Abstract:

MGG-01005 is an important gene associated with mycelia growth of Magnaporthe oryzae.In this study, a series of bioinformatics analysis including general physical and chemical properties, structural domains, and functional site prediction were carried out to construct the prokaryotic expression vector PETM13-MGG-01005. IPTG was used to induce the expression of recombinant protein, and the protein was purified by nickel ion affinity chromatography, anion exchange chromatography and molecular sieve chromatography. The results showed that the molecular weight of the protein was about 16 471.49 u, which encoded 153 amino acids. It contained the domain of Tctex-1, had no transmembrane structure and signal peptide, had no functional site, and had the active site of phosphoric acid, and was an unstable hydrophilic protein. The protein could be induced to express by 0.1 mmol·L ^-1 IPTG. The optimal eluent of affinity chromatography was divided into 20 mmol·L ^-1 Tris-HCl, 500 mmol·L ^-1 NaCl, and 80 mmol·L ^-1 imidazole. Anion exchange chromatography showed that the protein was resistant to low salt conditions. The molecular sieve chromatography has the conformation of uniform morphology and good symmetry, and the molecular weight of the protein corresponding to the maximum elution peak is about 35 ku, indicating that the protein exists in the form of dimer. In this study, a large amount of high purity protein was obtained in order to lay a theoretical and practical foundation for further exploration of the function of this protein and subsequent related studies on Magnaporthe oryzae.

Key words: Magnaporthe oryzae, prokaryotic expression, purification, bioinformatics analysis