孔雀草抗壞血酸過氧化酶基因之選殖與功能分析

Abstract

孔雀草(Tagetes patula)是一種常見的景觀花卉，已被證實為一種鎘的累積植物。我們過去的研究發現鎘會誘導孔雀草抗壞血酸過氧化酶(Ascorbate peroxidase, APX)活性增加APX是重要的抗氧化酵素，可分解H2O2，為了解APX對孔雀草鎘耐受性扮演的角色，本試驗進行孔雀草APX基因的選殖與功能分析。試驗中利用RACE-PCR方法，從孔雀草中選殖出一個由251個胺基酸組成的APX全長cDNA，命名為TpAPX1。該基因具有8個外顯子與7個內顯子，演化樹分析顯示其與草莓的APX親緣性最接近。TpAPX1在花有較高表現，老葉最少，受CdCl2 誘導，但受到銅與三價砷的抑制。功能分析部分，在大腸桿菌大量表現TpAPX1可表達出具有APX活性蛋白。次細胞定位分析顯示TpAPX1表現在細胞質。大量表現TpAPX1在阿拉伯芥中，可使轉殖阿拉伯芥在100μM CdCl2 與0.1 μM Methyl Viologen中，具有比一般植株(WT)較高的逆境抗性，顯示TpAPX1具有幫助植物抵抗氧化逆境的功能。

Tagetes patula, a common landscape plant, has been identified to be a cadmium (Cd)-accumulator. According to our previous study, the activity of ascorbate peroxi-dase (APX) in T. patula can be induced by CdCl2 treatment. APX that can detoxify H2O2 using ascorbate as a substrate plays a key role in protect on plants from oxidative stress. To evaluate the physiological role of APX in T. patula under CdCl2 treatment, rapid amplification of cDNA ends (RACE) method was used to clone the APX gene in T. patula. The full-length cDNA of TpAPX1 contains a 753 bp open reading frame which encodes 251 amino acids with an estimated molecular weight of 25 kDa. The genomic structure of TpAPX1 has 8 exons and 7 introns. Phylogenetic analysis revealed TpAPX1 has high similarity with Fragaria x ananassa APX. TpAPX1 was expressed in all tissues of TpAPX1 , and the highest and lowest mRNA levels were in flowers and roots, respectively. TpAPX1 mRNA level was induced by CdCl2 but repressed by CuSO4 and As2O3. The expression of TpAPX1 in E. coli validated that it can be translated as a protein with APX activity. PEG-mediated transient expression with a TpAPX1-GFP fusion construct in Arabidopsis protoplast shwowed that TpAPX1 localizes in cytosol. Ectopic expression of TpAPX1 in Arabidopsis increased Methyl Viologen and Cd2+ stress tolerance. In conclusion, cytosolic TpAPX1 plays an important role of oxidate stress tolerance in plant.