文心蘭南西品系過氧化氫酶啟動子Ppox3-19基因調控之功能分析

Abstract

本實驗室先前從文心蘭扣減式EST cDNA library中得到九個Class Ⅲ的過氧化氫酶基因，有些成員於不同逆境因子(H2O2 、4°C低溫、ABA) 處理下，會被誘導表現；為了解其調控機制，本研究選殖了出文心蘭過氧化氫酶Ppox3-19之1364bp啟動子(peroxidase promoter)，經分析發現有ABRE、GARE、以及與防禦反應相關之TC-rich repeats等cis-elements，將啟動子後接β-glucuronidase(GUS)報導基因並轉殖至阿拉伯芥，研究於不同處理(IAA、ABA、JA、GA3各100 μM，以及培養基中1 %、2 %、3 %、4 %、5 % 蔗糖)及不同大小啟動子片段啟動報導基因GUS之表現情形。於不同處理下，GUS組織染色結果顯示，報導基因皆會在七天大之阿拉伯芥轉殖株的保衛細胞表現，推測於啟動子上具有保衛細胞專一的cis-elements；另外，GUS螢光活性分析顯示，100 μM 之JA、GA3、ABA三種賀爾蒙會提高GUS表現，可達2至 2.5 nmole MU/min/mg protein。此外，隨著培養基中蔗糖濃度越高，GUS表現量會越多，其中5% 蔗糖的處理，GUS表現可達4.3 nmole MU/min/mg protein。 分析不同5’端長度之啟動子於阿拉伯芥轉殖株之表現，推測啟動子於-1343 bp~ -1291 bp處，應有保衛細胞專一性表現之cis-element；在-1291 bp 至-701 bp 除了具有葉肉細胞表現之cis-element 外，也可能具有增強GUS 表現量之cis-elements。 為了解Ppox3-19與ABA訊息傳遞之關係，將Ppox3-19全長轉殖入阿拉伯芥aba 及abi 突變株中，結果顯示abi1中無GUS表現，而abi2中仍有GUS表現於保衛細胞中，顯示Ppox3-19表現於保衛細胞需要ABI1存在於植物細胞中。 由阿拉伯芥轉殖株之結果，我們認為ABA 訊息傳遞路徑之參與是影響POX 基因啟動子於細胞中表現的重要因子，而POX啟動子的-1343 bp~ -1291 bp是決定保衛細胞專一性表達的重要cis-element。

Previous studies had shown that expression patterns under abiotic stress of nine class III peroxidase genes isolated from Oncidium substractive cDNA library. Here we cloned a 1.3 kb of Oncidium peroxidase promoter, named Ppox3-19 that was fused with a GUS reporter gene. By using transgenic Arabidopsis, the promoter activity was investigated under various concentrations of different hormones and sucrose. The results revealed that 7-day-old seedlings of transgenic Arabidopsis specifically exhibited higher GUS activities in guard cells in the presence of 5 % sucrose and 100 μM different hormones such as JA, GA3, and ABA. To further characterize the Ppox3-19, a series of deletions of the promoter fused with were established, and transformed into wild-type Arabidopsis. The results revealed that transgenic plants with the full-length of the promoter showed higher GUS activities in guard cells, rather than those with deletion constructs. We concluded that Ppox3-19 contains guard cell-specific cis-elements between -1341 bp and -1291 bp. The regulatory relationship between ABA and guard cells was also analyzed by transforming the Ppox3-19::GUS construct into Arabidopsis aba and abi mutants. Accordingly, it was found that GUS activities were detected in guard cells of abi2 but not in abi1, which indicates that Ppox3-19 be involved in downstream of ABI1 signaling. Thus, the promoter activity of the peroxidase pox3-19 by higher concentration (> 100 μM) of ABA, and a guard cell-specific cis-element may be present in the promoter region from -1343 bp to -1291bp.