Abstract: 【Objective】The objective of this study is to reveal the function of β₁-tub and β₂-tub in resistance of Gibberella zeae to carbendazim.【Method】α-, β₁-, β₂- and γ-tub of 5 carbendazim resistant strains, Js449 (EC₅₀=7.911 μg·mL^-1), Js462 (EC₅₀=6.515 μg·mL^-1), Js484 (EC₅₀=5.031 μg·mL^-1), Js506 (EC₅₀=8.455 μg·mL^-1) and Js519 (EC₅₀=6.280 μg·mL^-1), were cloned and sequenced, and alignment were carried out among these sequences with those of HG-1 (EC₅₀=0.552 μg·mL^-1), a carbendazim sensitive strain, respectively. Relative expression levels of β₁-tub and β₂-tub in Js506, a resistant strain, in response to carbendazim were detected using real-time quantitative PCR (qPCR). The overexpression vectors harboring β₁-tub and β₂-tub were constructed and transferred into HG-1. Hygromycin phosphotransferase gene and flanking sequences of target gene were fused by split PCR. The entire β₂-tub locus was deleted from Js506 and complementation was also performed by protoplast transformation and homologous recombination. Sensitivity to carbendazim, colony growth and pathogenicity of Js506, HG-1 and their mutants were tested. 【Result】  No mutation was detected in α-, β₁- and γ-tub of 5 resistant strains based on DNA sequence alignment with corresponding sequences of HG-1. The multiple sequence alignment for β₂-tub revealed a mutation (Phe 167 Tyr) at the codon 167 in Js449, Js462 and Js506 and at the codon 200 in Js484. A change at the codon 198 from Glu to Gln was also detected in Js519. Expression of β₁-tub was induced in Js506 by treatment of 5 μg·mL^-1 carbendazim at a significant level of 0.05. Carbendazim at 10 μg·mL^-1 did not exert an influence on expression level of b₂-tub in Js506 significantly. Overexpression of β₁-tub in HG-1 resulted in an increase of the value of EC₅₀ up to 2.839 μg·mL^-1 and enhanced resistance to carbendazim (P<0.05). No significant difference was presented in resistance level between the mutant with overexpressed b₂-tub and the wild strain. Two transformants, Δβ₂tub-Js506-1 and Δβ₂tub-Js506-2, were generated by knocking out assay and validation was carried out by hygromycin screening, PCR amplification and Southern blotting. Compared with the parent strain, both two deletion mutants of b₂-tub locus displayed supersensitivity to carbendazim, slower growth of colony, and reduced pathogenicity (P<0.05). Two complementation mutants of △β₂tub-Js506-1, β₂tub-Js506-C1 (EC₅₀=7.521 μg·mL^-1) and β₂tub-Js506-C2 (EC₅₀=7.243 μg·mL^-1), were identified and these biological characteristics were almost restored in the two transformants by genetic complementation. 【Conclusion】Resistance of 5 strains was correlated with point mutations at codons 167, 198 and 200 in the β₂-tub but not with mutations in the β₁-tub sequence. Treatment of culture with carbendazim induced β₁-tub transcript levels and overexpression of β₁-tub improved its resistance to carbendazim. β₂-tub is necessary for resistance of G. zeae to the fungicide, and both two tubulin genes are able to affect the resistance.

Key words: Gibberella zeae (Schwein.) Petch, tubulin gene, carbendazim, overexpression