Prokaryotic Expression, Purification and Characterization of a Novel Rice Seed Lipoxygenase Gene OsLOX1

Lipoxygenase （LOX, EC1.13.11.12） is a key enzyme during the degradation of lipids in animals and even plants, and also the first key enzyme responsible for the biosynthesis of jasmonate. To purify and characterize the OsLOX1 gene from rice seeds, the entire coding region of the OsLOX1 gene was inserted into an expression vector pET30a（＋） and transformed into Escherichia coil BL21 （DE3）. Expression of the fusion protein was successfully induced by isopropyl-β-D- thiogalactopyranoside （IPTG） and the purified recombinant protein was obtained by His.Bind Kits. Further assay showed that the purified recombinant protein exhibited the LOX activity. The optimum pH was 4.8 （acetate buffer） and the optimum temperature was 30℃ for the above enzyme. Thus, the recombinant might confer an available usage for the synthesis of jasmonate in vitro, and also provides a possibility for elucidating the inter-relationship between the primary structure of the plant seed lipoxygenase protein and its physiological functions.

Effects of Lipoxygenase Null Genes of Soybean in Controlling Beany-flavor of Soymilk and Soyflour

The flavor of the soymilk and soyflour obtained from the lipoxygenase mutant isolines was concentrated by simultaneous distillation and extraction (SDE), and its constituents were identified by gas chro-matography (GC) and gas chromatography-mass spectrometry. Results showed that the same 24 flavor constituents were isolated in both soymilk and soyflour, and most of them were aldehydes and alcohols. Lox2 was most responsible for the production of the volatile and beany-flavor components, and Lox1 less responsible. Lox3 was least responsible and can reduce the yield of hexanal. Either Lx1 or Lx2 could significantly reduce the volatile and beany-flavor, and Lx3 could significantly increase the yield of hexanal. Primary and secondary interactions existed among the null mutant genes, and the major effects and interactions could be affected by processing conditions. The isoline with triple lipoxygenase null genes yielded the least volatile and beany-flavor components, and the isoline without the lipoxygenase gene Lx3 produced the greatest amount of the volatile and beany-flavor components. The amounts of volatile and beany-flavor components produced by the other isolines were between that of the isoline with triple lipoxygenase null genes and the isoline without lipoxygenase gene Lx3. According to the correlation analysis, the hexanal amount could be used as an index in evaluating the importance of lipoxygenase isozymes in the yield of beany-flavor compounds, and the effects of the different types of lipoxygenase null mutants in controlling beany-flavor compounds. The cultivars with triple lipoxygenase null genes will be a quality raw material for soy food processing.

Role of LOX3 Gene in Alleviating Adverse Effects of Drought and Pathogens in Rice

Lipoxygenase 3 (LOX3) is a major component of the LOX isozymes in mature rice seeds. To investigate the role of LOX3 gene under stresses, a plant expression vector containing antisense cDNA of LOX3 was constructed. Rice varieties Wuyunjing 7 and Kasalath were transformed by the Agrobacterium-mediated method and transgenic rice plants were generated. PCR and Southern blot results showed that the antisense LOX3 gene was integrated into the rice genome. Analyses of embryo LOX3 deletion and semi-quantitative RT-PCR confirmed the antisense suppression of LOX3 gene in transgenic plants. The T2 antisense plants of LOX3 were sensitive to drought stress, rice blast and bacterial blight compared with non-transgenic plants. These results suggest that the LOX3 gene might function in response to stresses.

Effects of abiotic stress and hormones on the expressions of five13-CmLOXs and enzyme activity in oriental melon(Cucumis melo var.makuwa Makino)

Lipoxygenases（LOXs） are a group of non-heme,iron-containing enzymes and extensively involved in plant growth and development,ripening and senescence,stress responses,biosynthesis of regulatory molecules and defense reaction.In our previous study,18 LOXs in melon genome were screened and identified,and five 13-LOX genes（CmLOX08,CmLOX10,CmLOX12,CmLOX13 and CmLOX18） were predicted to locate in chloroplast.Phylogenetic analysis result showed that the five genes have high homology with jasmonic acid（JA） biosynthesis-related LOXs from other plants.In addition,promoter analysis revealed that motifs of the five genes participate in gene expression regulated by hormones and stresses.Therefore,we analyzed the expressions of the five genes and LOX activity in leaves of four-leaf stage seedlings of oriental melon cultivar Yumeiren under abiotic stress：wounding,cold,high temperature and hydrogen peroxide（H_2O_2）,and signal molecule treatments：methyl jasmonate（MeJA）,abscisic acid（ABA） and salicylic acid（SA）.Real time qPCR revealed that wounding and H_2O_2 induced the expressions of all the five genes.Only CmLOX08 was induced by cold while only CmLOX13 was suppressed by high temperature.ABA induced the expressions of CmLOXIO and CmLOX12 while inhibited CmLOX13 and CmLOX18.MeJA increased the 3 genes expressions except CmLOX08 and CmLOX13,whereas SA decreased the effect,apart from CmLOX12.All the abiotic stresses and signal molecules treatments increased the LOX activity in leaves of oriental melon.In summary,the results suggest that the five genes have diverse functions in abiotic stress and hormone responses,and might participate in defense response.The data generated in this study will be helpful in subcellular localization and transgenic experiment to understand their precise roles in plant defense response.