Deletion and Characterization of the Water-Deficit-Stress Induced Ip3 Promoter from Loblolly Pine

利用消去法探討及研究水分逆境誘導之德達松lp3基因啟動子

10.29950/JHKUM.200407.0006

王昭泰(Jau-Tay Wang)；Veera Padmanabhan；Jean H. Gould；Ronald J. Newton

水分逆境 ； 聚乙婦甘油醇 ； 氧化逆境 ； 離層酸 ； fluridone ； water deficit stress (WDS) ； polyethylene glycol (PEG) ； oxidative stress ； abscisic acid (ABA) ； fluridone

興國學報

3期（2004 / 07 / 01）

69 - 84

英文

利用差異性篩選及表現表現分析法，我們已經描述了從德達松的水分逆境（WDS）狀況下誘導的基因cDNA lp3。為了更加了解1p3基因在水分逆境下的調控狀況，我們分離及克隆了1p3基因上游調節部份的片段，接於uidA報告基因的前方。藉著使用消去一連串的1p3啟動子，我們研究1p3全長啟動子（-1026bp）及四個不同5'消去之構築（-745，-589 - 279 and - 115bp）並將其接於ß-葡糖醛酸糖酶（uidA, GUS）前，轉殖於菸草上。轉殖基因之菸草（TO）含有不同長度啟動子構築之一，使用不同濃度的聚乙烯醇（PEG）、甲基茉莉酸鹽（MeJa）、離層酸（ABA）及離層酸的抑制劑fluridione處理。對根、莖及葉使用螢光言十進行定量分析；組織化學染色法進行定性分析（Jefferson,1987）。在兩項分析中發現，聚乙烯醇，離層酸及fluridone濃度增加時，GUS活性亦增加。GUS活性表現在全長之1p3啟動子構築下， PEG與fluridone處理後表現最強，離層酸及甲基茉莉酸鹽表現較弱。啟動子片段長度為-1026及-745bp，對於水分逆境、離層酸及fluridone的處理產生較強的反應。啟動子片段長度為-589及-279bp，對水分逆境、離層酸及fluridone的處理產生的反應較弱。啟動子片段長度為-115bp，對水分逆境、離層酸及fluridone的處理全無反應。實驗資料顯示，啟動子區域介於-745及-115之間的區域對水分逆境、離層酸及f1uridone處理之反應至為重要。結果顯示，1p3啟動子在環境逆境的情況下，在松樹中具有調節的功能。

By differential screening and expression analysis, we have previously characterized a water deficit stress (WDS) induced cDNA Ip3 from loblolly pine (Pinus taeda L.). In order to understand the regulatiol1 of Ip3 under water deficit stress, we isolated and cloned its upstream regulatory sequences in front of a uidA reporter gene Using deletion series of the Ip3 promoter, we studied the expression of the whole Ip3 promoter (-1026 bp) and four 5, deletion constructs (-745, -589 -279 and -115 bp) fused to the ß-glucuronidase (uidA, GUS) reporter gene in tobacco. Transgenic tobacco (TO) plants containing one of the 4 deletion constructs were treated with different concentrations of polyethylene glycol (PEG), methyl jasmonate (MeJa), ABA and fluridone, an inhibitor of ABA biosynthesis. Roots, stems and leaves of the plants were assayed for GUS activity by using the quantitative fluorimetric assay and qualitative histochemical staining (Jefferson, 1987). 111 both assays, an increase of GUS activity was observed when the concentration of PEG, ABA or fluridone was increased Expression of GUS from the complete Ip3 promoter was strongest in PEG and fluridone treatments, but less intense with ABA and MeJa. The promoter fragments -1026 and -745 bp produced strong responses to WDS. ABA and fluridone. Fragments -589 and -279 bp produced a weak response to WDS, ABA and fluridone. The promoter fragment -115 did not have expression in WDS, ABA or fluridone treatments. These data show that the region between -745 and -115 is necessary for ABA, f1uridone and WDS dependent expression. Our results suggest that Ip3(P) has a regulatory function in pine during environmental stress.

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1. 王昭泰(2006).The Cloning of Water-Deficit-Stress Induced lp3 Gene from Loblolly Pine.興國學報,6,69-92.