Cloning and Expression Analysis of FvALT from Fusarium verticillioides

doi:10.13560/j.cnki.biotech.bull.1985.2024-0432

Abstract

Abstract:

【Objective】 Fusarium verticillioides is the main pathogen of maize tassel rot, thus exploring the biological function of FvALT will provide a theoretical basis for the development of novel targeted fungicides. 【Method】Physiological and biochemical characteristics and homology of FvALT were analyzed using online tools such as ProtParam, ProtScale, WoLF PSORT and Clustal X. The protein structure was obtained using SOMPA and SWISS-MODEL, and the binding ability of FvALT to substrates was analyzed using AutoDock. The target proteins were then obtained by prokaryotic expression and affinity nickel column chromatography, and the binding ability of FvALT to the substrate was verified by fluorescence titration technique.【Result】FvALT is a hydrophilic protein and localized in the cytoplasm, its protein secondary structure is dominated by α-helix and irregular coiling, and is highly conserved in Fusarium, with a typical PTZ00377 superfamily structural domain. The optimal conditions for the induction of FvALT are 0.4 mol/L IPTG, 16℃ for 16 h. FvALT has strong binding effects with α-ketoglutarate, and the binding sites are ARG³¹⁰, SER¹⁴⁹, SER²⁹⁸, SER³⁰⁰ and ASP²⁵⁸. FvALT has a strong binding effect with α-ketoglutarate, and the binding sites are ARG³¹⁰, SER¹⁴⁹, SER²⁹⁸, SER³⁰⁰, and ASP²⁵⁸.【Conclusion】FvALT has a typical alanine aminotransferase profile and binds specifically to the substrate alpha-ketoglutarate.

Key words: Fusarium verticillioide, FvALT, bioinformatics analysis, prokaryotic expression, molecular docking

HAO Nan, GENG Shan, ZHAO Yu-wei, HOU Zhi-han, ZHAO Bin, LIU Ying-chao. Cloning and Expression Analysis of FvALT from Fusarium verticillioides[J]. Biotechnology Bulletin, 2024, 40(12): 256-263.

Figures/Tables 5

Fig. 1 Advanced structure of FvALT protein of F. verticillioides A: Secondary structure. B: Tertiary structure. C: Evaluation of tertiary structure

Fig. 2 Protein sequence alignment and phylogenetic analysis of ALT A: Homologous analysis of ALT proteins in fungi, plants, and animals. B: Amino acid alignment of ALT proteins in fungi

Fig. 3 Expression of FvALT in E. coli BL21（DE3） M: Standard of protein molecular weight. 1: Strains without IPTG induction. 2: Precipitation of lysed strain with IPTG induction. 3: Lyse the strain supernatant with IPTG induction. 4: Flow-through fluid. 5: 100 mmol/L imidazole elution. 6: 150 mmol/L imidazole elution

Fig. 4 Analysis of the binding ability of FvALT to substrate in F. verticillioides

Fig. 5 Analysis of the binding sites of FvALT in F. verticillioides and α-ketoglutarate

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