The Binding Kinetics of Lysinibacillus sphaericus Cry48Aa/Cry49Aa Toxin to Brush-border Membrane Fractions of Culex quinquefasciatus Larvae

doi:10.16409/j.cnki.2095-039x.2016.03.006

Abstract

Abstract: The Cry48Aa/Cry49Aa mosquitocidal two-component toxin was recently characterized from Lysinibacillus sphaericus strain IAB59, with high toxicity against binary toxin (Bin) susceptible and resistant Culex spp. However, its action mode against the target mosquitoes has not been elucidated. In this work, the cry48Aa and cry49Aa genes were individually cloned and expressed in the crystal minus B. thuringiensis. The purified Cry48Aa/Cry49Aa toxin showed similar high toxicity against Bin-susceptible and resistant C. quinquefasciatus. Direct-binding assays and competition assays were used to identify specific binding between the biotinylated toxin and brush-border membrane fractions (BBMF) of the susceptible and resistant C. quinquefasciatus larvae. The results showed that Cry48Aa had a higher binding affinity, with a dissociation constant (Kd) of (9.5±1.8) nmol/L and (13.9±2.3) nmol/L, compared to the Kd of (25.4±3.8) and (28.1±4.2) nmol/L for Cry49Aa. In heterologous competition binding assays, Cry48Aa toxin could effectively compete with Cry49Aa toxin for binding sites on BBMF from both mosquito larvae, with IC₅₀ of (22.1±3.7) nmol/L and (15.4±2.6) nmol/L, respectively, whereas Cry49Aa (with IC₅₀>17 μmol/L) could not compete and block the binding sites of Cry48Aa toxin. These results provide important information for the preliminary action mechanism of Cry48Aa and Cry49Aa.

Key words: Culex quinquefasciatus, Cry48Aa/Cry49Aa mosquitocidal toxin, brush-border membrane fractions, binding kinetics, Lysinibacillus sphaericus

CLC Number:

S476.11

GUO Qingyun, CAI Quanxin, HU Xiaomin, YAN Jianping, YUAN Zhiming. The Binding Kinetics of Lysinibacillus sphaericus Cry48Aa/Cry49Aa Toxin to Brush-border Membrane Fractions of Culex quinquefasciatus Larvae[J]. journal1, 2016, 32(3): 318-325.

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