Cloning and Expression Analysis of Ascorbate Peroxidase Gene from Eggplant under Flooding Stress

茄子淹水逆境下抗壞血酸過氧化酶基因之選殖與表現分析

林冠宏(Kuan-Hung Lin) ； 羅筱鳳(Hsiao-Feng Lo) ； 林俊宏(Chun-Hung Lin) ； 詹明才(Ming-Tsir Chan)

《Botanical Studies》 48卷1期 (2007/01) Pp. 25-34

摘要

我們先前發現淹水造成茄子根部抗壞血酸過氧化酶的活性增加，本研究目的為選殖抗壞血酸過氧化酶基因，並測定淹水逆境下茄子不同組織抗壞血酸過氧化酶基因表現之調節情形。即時聚合鏈反應結果發現，茄子不同組織的表現圖譜顯示其變異性廣，其中以EG117淹水72小時之抗壞血酸過氧化酶轉錄子最多，每種組織與遺傳型之差異性表現直接與淹水逆境反應有關。在NCBI資料庫篩選並比較抗壞血酸過氧化酶的基因序列，根據番茄與馬鈴薯設計消退引子組(degenerate primer sets)，利用逆轉錄即時聚合鏈反應方法擴增茄子抗壞血酸過氧化酶的cDNA，並以5´與3´快速擴增cDNA端技術完成全長之抗壞血酸過氧化酶cDNA，cDNA系的開放編閱架構(open reading frame)長度為753鹼基對，譯碼出細胞液之抗壞血酸過氧化酶，茄子抗壞血酸過氧化酶的序列與馬鈴薯、番茄、番椒與菸草的抗壞血酸過氧化酶序列分別有96%、95%、93%與91%的相似度；以Neighbor-Joining法作導譯胺基酸序列(deduced amino acid sequence)之演化分析，植物的cAPXs分為兩主群，茄子cDNA與馬鈴薯的親緣關係比番茄更近，南方墨點分析顯示茄子譯碼抗壞血酸過氧化酶的基因有兩套。這些結果顯示茄子的cAPX可能與過氧化氫解毒有關，因此幫助克服淹水誘導之逆境。

關鍵字

抗壞血酸過氧化酶 ； 即時聚合鏈反應 ； 淹水 ； 茄子 ； 演化

並列摘要

Previously, we found that flooding leads to an increase in APX enzymatic activity in the roots of eggplants. The objectives of this work were to clone the ascorbate peroxidase (APX) gene, and measure the regulation of APX gene expression in different tissues of eggplant under flooding stress conditions. Different tissues from eggplants displayed wide variations in their expression profiles using Real-Time PCR. The highest level of APX transcripts were detected in roots of EG117 at 72 h of flooding treatment. The differential expressions of each tissue and genotype were directly associated with flooding stress responses. After screening and comparing APX gene sequences at the NCBI database, the degenerate primer sets designed from tomato and potato were used to amplify the APX cDNA of eggplant with the reversetranscription PCR method. The completion of a full-length of APX cDNA was performed using 5' and 3' rapid amplification of cDNA ends (RACE) technique. The open reading frame of cDNA clone was 753 base pair long encoding a cytosolic APX (cAPX). The sequence of eggplant APX gene had 96%, 95%, 93% and 91% homology to that from the potato, tomato, pepper and tobacco APX gene, respectively. A phylogenetic analysis of the deduced amino acid sequence of APX by Neighbor-Joining method indicated that the plant cAPXs diverge into two major clusters, and eggplant cDNA is more closely related to potato than to tomato. Southern blot analysis revealed that the eggplant gene encoding APX had two copies. These results indicate that the cAPX of eggplant may be involved in hydrogen peroxide-detoxification and thus helps overcome the stress induced by flooding.

並列關鍵字

Ascorbate peroxidase ； Eggplant ； Flooding ； Phylogenesis ； Real-Time PCR