阿拉伯芥AtMAPRs基因靜默轉殖株之建構與性狀分析

Abstract

我們在阿拉伯芥中發現四個與豬的 MAPR (membrane associated progesterone receptor conponent 1) 有 30~40% 相似度的同源性蛋白質，分別命名為 AtMAPR2、AtMAPR3、 AtMAPR4 與 AtMAPR5。在先前的研究指出 AtMAPR5 (putative membrane steroid binding protein, MSBP1) 可能為控制細胞延長之負調控因子，但對於其他 AtMAPRs 蛋白質的功能仍然缺乏全面性的了解。我們利用農桿菌基因轉殖的方式得到 AtMAPR4 基因靜默 (RNAi) 轉殖株及 AtMAPR4 基因過度表現 (overexoression) 轉殖株，由初步的性狀觀察發現 AtMAPR4 基因過度表現突變株在抽花序的時間有提早的現象；相反的 AtMAPR4 基因靜默突變株在抽花序的時間也有延遲的現象。由於先前我們也發現 AtMAPR4 基因在阿拉伯芥生長晚期，具有花的特異性表現，因此推測 AtMAPR4 可能與開花時間有關。另一方面，本實驗室先前的酵母菌雙雜合實驗結果顯示，AtMAPR5 可能與 RING-type 泛素接合酶 (At3g58030) 產生交互作用，且經序列比對後又發現另一個與其相似度高的 RING-type 泛素接合酶 (At2g42030)，因泛素接合酶具有辨識專一性目標蛋白質的功能，推測其可能與其他 AtMAPRs 蛋白質產生交互作用，因此我們將兩個 RING-type 泛素接合酶的基因選殖出來並在大腸桿菌中表現，作為將來進行與 AtMAPR 蛋白質交互作用實驗及 RING-type ubiquitin ligase 活性分析。

Four AtMAPRs (AtMAPR2, AtMAPR3, AtMPAR4, and AtMAPR5) in Arabidopsis sharing 30-40% similarity with porcine MAPR (membrane associated progesterone receptor component 1) have been identified. AtMAPR5 (putative membrane steroid binding protein, MSBP1) is proposed as a negative regulator of cell elongation (Yang et al., 2005). However, the functions of the other AtMAPRs are still unclear. By Agrobacterium-mediated transformation, we attempted to establish two transgenic plants where AtMAPR4 gene was either knocked-down by RNAi technology or overexpressed. Transgenic plants overexpressing AtMAPR4 showed that their bolting time was earlier than wild-type, whereas transgenic plants with knocked-down AtMAPR4 showed that they delayed flowering. We also found that AtMAPR4 was expressed higher in flowers by analyzing the tissue-specific expression in 35-day-old wild-type plants previously. It suggested that AtMAPR4 might be involved in the flowering. The second part of this research followed previous yeast two-hybrid assay experiment where AtMAPR5 might interact with RING-type ubiquitin ligase (At3g58030). Another RING-type ubiquitin ligase (At2g42030) shares high similarity with At3g58030. RING-type ubiquitin ligase could recognize target protein by specific domain. These two RING-type ubiquitin ligase were cloned and heterologously expressed in E.coli. They will be used in further pull-down experiment and ubiquitination assay.