分離及鑑定一個axe1的抑制基因SUX4

Abstract

在植物生命週期裡，由營養生長轉為繁殖生長而開花是個重要的生長發育轉變，此轉變受到各種環境因子和植物內在因子所調控。在阿拉伯芥中，HDA6是一種屬於RPD3家族的去乙醯化酵素，其突變株axe1-5具有延遲開花的性狀，而控制開花的主要負調控者FLOWERING LOCUS C (FLC)的表現量較野生型阿拉伯芥高。本實驗將axe1-5種子處理EMS藥劑誘發突變後，篩選出一提早開花的突變株為實驗材料，將其命名為SUPPRESSOR OF AXE 4 (SUX4)。 sux4axe1-5突變株在長日照和短日照下皆呈現提早開花，而將sux4axe1-5和野生型阿拉伯芥回交後得到sux4突變株，其在長日照和短日照下也皆是提早開花，而其中FLC表現量並無變化，但下游基因SOC1和FT皆提高表現，顯示SUX4藉由調控SOC1和FT來影響開花時間。利用染色質免疫沈澱(Chromatin immunoprecipitation)分析FLC基因，在axe1-5突變株中FLC基因的組蛋白三乙醯化(H3ac)程度提高，但在sux4突變株中並無改變，由此結果推測SUX4作用於HDA6下游去影響阿拉伯芥開花時間。 此外，由於siz1-3突變株具有與sux4axe1-5植株很相似的早開花性狀，因此也列為本研究之研究項目。在阿拉伯芥中，SIZ1是一個SUMO E3 ligase，具有輔助連接SUMO至目標蛋白的功能。過去研究證實SIZ1可對FLD蛋白做SUMO修飾，進而提高了FLC染色質上組蛋白四乙醯化(H4ac)的程度。本研究利用雙分子螢光複合物分析(BiFC)而發現，SIZ1和HDA6/HDA19分別都具有蛋白質交互作用，而FLD和HDA6/HDA19也被前人證實會形成共同抑制物複合體，經上面結果推測SIZ1蛋白可對HDA6/HDA19做SUMO修飾，進而調控FLC。

The floral transition is major developmental switch in the plant life cycle, which is regulated by both environmental and endogenous cues. HDA6 is a RPD3-type histone deacetylase (HDAC) in Arabidopsis thaliana. The Arabidopsis HDA6 mutant, axe1-5, showed a late-flowering phenotype, in which FLC was up-regulated. By screening plants derived from EMS mutagenized axe1-5 seeds, we isolated an early-flowering mutant, suppressor of axe1 (sux4). sux4axe1-5 plants flowered early in both long day (LD) and short day (SD). By backcrossing sux4axe1-5 with Col, sux4 mutant was isolated. sux4 also displayed early flowering in both LD and SD. In sux4 plants, FLC expression was not changed but SOC1and FT expression was increased, suggesting that SUX4 regulate flowering by affecting SOC1and FT expression, independently of FLC. In ChIP assay, histone H3 hyperacetylation is deposited on FLC chromatin in axe1-5, but sux4 mutation does not affect H3 acetylation caused by axe1-5, suggesting that SUX4 acting downstream of HDA6 to regulate flowering. We also analyzed the siz1-3 mutant, which has an early-flowering phenotype similar to that of the sux4axe1-5 plant. AtSIZ1 is a SUMO E3 ligase which conjugates small ubiquitin modifier peptides to a target protein. It has been demonstrated that SIZ1-mediated SUMO modification of FLD represses H4 deacetylation of FLC chromatin. In this study, we found that SIZ1 interacts with HDA6/HDA19 by BiFC assay. These results suggest that SIZ1 might mediate sumoylation on HDA6/HDA19 to affect histone acetylation on FLC chromatin.