠結合特定DNA序列的轉錄因子。在植物中，依照MYB的DNA結合區域 (Binding motif) 數目，將MYB家族分成三大次家族，分別為含有三個DNA結合區域的R1R2R3、兩個DNA結合區域的R2R3及一個DNA結合區域的R1/2次家族。在先前的水稻研究中，發現三個R1/2 MYB蛋白 (OsMYBSs) 參與了糖與賀爾蒙GA調控水稻α-amylase基因的表現。後來在大麥中，相似的R1/2 MYB轉錄蛋白HvMCB1及HvMYBS3也先後被鑑定出參與了種子萌發時期基因表現的調控機轉。然而，截至目前為止，MYBSs在植物體中的生理功能仍然不甚了解，因此在本研究中，選擇以阿拉伯芥為材料，希望針對MYBS做一連串生理功能的探討。 在本論文中，我們分離出四個阿拉伯芥的MYBSs基因，分別命名為AtMYBS1-1、AtMYBS1-2、 AtMYBS2-1與AtMYBS2-3，經由RT-PCR的分析，發現它們幾乎在各個組織器官中都有表現，雖然表現的狀況不盡相同。為了進一步研究MYBSs基因的生理功能，我們分別對阿拉伯芥進行gain-of-function及loss-of-function的研究。我們構築了完整長度的cDNA，利用農桿菌轉殖的方式，建立能夠大量表現MYBSs的阿拉伯芥轉殖株。另一方面，從SALK institute訂購MYBSs基因被T-DNA插入的突變種子，然後篩選得到同型合子的突變植株，這些大量表現與突變植株將有助於往後的研究。 此外，從本研究中，利用RT-PCR的方式，確認AtMYBSs基因的表現是受糖調控的。同時AtMYBSs，如同水稻與大麥中的MYBSs一樣，皆能夠辨認水稻αAmy3啟動子中糖調控序列上的TA-box，顯示了AtMYBSs基因可能參與了植物中糖代謝的調控機轉。 MYB proteins are transcription factors that contain conserved DNA binding domains, and comprise a large gene family which can be grouped into several subfamilies by phylogenic criteria. In plants, there are three types of MYB proteins, R1R2R3, R2R3, and R1/2. Previously, we identified three novel R1/2 type MYB proteins in rice, OsMYBS1, OsMYBS2, and OsMYBS3. They contain a single repeat of MYB DNA binding domain, and are involved in sugar- and hormone-regulated α-amylase genes expression mechanism. On the other hand, similar proteins in barley, HvMCB1 and HvMYBS3 have been reported that they are important transcription factors during barley seed development. However, the physiological functions of MYBSs in plants are still not well known. As Arabidopsis is a powerful model plant, supported by robust bioinformatics and comprehensive mutant collection, we use Arabidopsis to further investigate biological roles of MYBSs in plants. In this study, four different full-length AtMYBS cDNAs were isolated from Arabidopsis, and these genes were expressed ubiquitously in roots, leaves, flowers and siliques with their own unique expression pattern. To study physiological roles of these genes, a gain-of-function strategy was applied by generating over-expression of AtMYBSs transgenic Arabidopsis plants; meanwhile, loss-of-function method was used by screening T-DNA insertion mutants. MYBSs over-expression transgenic lines and T-DNA mutant lines has been identified in this work. These plant materials will be valuable for function analysis on AtMYBSs in the near future. The other part of this study was found gene expressions of AtMYBSs are sugar respondent. Like MYBSs in rice and barley, the TA-box of sugar response element is specifically recognized by AtMYBSs, implying AtMYBSs may play roles in regulation of plant sugar metabolism. |
