Preliminary Study Of Arabidopsis DOX1 And DOX2

Plants maintain their physiological activity by using escape ,avoidance and tolerance under different kinds of biotic stress and abiotic stress , which involve lots of physiological and biochemical processes. The oxidation of polyunsaturated fatty acids plays an important role in the regulation of gene expression under stress. There are three approaches in fatty acid oxidation, includingα-,β-andω-oxidation. And fatty acidα-dioxygenase not only catalyzed the initial reaction inα-oxidation of long-chain fatty acid ,but also participated in the plant's anti-bacterial process.There are two copies of Dox were obtained in Arabidopsis, Dox1 and Dox2. In order to elucidate the biochemical function and their roles in the process of disease resistance, the following studies were carried out:1.The CDS of Dox1 was amplified by RT-PCR ,and subcloned into expression vector pMAL-c4x. The recombinant protein was expressed in BL21 (DE3)RIPL codon + strain and purified using amylose resin column. The results of SDS-PAGE indicate: The fusion protein mainly exsited as soluble protein, and its yield accounted for 3.7% of the total bacterial proteins. The purity of recombinant protein was about 45% . The peroxidase activity of soluble MBP-fused DOX1 wasn't detected by the guaiacol method. The alpha-dioxygenase activity of the purified protein was tested using 2,4-DNP, result showed that the soluble fusion protein had detectable alpha-dioxygenase activity.2.GST fusion DOX1 was expreassed in BL21 (DE3)-RIPL codon+ Strain too, but it mainly existed in inclusion body. So we choose tapping to purified the recombinant protein.And finally we obtained the polyclonal antibody by immunized mouse. The result of western blot showed that the preparation og DOX1 antibody is succeed.3.The function of one protein was related to its sub-cellular location. In order to study the sub-cellular location of DOX, we constructed plant expression vector pBI–DOX-GFP. The T0 seed of transgenic plants were obtained by floral-dip.4.The phenotype of T-DNA inserted mutation lines of Dox1 and Dox2 was observed. The mutation lines of Dox1 are more sensitive to environmental stimulus than Dox2. And the mutation of Dox2 can dramatically affect the growth and development of Arabidopsis thaliana. The study laid a foundation for the subcellular localization of the DOX1 and DOX2 and their action pattern in the process of plant development and anti-stress.