Gene Cloning Of Rice OsHO2and Preliminary Study On Enhanced Tolerance Of Transgenic Arabidopsis Plants Overexpressing SE5(OsHO1) To Herbicide Paraquat

Heme oxygenase (HO, EC1.14.99.3) catalyzes the oxygenation of heme to biliverdin, carbon monoxide (CO) and Fe2+. Recently, increasing evidences have shown that, in addition to its role in plant growth and development such as involved in phytochrome synthesis and lateral adventitious root formation processes, HO plays an important role in multiple stress responses, including NaCl stress and paraquat exposure. However, the OsHO2gene has not been identified, and the function of OsHO1gene in stress response needs to be further studied.In present study, rice (Oryza sativa) OsHO2gene was isolated. It was found that OsHO2contained four exons and three introns within genomic DNA sequence and encoded a polypeptide with331amino acids. The OsHO2transit peptide encoded a protein of47amino acids. OsHO2had a conserved HO signature sequence and showed a high similarity to HOs in other plants. However, OsHO2had no conserved His site to bind heme, and couldn’t catalyze it to BV, CO and Fe2+. With the construction of the gene of mature protein regions with prokaryotic expression vector pET-28a (+), the mOsHO2was successfully expressed. The soluble protein fraction was applied to a Ni-affinity chromatography column and the fused protein was isolated according to the manufacturer’s protocol. The results of the HO activity analysis showed that the fused protein had no HO activity, indicating that OsHO2may not be a real heme oxygenase. Moreover, construction of the OsHO2transit peptide with GFP protein showed the localization of OsHO2was in the cytoplasm membrane. Results from RT-PCR analysis revealed that the transcripts of OsHO2in leaves ranked the highest, followed by stems, germinating seeds and roots. Additionally, the transcripts of OsHO2could be significantly induced by some pro-oxidant compounds, including paraquat and NaCl, indicating its role in abiotic stress responses.In addition, we identified the protective role of SE5(OsHO1) against paraquat-induced oxidative damage by using transgenic Arabidopsis plants overexpressing OsHO1(35S:OsHO1). Compared with wild-type,35S:OsHO1displayed enhanced tolerance to paraquat stress as evidenced by improved seed germination rate, fresh weight and chlorophyll content. Moreover, lower level of reactive oxygen species (ROS) was observed in35S:OsHO1compared with wild-type in the presence of paraquat. Results from real-time PCR and enzyme activity analysis demonstrated that35S:OsHO1alleviated paraquat-induced oxidative damage via increasing activities and transcript levels of antioxidative enzymes including catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). Moreover, the oxidative damage induced by paraquat could be rescued by hemin and CO, but aggraviated together with ZnPP. Together, our results showed that OsHO1played a role in improving plant tolerance to paraquat stress.