Gene Cloning, Function Analysis Of Heme Oxygenase 1 (OsHO1) In Oryza Sativa And The Biological Function Of Its Substrate Hemin And Hematin

Heme oxygenase (HO, EC 1.14.99.3) catalyzes the rate-limiting step of conversion from heme to biliverdin (BV), carbon monoxide (CO) and ferrous iron (Fe2+). Ample studies showed that HOs might be a key factor of the signaling system in animals. In recent studies, plant HOs were confirmed to be involved in stomatal closure, inducing lateral and adventitious root formation, and counteracting oxidative damage as well as participating in the phytochrome synthesis and photoperiods responses.In the present study, a series of studies based on the materials of rice (Oryza sativa L.) seedlings have been conducted in the gene isolation, sequence analysis, and expression analysis. According to the SE5 gene sequence reported previously, degenerate primers were designed to amplify coding regions (CDS) of the rice heme oxygenase (OsHO1) gene. The length of its open reading frme (ORF) is 870 bp, and encodes a peptide containing 289 amino acids. The multi-alignment analysis revealed that OsHO1 contains histidine heme ligand and HO signature sequence which is the similar to the other plant HOs. It is predicted that the cleavage of the transit peptide is 64 amino acids. With the construction of the gene of mature protein regions with prokaryotic expression vector pET-28a (+), it was succeded to express the mOsHO1 fusion with the 6×His. The soluble protein fraction was applied to a Ni-affinity chromatography column and the fusion protein was isolated according to the manufacturer’s protocol. With the detection of the HO activity of fusion protein, the results showed that the Vmax value for the complete reaction was estimated as 63.3 nmol BV Ixa h"1 mg protein-1 with a Km for heme of 2.9μM. The optimum pH value of the fusion protein is 7.0. Temperature dependence of the HO reaction was measured between 10℃and 45℃, and the HO enzyme activity increases with the rising temperature. Besides, construction of the OsHO1 transite peptite with GFP protein showed the localization of OsHO1 is in chloroplast at least. Expression analysis showed that SE5 gene can be detected in all of the organs examined. However, the relative levels in each organ varied. For example, the relative expression of OsHO1 gene exhibited higher level in sterms, leaves and germinating seeds than that in roots and embryos. The similar results were also proven by western blotting using the anti-mOsHO1 antibody. When 2-week-old rice seedlings were treated with the abiotic stress such as NaCl and PEG treatment, the OsHO1 gene was induced both in roots and leaves. These results suggested that OsHO1 might be induced by abiotic stress and play a role in oxidative stress.In a subsequent experiment, we inserted SE5 gene into the binary vectory in the sense. and antisense orientations (named pVec8_Ubi-RNAi-HO1) on the principle of RNA interference (RNAi). Meanwhile, we also constructed the SE5 gene overexpression vector pCAMBIA1302-SE5 and pVec8_Ubi-SE5. Using the optimized agrobacterium-mediated transformation procedure, the overexpression vector was transformed into rice and Arabidopsis. The results of PCR showed that the targeted gene had been intergrated into the rice and Arabidopsis genome. The inhibtion of seed germination and oxidative damage triggered by paraquat were alleviated in the SE5 overexpression transgenic Arabidopsis when compared with those of the wild-type Arabidopsis.It is found that both hemin and hematin, the group substituent of heme, could counteract the inhibition of seed germination (such as germination rate, germination energy, and germination index) caused by salinity or osmotic stress with the pre-incubation and co-treatment. Furthermore, the effects may be contributed to the improvement of the amylase activity, the content of reducing sugar and soluble sugar. When adding CO scanvenger Hb or the potent inhibitor of HO ZnPP (zinc protoporphyrin), the effects of hemin and hematin were reversed, respectviely. It thus suggested that hemin and hematin induce the HO expression to produce CO, then improving the seed germination. It was further noticed that, pretreating wheat seeds with MB (methylene blue), an effective scavenger of NO (nitric oxide), could reverse the decrease of TBARS (thiobarbituric acid reactive substances) contents induced by hemin or SNP (sodium nitroprusside). Together, all of these suggested that endogenous NO might play a key role in hemin-or hematin-mediated cytoprotective responses.