Expression Analysis And Characterization Of Ostryopsis Davidiana NADP-dependent Isocitrate Dehydrogenase In Ecotomycorrhiza

Ostryopsis davidiana is pioneer shrub species in extremely arid degraded mountain ecology system. There is an important relationship with mycorrhizal fungi. The formation of ectomycorrhiza can not only change gene expression of plants and fungi, but also produce abnormal function of proteins and promote the metabolism of each other. In this study, O. davidiana mycorrhizal seedlings were obtained using the tissue culture mycorrhizal synthesis techniques. NADP-isocitrate dehydrogenase(IDH) was extracted from the mycorrhizal tissue, root tissue and fungus respectively. The enzymes characteristics of three sources were studied. The total RNA from three materials was extracted. NADP-IDH gene expression was compared by semi-quantitative PCR during mycorrhizal synthesis of a host plant roots.The main results were as follows:1. After inoculated three strains of Cg (CgO5, CgSO1, CgSO11) in the tissue culture bottles of O. davidiana for six months, the results showed that the CgO5is the best mycorrhizal fungi strain of O. davidiana.2. The three sources of IDHs were purified by ammonium sulfate precipitation and glucan gel chromatography and tested by SDS-PAGE electrophoresis. The IDHs subunit relative molecular mass of mycorrhizal tissue, root tissue and fungus were about46kD. Specific activity for IDH of mycorrhizal tissue, root tissue and fungus were121.12U/mg,115.24U/mg and102.29U/mg, respectively. The optimal pH values for IDH of mycorrhizal tissue, root tissue and fungus were pH8.0, pH7.5and pH8.5, and their optimal temperatures were40℃,50℃and45℃, respectively. The activity of three IDHs was entirely dependent on the binding of a divalent metal ion. The maximum activity of IDHs was observed in the presence of Mn2+or Mg2+. The activity of IDHs was dramatically inhibited by other metal ions, such as Ca2+, Zn2+and Ni2+. The Km values for isocitrate of mycorrhizal tissue, root tissue and fungus were3.12μM,26.31μM and50μM, respectively. The Km values for NADP+were20.83μM,52.63μM and27.7μM, respectively.3. The total RNA was extracted from the mycorrhizal tissue, root tissue and fungus by CTAB method respectively. The A260/A280value was1.8-2.0. The concentration was about0.5ug/μ1. It shows that there is no DNA interference in total RNA.1%agarose gel electrophoresis shows that the total RNA was complete. By the Semi-quantitative PCR test, the results showed the NADP-IDH was expressed in mycorrhizal tissue, root tissue and fungus, and the expression quantity were significant difference, the highest amount of expression in the mycorrhizal tissue.