Fatty acid hydroperoxide lyase (HPL) is a cytochrome P-450 enzyme that
cleaves fatty acid hydroperoxides to form short-chain aldehydes and oxo-acids,
and further to produce vola-tile flavor molecules and potential signal
molecules (Noordermeer, 2000). The aldehydes which formed by HPL are involved
in wound healing and pest (Bate, 1998). They are also important constituents
of the characteristic flavors of fruits, vegetables, and green leaves
and are widely used as food additives (Whitehead, 1995). In this paper,
rice HPL gene was cloned and then expressed in E. coli,
further research will be done in future.
Using a recently cloned barley HPL (GenBank accession number: AJ318870)
as probe, a contig which was highly homologous to barley HPL was
found from China rice genome database(http://btn.genomics.org.cn/rice/).
Based on a polymerase chain reaction cloning strategy, a novel rice HPL
gene was isolated and named OsHPL1 (GenBank accession number: AY340220).
OsHPL1 was the first HPL gene isolated from rice(Oryza
sativa L), and it's open reading frame is 1461bp, encode a 487-amino
acid polypeptide with a predicated size of 55 KD. The putative protein
has a conserved motif LP x R x IPGSYG x P x GP characteristic of CYP74
subfamily of P450 protein, and also conserved A,B,C and D domains for
many cytochrome P450 families. The derived amino acid sequence of OsHPL1
has an identify of 51-72% with the HPL from barley, banana and alfalfa.
These results ensured that OsHPL1 encodes hydroperoxide lyase.
Furthermore, OsHPL1 entire open reading frame was subcloned into
an expression vector pET-30a(+)(Novagen) and then expressed in E. coli
strain BL21(DE3) with IPTG inducer in 28C. Analysis of the protein from
transformed cells by SDS-PAGE showed a detectable supplementary band with
the expected molecular weight (Fig. 1). The active protein was present
in both the soluble and the solubilized membrane fraction in the E.
coli cell. The fusion protein showed to act on both13-and 9-hydroperoxides,
with a preference for the former. Genomic DNA isolated from mature leaves
were digested with Dra I ,BamH I, EcoROuand EcoR V
respectively and transformed to Hybound-N+ membrane. The hybridization
was carried out with a probe made from coding region of OsHPL1,
and signal detection was performed by ECLTM kit(Amersham) with
middle stringency(The member was washed in 2 x SSC twice for 15 min).
The result displayed that OsHPL1 has several copies in rice genome
(Fig. 2). When
used the 5'-UTR of OsHPL1 as probe, the same result was obtained.
In addition, the rice genomic database was searched with the OsHPL1
as a query. The BLAST result indicated that there have several contigs
partly homologous with OsHPL1. Therefore, It can't exclude the
possibility that OsHPL1 belongs to a muti-gene family.
Total RNA was also isolated from rice various tissues, and the first-strand
cDNA were synthesized with RNA LA PCR kit (TaKaRa). Using first-strand
cDNA as the templates and Actin as control gene, result of RT-PCR
demonstrated that OsHPL1 was expressed higher in floral and embryo,
while lower in stem and seedling (Fig. 3).
The 800bp upstream of first start codon of OsHPL1 was isolated
by PCR method, and predicted as promotor by software(Promotor Prediction).
Sequence analysis showed that there were answer motif in promoter sequence,
OsHPL1 maybe be regulated by SA. ABA and has relation with cold
and drought stress.
Bate, N.J., S. Sivasankar and C. Moxon, 1998. Molecular characterization
of an Arabidosis gene encoding hydroperoxide lyase, a cytochrome
P450 that is wound inducible. Plant Physiol, 117: 1393-1400.
Cass, B.J., F. Schade and C.W. Robinson, 2000. Production of tomato flavor
volatiles from a crude enzyme preparation using a hollow-fiber reactor.
Biotechnol Bioengineer, 67(3): 372-377.
Noordermeer, M.A., A.J.H.V. Dijken and S.C.M. Smeekens, 2000. Characterization
of three cloned and expressed 13-hydroperoxide lyase isoenzymes from alfalfa
with unusual N-terminal sequences and different enzyme kinetics. FEBS
Lett, 267: 2473-2482.
Noordermeer, M.A., W.V.D. Goot and A.J.V. Kooij, 2002. Development of
a biocatalytic process for the production of C6-aldehydes from vegetable
oils by soybean lipoxygenase and recombinant hydroperoxide lyase. J Agricul
Food Chem, 50: 4270-4274.
Whitehead, I.M., B.L. Muller and C. Dean, 1995. Industrial use of soybean
lipoxygenase for the production of natural green note-favor compounds.
Cereal Foods World, 40: 193-197.