Cloning And Expression Of OSHPL1 Gene Encoding Rice Hydroperoxide Lyase

Fatty acid hydroperoxide lyase (HPL) is a novel CytP-450 enzyme that cleaves fatty acid hydroperoxides to form short-chain aldehydes and oxo-acids, and further to produce volatile flavor molecules and potential signal molecules. The aldehydes which formed by HPL are involved in plant resistant to disease and insect herbivores. They are also important constituents of the characteristic flavors of fruits, vegetables, and green leaves. Now the research on HPL focus on dicotyledon, and the research on HPL of monocotyledon still haven't been reported. In this paper, the research on cloning of rice HPL and enzyme activity assay were carried out and several result were obtained.1. Enzyme activity assay of rice HPLBased on the method of measure HPL Enzyme activity in other plants, the means on preparation of hydroperoxides and measurement rice HPL activity were set up. The 9-HPL arid 13-HPL activity of several rice varieties with different storability and different dormancy were tested. The results showed that:9-HPL has maximum activity in seeds in 2-3 days after germination, and then began to decrease. The tendency is the same as LOX activity in germination stage.9-HPL activity has no remarkable change in seeds between varieties during dormancy, but 13-HPL activity is positive correlativity with dormancy. After dormancy was broken, 13-HPL activity become no remarkable difference among varieties, but 9-HPL activity is positive correlativity with dormancy.2. Cloning of rice HPL geneUsing a polymerase chain reaction-based cloning strategy, we isolated a HPL gene from rice (OsHPLl) that is high homologous to a recently cloned HPL from barley. OsHPLl was the first HPL gene isolated from rice, it's CDS is 1461bp, encodes a putative55KD protein. The deduced protein sequence indicates that this gene encodes a cytochrome P-450.Furthermore, OsHPLl was cloned into an expression vector pET-30a and then expressed in Escherichia coli induced by IPTG. The recombinant enzyme showed to act on both 13-and 9-hydroperoxides, with a preference for the former. The result of southern blot indicated that OsHPLl has several copies in rice genomic. Expression profile analysis using RT-PCR indicated that OsHPLl was expressed higher in floral and embryo, lower in stem than other tissues. The 500bp upstream of first start codon was isolated, and be predicted as promoter by software. Sequence analysis of promoter showed that the tissue specific expression motif was found. Because there are answer motif in promoter sequence, OsHPLl maybe be regulated by SA, ABA and has relation with cold and drought stress.