| 摘要: | 水稻中OsMYBSs屬於R1/R2這類的MYB轉錄因子，已知會參與在糖訊息的途徑中，藉由結合在啟動子之TA-Box（TATCCA）序列，調控下游基因αAmy3的表現。利用水稻OsMYBSs的蛋白質氨基酸序列，與阿拉伯芥蛋白質資料庫進行比對，得到四個相似度極高且同時具有R1/R2家族高度保留區的序列，分別命名AtMYBS1、AtMYBS2、AtMYBS3與AtMYBS5。本論文主要利用reverse genetics的方式，以gain-of-function（大量表現AtMYBSs）以及loss-of -function（T-DNA插入之atmybs突變株）做為研究材料，來研究AtMYBSs在阿拉伯芥中的功能。同時考量研究轉錄因子時，常因為有基因redundancy的現象而增加研究的複雜度，我們利用Chimeric repressor silence technology（CRES-T）此項技術解決基因redundancy的問題。大量表現AtMYBS-SRDX的轉殖株也做為我們的研究材料，利用觀察這些植株的表徵，來推測AtMYBSs在阿拉伯芥中扮演的功能為何。在一般正常生長情況（16h light/8h dark）下，突變株atmybs2、atmybs3及tamybs5與wild-type植株外表性狀並未有明顯的差異。而且AtMYBS並未參與在澱粉代謝的途徑中。在種子萌芽時期，atmybs2的突變株對於高濃度糖與植物荷爾蒙ABA具有不敏感的外表型，atmys2突變株對於熱逆境及鹽害逆境並未有較高的抗性，顯示AtMYBS2在種子萌芽的階段可能參與在糖與ABA調控的途徑中。DIN3 (dark-induced 3)和DIN4 (dark-induced 4)基因為branched-chain ?-ketoacid dehydrogenase的酵素複合體的成員蛋白質，參與在支鏈胺基酸的代謝途徑中，其啟動子的位置上具有TA-box like的序列，在幼苗時期經由qRT-PCR分析，發現AtMYBS2負調控DIN3和DIN4基因的表現。CwINV4 (cell wall invertases)啟動子的位置上也有TA-box，也被AtMYBS2負調控著，綜合以上結果，推測AtMYBS2參與在糖訊息與植物荷爾蒙ABA傳遞途徑中，藉由結合在TA box的序列而調控下游基因的表現，而影響阿拉伯芥種子的萌芽。 MYB transcription factors are one of the largest gene families in plants. There are three types of MYB proteins, R1R2R3, R2R3, R1/2. Previously, three novel R1/2 type MYB proteins in rice, OsMYBS1, OsMYBS2, and OsMYBS3, which are reported involved in sugar- and hormone-regulated α-amylase genes expression during seed germination. In order to further investigate the biological roles of MYBSs in plants, Arabidopsis is applied in this study because of its powerful bioinformatics and comprehensive mutant lines. Here, both gain-of-function and loss-of-function strategies were applied by for studying the physiological roles of AtMYBSs. Gene redundancy interfere functional analyses of MYBSs transcription factors. Therefore, Chimeric REpressor Silencing Technology (CRES-T), fused the short transcriptional repressor domain (SRDX) to AtMYBSs, is also used. There are no obvious different phenotypes and leaf starch content existing in gene knock-out of AtMYBSs in Arabidopsis. The atmybs2 presents glucose and abscisic acid (ABA)-insensitive phenotype during seed germination stage. The atmybs2 also presents ABA-insensitive in the seedling root growth. But the atmybs2 is not insensitive to heat stress or slat stress. The DIN3 (dark-induced 3) and DIN4 (dark-induced 4) which are subunits of a branched-chain ?-ketoacid dehydrogenase functions involved in amino acid degradation, both are downstream regulated genes of AtMYBS2. In addition to, one of cell wall invertases, CwINV4, is also a target gene of AtMYBS2. TA-box is identified on promoter region of these 3 AtMYBS2-responsive genes. According to these results, we conclude that AtMYBS2 may involve in seed germination pathway by regulating the TA-box containing gene(s) expression. |
