阿拉伯芥中受乾旱誘導之轉錄因子At2g20880功能分析

Abstract

當植物暴露在不同逆境時會有大量的基因被誘導出來，而這些基因產物具有不同的功能以不同方式幫助植物對抗逆境。我們從Shinozaki在2002年發表的微矩陣序列分析資料中得到At2g20880基因的表現量在乾旱逆境下比正常生長狀況高出24倍。At2g20880轉錄因子被歸類為DREB subfamily一員，功能大多跟植物對抗逆境有關。利用RT-PCR發現At2g20880在根、莖、葉、花都有表現，但是在花的表現量較低。利用northern blot及RT-PCR確定在兩週大的阿拉伯芥野生型植株中At2g20880基因的表現量只有在乾旱逆境才會增加，不會受到高溫逆境及ABA的誘導而大量表現。我們利用RNAi（RNA interfering）方式專一性地抑制At2g20880基因，並確定At2g20880基因表現量下降；另外，將At2g20880基因全長構築於pB2GW7載體持續大量表現At2g20880基因。比較在正常生長狀況下，兩週大野生型、At2g20880突變株以及At2g20880大量表現轉植株的外表型態並無太大差異。接著觀察兩週大野生型以及At2g20880大量表現轉植株的耐旱能力，發現在乾旱環境下，轉植株的生長狀況比野生型好，存活率也比較高，證明在阿拉伯芥大量表現At2g20880基因可提高植物對乾旱的忍受力。ABA處理後，At2g20880基因表現量沒有上升；讓野生型與At2g20880大量表現轉植株生長在有ABA的情況下， 兩者之種子萌發率及根部生長狀況沒有差異，表示At2g20880大量表現轉植株對ABA敏感性沒有改變。將At2g20880基因全長構築於GFP蛋白表現載體（pK7WGF2）中。利用CaMV 35S啟動子表現GFP及At2g20880蛋白質。以基因槍將載體打入洋蔥表皮細胞，發現At2g20880在細胞質中有表現。由本篇實驗結果得知At2g20880會參與乾旱訊息傳導路徑，屬於ABA-independent pathway，在阿拉伯芥大量表現此基因可以提高植物耐旱能力，因此推測At2g20880為乾旱反應的正向調控者。

Plants often meet many biotic and abiotic stresses throughout their life cycle. Assortments of genes with diverse functions are influenced by dehydration stresses. Most of their gene products may function in stress responses and tolerance at the cellular level. The predominant mechanism for controlling plant gene expression is regulated at the transcriptional level and is mediated by transcription factors (TFs). According to the microarray data of (Kamiya et al., 2002), we selected a dehydration-induced gene At2g20880 to study more detail in this paper. At2g20880 contains one highly conserved AP2 domain and belongs to DREB subfamily. Using RT-PCR, we found At2g20880 gene was expressed in root, stem, leaf, but has lower expression levels in flowers. We found that this gene was highly inducible by dehydration but not by high temperature and abscisic acid. The At2g20880 RNAi transgenic plants and At2g20880-overexpressing plants exhibited the same phenotype as wild-type under normal growth conditions. At2g20880-overexpressing plants, however, survived better than wild-type under water deficient condition. The expression of At2g20880 gene was not influenced by ABA treatment; and there is no difference in seed germination rate and root growth between At2g20880-overexpressing transgenic plants and wild type in the presence of ABA, suggesting that the ABA sensitivity of At2g20880-overexpressing transgenic plants was not altered. Localization study using a fusion protein consisting of the full length of At2g20880 coding region and GFP fusion under the control of 35S promoter revealed that the GFP fluorescence was detected in the cytoplasm but not in the nucleus. In this study, we found At2g20880 is involved in dehydration-induced signaling transduction in an ABA-independent manner. Overexpressing At2g20880 gene enhanced dehydration tolerance in Arabidopsis. Our study provides evidence that At2g20880 is a positive regulator in dehydration stress response.