| Lipoxygenase is a class of non-heme iron containing dioxygenase which catalyzes the hydroperoxidation of polyunsaturated fatty acids and its esters containing a 1,4-pentadiene structure into conjugated hydroperoxy fatty acids. Lipoxygenase is widely distributed in higher plants and it has been suggested that LOXs might be involved in plant growth, development, maturation, senescence, wound responses, or pest resistance, especially involved in hydroperoxide products and ethylene biosynthese during maturation and senescence. Three isozymes, namely LOXl, L0X2 and LOX3, are found in rice embryos. LOX-3 is the major component of isozymes in rice grain, accounting for 80-90% of total LOX activity in ungerminating mature seeds. In the present study, we obtained transgenic rice plants transformed with LOX3 cDNA and promotor. Furthermore, the functions of LOX3 gene and promotor were discussed by identifying and analyzing transgenic rice plants. The main results were as follows.1. Plant expression vectors containing sense and antisense of embryo LOX3 cDNA were constructed. The promoter of LOX3 gene was cloned by PCR strategy from Daw Dam, which lacks LOX3 in its seed, and Koshihikari, which has normal LOX3 activity, respectively. Then the LOX3 promoter- reporter fusion gene was constructed and introduced into rice by Agrobacterium-mediated transformation.2. Transgenic rice plants transformed with sense and antisense LOX3 cDNA and promotor were generated.3. The gene was introduced into the rice genome identified by PCR and Southern. Through the analysis of embryo LOX3 deletion and semi-quantitive RT-PCR, it was demonstrated that the expression of LOX3 gene was suppressed in transgenic plants with antisense gene.4. The expression of reporter gene could be all driven by the LOX3 promoter from both Koshihikari and Daw Dam, indicating that null-allele of LOX3 gene in Daw Dam did not resulted from its promoter activity. Reporter gene expression could be detected in roots, stem, leaf, anthers, seed capsules and embryos of transgenic plant, moreover detected in the embryo and plumule, but not in endosperm of transgenic germinating seeds. The activity of LOX3 promoter was significantly stronger than that of CaMV 35S promoter. Thus the LOX3 promoter may be a efficient and useful promoter.5. Two 5'-deletion of LOX3 promoter were used to detect the activity of important cis-element in promoter. The results indicated that the activity of these two 5'-deletion was significantly stronger than that of the whole promoter, suggesting that an unknown negative regulatory element would exist in the deletion regions. Furthermore, the activity between these two 5'-deletion was also markedly different, therefore an important regulatory element might be present in the distinct regions.6. Using transgenic T1 plants, the inducible expression properties of LOX-3 promoter was analyzed. The GUS activity driven LOX-3 promoter was enhanced in the leaf, stem and root of transgenic Tl plants especially after ABA treatment, then aging and NaCl treatment. These results indicated that LOX-3 promoter was inducible and the stress-induced elements played a role in the adversity stress.7. T2 transgenic plants with antisense LOX3 gene were treated with abiotic stress including salt and drought, and biotic stress such as rice blast, stripe and bacterial blight. Compared with non-transgenic plants, transgenic plants containing antisense LOX3 gene were susceptible to abiotic stress i.e. salt and drought, furthermore most sensitive to rice blast and more to bacterial blight among biotic stresses. Response of LOX3 promotor to adversity stress demonstrated that embryo LOX3 gene had some function in the response to adversity stress.
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