Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (15): 2971-2979.doi: 10.3864/j.issn.0578-1752.2014.15.007

• PLANT PROTECTION • Previous Articles     Next Articles

Function Analysis of BcKMO Gene in Growth, Development and Pathogenicity of Botrytis cinerea

 LI  Pei-Fen, ZHAO  Fu-Xin, DONG  Li-Ping, ZHENG  Hui-Xin, ZHAO  Bin, ZHANG  Jing, SI  He-Long, XING  Ji-Hong, HAN  Jian-Min, DONG  Jin-Gao   

  1. Laboratory of Mycotoxin and Molecular Plant Pathology, Agricultural University of Hebei, Baoding 071001, Hebei
  • Received:2014-01-27 Online:2014-08-01 Published:2014-04-23

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Abstract: 【Objective】The objective of this study is to investigate the function of BcKMO gene in growth, development and pathogenicity of Botrytis cinerea, to further clarify the molecular mechanism of the BcKMO gene in development and pathogenicity of the fungus and provide a theoretical basis for the gray mould control. 【Method】 Bioinformatics methods were used to analyze the BcKMO gene and its encoding product. The BcKMO gene coding region was amplified and cloned into pBARKS1-eGFP, and pBARKS1-BcKMO-eGFP was constructed. The protoplasts of mutant BCG183 were prepared and transformed with pBARKS1- BcKMO-eGFP by PEG-mediated method. PCR, Southern blotting and real-time PCR were used to identify the transformant. Phenotype including colony morphology, mycelium morphology, growth rate, conidial yield, and so on, and pathogenicity assays of wild-type strain BC22 (WT), mutant BCG183 and complemutant BCG183/BcKMO were performed to verify the function of the BcKMO gene in growth, development and pathogenicity. 【Result】The BcKMO gene encodes kynurenine 3-monooxygenase (KMO) having monooxygenase FAD conserved domain and four Aromatic-ring hydroxylase-like motifs. The BcKMO protein displayed high homology to rossmann-fold NAD(P)(+)-binding protein (gi156049701) of Sclerotinia sclerotiorum and FAD-dependent oxidoreductase (gi512192943) of Ophiostoma piceae. The expression vector of the BcKMO gene, pBARKS1-BcKMO-eGFP, was successfully constructed and transformed into the protoplasts of mutant BCG183. Results of PCR, Southern blotting and Real-time PCR showed that the BcKMO gene complementing mutant (BCG183/BcKMO) was successfully obtained. Phenotype assay showed that the mutant BCG183 growed slowly, did not produce conidia and sclerotia, and hyphae slender, but the phenotypes of the BCG183/BcKMO were all similar to that of WT. Compared with WT and the BCG183/BcKMO, the pathogenicity of the mutant BCG183 on kidney bean leaves and cucumber leaves were dramatically increased.【Conclusion】The BcKMO gene is a negative regulation in growth and development, and a positive regulation in pathogenicity of B. cinerea.

Key words: Botrytis cinerea , BcKMO , mutants , growth and development , pathogenicity

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