Study On Fatty Acid Alpha-DOX Gene In Arabidopsis Thaliana,Triticum Aestirum And Zea Mays

The plants in nature are often subjected to various natural hazards, such as freezing, drought and salinity, etc. In order to reduce these disaster, plants must be made to adapt to the environment, its body structure and biochemical function will change. The molecular mechanisms also be different. Reseach data shows that the plants dioxygenase is involved in the oxidation process and related to plant stress response. Our group have obtained two genes of fatty acid alpha-DOX from Arabidopsis, Alpha-dioxygenase1(AtDOX1) and Alpha-dioxygenase2(AtDOX2) , and expressed and purifed them using E.coli system. Furthermore, based on EST assembling we cloned the gene of Triticum aestirum DOX (TaDOX). To further clarify the biological function of DOX , this paper carried out the following studies:1. The encoding sequence of Arabidopsis AtDOX2 was cloned into pPIC9k to obtain pPIC9k-AtDOX2. The recombinant vector was linearized and electroporated into Pichia pastoris strain GS115. After G418 selection, PCR analysis and optimization of methanol inducing time,the high level expression strain of pPIC9k-AtDOX2/GS115 was obtained.2. The recombinant AtDOX2 was expressed by methanol inducing.SDS-PAGE analysis revealed that, the expression level of recombinant protein reach to top at 96 hours after inducing by 0.5% methanol and the target protein accounts for 15% of the extracellular total protein. We purified the recombinant protein by His-tag column, and the purity of recombinant protein was more than 60%. The peroxidase activity of AtDOX2 was tested by the ABTS assay, the result showed that the recombinant protein has peroxidase activity, and the peroxidase activity of AtDOX2 can be activated by Ca²⁺and Mg²⁺ and inhibited by EDTA, imidazole and Mn²⁺. Theα-dioxygenase activity was tested by 2, 4-DNP assay, the result showed that the recombinant protein has dioxygenase activity, and the dioxygenase activity of AtDOX2 can be activated by Ca²⁺ too.3. We analysed the expression pattems of TaDOX in leaves and roots under stress of 250mmol/L NaCl and 16%PEG8000 by real-time quantitative PCR.The results showed that in leaves the expression level of TaDOX had significantly decreasing both subjected to PEG and NaCl stress; in roots the expression had not remarkably change under PEG stress and had a increasing under salt stress.4. The ZmDOX in Zea mays was cloned and analysed with Bioinformatics methods. The hydrophobicity anlysis result shows there has not significant tran-membrane signal region. There is 92% similarity between Zea mays and Oryza Sativa, and has 91% between Zea mays and Triticum aestivum.The similarity reduces to 62% and 59% compare with AtDOX1 an AtDOX2 of Arabidopsis thaliana, respectively. Monocotyledon plant only has one DOX, and all monocotyledon DOX belong to the same phylogenetic clan.The study laid a foundation for clarify the biochemical function of the DOX in plants.