Isolation And Characterization Of Rice OsLOX1 Gene

In developing rice seeds, there are three distinct lipoxygenase, LOX-1, LOX-2 and LOX-3. For the complicated and important physiological role, lipoxygenase was extensively studied and paid attention to, in recently. However, the physiological role of plant LOXs is still unclear so far, especially in the case of rice LOX-1.In this report, we cloned a novel rice lipoxygenase gene by amplification of RT-PCR and approach of RACE, identified the biochemical characterization of the OsLOX1, and analyzed the structures of the gene. The results showed that the OsLOX1 may correspond to the rice LOX-1. To elucidate the relation between resistance to insect and the physiology role of the OsLOX1, we produced the constructs of sense- and antisense-OsLOX1, then transformed to the rice plants by Agrobacterium-mediated method.The results were as followings:(1) Lipoxygenase activity in maturing and germinating rice seeds. Using the mutant rice cultivar BeiLu PL2 (lack LOX-3) characterized by our laboratory, and Koshihikari (normal LOX-3 activity), we studied LOX content and activity in maturing and germinating rice seeds with and without LOX-3. In maturing seeds, the LOX-3 activity was distinctly higher than the activity of other two isozymes. Whereas, the LOX-2 activity was the main activity of LOX in new germinating seeds. But, the LOX-1 activity was very low in two development stages.(2) A novel and simple CTAB-LiCl-based extraction method for high-quality and total RNA of rice embryo samples is developed. This method can efficiently eliminate the interference of polysaccharide and lipids rich in rice (Oryza sativa L.) embryo obtained under room temperature without using liquid nitrogen. The results of ultraviolet spectrophotometer and agarose gel electrophoresis analysis show that the obtained RNA has no obvious degradation and a good purity sufficient for further RT-PCR and RNA gel blotting. Therefore ,it is also especially useful for the RNA extraction of plant material plenty of polysaccharide and lipids.(3) The full length cDNA of a novel dual position specific LOX was isolated from developing rice seeds, designed as OsLOX1 (GenBank accession number DQ389164). A homology search against the rice nucleotide database revealed the OsLOX1 gene to be on rice chromosome 3. The restriction enzyme map of the reported genomic sequence agreed with the result of the Southern blot analysis for the OsLOX1. The OsLOX1 transcripts were detected in immature seeds and germinated seedling with low abundance. In contrast, OsLOX1 mRNA accumulated rapidly and transiently in response to wounding or nitric oxide, and reached a peak of expression after 3 h of wounding or nitric oxide treatment. Taken together, these results suggested that the main physiological role of OsLOX1 may be involved in the defense system of rice plants.(4) In the previous work, a novel lipoxygenase gene from rice, named as OsLOX1, was isolated. Moreover, the OsLOX1 may have a potential dual positional-specific lipoxygenase activity, when the phylogenetic analysis of plant LOXs was performed. Here, the expression, purification and characterize of OsLOX1 were reported. The entire coding region of the OsLOX1 gene was inserted into an expression vector, pET30a(+) and transformed into Escherichia coli BL21 (DE3). The fusion protein was successfully expressed induced by IPTG and the purified recombinant protein was obtained by His·Bind(?) Kits. Further assay shows that the purified recombinant protein exhibited the LOX activity. The pHopt and Topt of above enzyme were pH4.8 (acetate buffer) and 30℃, respectively. Thus, this recombinants might confer an available usage for the synthesis of JA in vitro, and also provide a possibility for elucidating the inter-relationship between primary structure of the plant seed lipoxygenase protein and its physiological functions. Taken together, our data showed that the OsLOX1 may correspond to the rice LOX-1.(5) By Agrobacterium-mediated method, two binary vectors pLOX1S (35SPro.5'+OsLOX1 cDNA+NosTer3') and pLOX1A(35SPro.5'+ antisene-OsLOX1 cDNA+NosTer3') were transferred into japonica rice cvs. Beilu PL2. Total 46 independent transformed plants were got on regeneration medium. PCR analysis and Hygromycin selection revealed that 32 plants were positive and the transform frequency is 69.6%. Southern blot further confirmed the integration of foreign gene. Semi-quantative RT-PCR analysis and the LOX activity identification also confirmed the transcripts of the OsLOX1 gene in transgenic rice plants. Insect bioassays showed that some transgenic rice plants (some sense vectors) significantly increased their resistance to the BPH (brown planthopper) attack, and the others transgenic rice plants (the others sense vectors and all the antisense vectors) decreased their resistance to the BPH attack.