Expression, Purification Of Arabidopsis Fatty Acid Alpha DOX1 And DOX2, And Alternative Splicing Of DOX1

Many plant molecular studies have focused on resistance related genes. Fatty acid Alpha dioxygenases are very important genes related to pathogen resistance, and they have been found and cloned from many different species. Alpha-Dioxygenases constitute a family of fatty acid-metabolizing enzymes recently discovered in plants. Lipid derived signal molecules, which involve in plant resistance and developmental regulation, can be generated during fatty acid alpha oxidation. There are two copies of DOX in Arabidopsis, which are named DOX1 and DOX2, respectively. In order to characterize their biochemical functions and expression patterns, studies have been made as follows:Bioinformatic analysis of Arabidopsis DOX1 and DOX2. 78 rare codons of E.coli were found in the DOX1 ORF using Rare Codon Caltor software, which accounts for 12% of the DOX1 total codons; and 88 in the DOX2, which accounts for 14% of the DOX2 total codons. The hydropathy profile of DOX1 and DOX2 indicate both proteins may have transmembrane regions.DOX1 and DOX2 were obtained and expression vectors were constructed. First, the DOX1 was cloned by RT-PCR, and DOX2 was obtained from ABRC. The encoding sequences of Arabidopsis DOX1 and DOX2 were inserted into pGEX-5X-1 to obtain pGEX-DOX1 and pGEX-DOX2, respectively.The DOX1 and DOX2 were expressed in E.coli, and the peroxidase activity of purified proteins were tested. The recombinant vectors were transformed into E.coli BL21 (DE3)-RIPL codon+, respectively. Fusion proteins about 98kD were detected by SDS-PAGE and Western blot in the induced recombinant BL21 (DE3)-RIPL codon+ strain. The target protein DOX2 was purified by the GST chromatography column, and the peroxidase activity of the purified protein was tested using the guaiacol method, the result shows the soluble fusion protein has no detectable peroxidase activity.An alternative splicing form of DOX1 has been found. We got two forms of DOX1 after SA treatment, one form is the same as the published sequence; the other has a retained intron. The new transcript generates a truncated target protein. The biological function of the alternative splicing is unknown yet, which needs further research.It will be helpful to further research the function of Arabidopsis alpha DOX .