CBF4::LUC轉殖株的建立及CBF4啟動子的片段分析

Abstract

在植物生長的過程，會遇到許多不同的非生物性逆境，這些逆境包括了低溫，高溫，乾旱以及高鹽逆境。而乾旱逆境不但對植物同時也對人類影響甚鉅，乾旱逆境會使植物脫水而受到傷害，為了抵抗乾旱逆境，植物在缺水時有相對應的生理反應：包括了氣孔的關閉、葉片面積的減少、以及根的延伸，這些生理反應使得植物體能夠減少水分的散失或是增加水分的獲取。而在分子層次，植物在乾旱逆境下亦有複雜的訊息傳導路徑。在這些路徑中，有一群DREB (DRE-binding protein)轉錄因子，受到乾旱的誘導時，經由DRE (drought-responsive element)來誘導下游的基因表現。DREB subfamily可再分成數群包含CBF (CRT-binding factor)/DREB1和DREB2。DREB2主要受到了乾旱的誘導。而CBF/DREB1則受到低溫逆境的誘導。而除了CBF1, 2和3，DREB1中的DREB1D (CBF4)在2002年被釣取出來，CBF4不受到低溫逆境的誘導，而是被乾旱逆境所誘導。雖然已知CBF4會受到乾旱的誘導且參與在與ABA有關的訊息傳導路徑，但是在CBF4的上游，還有哪些蛋白質調控其表現則尚未可知。為了研究CBF4的上游調控者，本實驗室根據1997年Ishitani等人篩選突變株的方法，首先建立CBF4 promoter::LUC轉殖株的T-DNA突變庫，並利用冷光強度的改變來篩選CBF4表現異常的轉殖株。另一方面，進行CBF4啟動子的片段分析探討CBF4啟動子上與乾旱逆境有關的cis-element，以期在未來進行酵母菌單雜合釣取CBF4上游的調控者。在TAIR網站的序列庫，並沒有標出精確的CBF4 5’ UTR序列，為了得到正確的CBF4啟動子範圍，首先進行5’-RACE。根據5’-RACE結果，CBF4 5’ UTR長度為84bp，依此設計引子，得到CBF4啟動子區域 (start codon上游1236bp)。將此片段構築於含有LUC報導基因之質體，轉入阿拉伯芥內得到CBF4 promoter::LUC轉殖株。之後以北方墨點法及RT-PCR檢測轉殖株在乾旱逆境下LUC的表現量，並進行冷光實驗檢測轉殖株在乾旱逆境後是否能發出冷光，結果顯示轉殖株在乾旱逆境下能表現LUC報導基因並發出冷光。利用南方墨點法挑選T-DNA單一插入的轉殖株，以作為未來研究的材料。在CBF4啟動子片段分析方面，先以PLACE生物資訊網站分析CBF4啟動子，並標出與乾旱逆境有關的cis-element。根據cis-element的分佈，以PCR方式得到不同長度的CBF4啟動子片段，構築於 GUS報導基因質體 (pKGWFS7)。並轉入阿拉伯芥內獲得轉殖株。另外也利用CBF4 promoter::GUS轉殖株觀察CBF4在乾旱逆境下，CBF4的器官表現位置，根據染色結果，在乾旱逆境下，CBF4會表現在cauline leaf。

Plants suffered from many abiotic stresses in environment, including cold, heat, drought, or high salt. Drought stress, one of the most influential stresses to plants, and also to human, causes plants damage by dehydration. To resist drought stress, some physiological responses are induced in plants, such as stomata closure, decreased leaf area, leaf abscission and extension of roots to deeper, moist soil. The purpose of these responses is to reduce water loss or to increase water uptake. At the molecular level, plants also exhibited a complex signaling transduction under drought stress. DREB (DRE-binding protein) subfamily contains genes of transcriptional factors which bind to DRE (drought responsive element) cis-element of downstream genes and to induce gene expression. DREB subfamily is consisting of DREB1/CBF (C-repeat binding factor) and DREB2. DREB2 was induced by water deficient, while DREB1 /CBF was induced by low temperature. DREB1D (CBF4) was cloned in 2002. However, CBF4 was not induced by cold, but by drought. Although CBF4 is induced by water deficiency and is involved in ABA-dependent signaling transduction pathway, the upstream regulators of CBF4 are still unclear. To study the upstream regulator of CBF4, we used mutant screening method according to Ishitani (1997). We established a CBF4 promoter::LUC mutant pool, and will screen mutants which exhibit deregulated CBF4 expression by altered luminescence. Also, we would like to perform CBF4 promoter deletion assay to study which fragment of CBF4 promoter is most relevant to the induction of CBF4 genes, so that the responsive element will be used in the yeast one-hybrid assay. CBF4 5’ UTR sequence is still unclear in TAIR sequence database. To obtain precise CBF4 promoter length, 5’-RACE was performed to obtain CBF4 5’ UTR sequence. It was shown that CBF4 5’ UTR region is 84bp upstream of ATG. We establish a CBF4 promoter::LUC construct and transformed Arabidopsis by Argobacterium infection. LUC expression could be detected in the CBF4 promoter::LUC transgenic plants after drought treatment by Northern blot and RT-PCR. CBF4 promoter::LUC plants can also exhibit luminescence under drought stress. Southern blot was also performed to pick the CBF4 promoter::LUC transgenic plant that was T-DNA single-inserted. According to the promoter analysis by PLACE website, we constructed different length of CBF4 promoter fragments and acquired CBF4 promoter::GUS transgenic plants which contain various length of promoter sequence. In GUS staining test, CBF4 was expressed in cauline leaf under drought stress.