34. Cloning and expression of OsHPL1 gene encoding rice hydroperoxide lyase

National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University; Jiangsu Research Center of Plant Gene Engineering, Nanjing 210095, China
* corresponding author: Email: wanjm@mail.njau.edu.cn, Tel: +86-25-84396516, Fax: +86-25-84396516

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.


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