Construction of T-DNA insertion mutants of Botrytis cinerea via Agrobacterium tumefaciens mediated transformation and sequence analysis of insertion site

Abstract:

Abstract：【Objective】Construction of Botrytis cinerea T-DNA insertion mutant library through Agrobacterium-mediated transformation and for further understanding of the molecular mechanisms underlying the pathogenicity of Botrytis cinerea.【Methods】Agrobacterium containing the binary vector pCMBIA1390 was used for transformation of Botrytis cinerea. Hygromycin resistant transformants were screened out and subjected to biological and morphological observation. Detached tomato leaves were used for pathogenicity assay. The flanking sequence of T-DNA inserted in mutant genome was cloned and analyzed by using TAIL-PCR.【Results】A variety of mutants were obtained and important phenotypes including reduction in growth rate, loss or reduction in conidiation and loss of pathogenicity were screened out. The T-DNA flanking sequence of one of the mutants was successfully amplified with TAIL-PCR.【Conclusion】Agrobacterium-mediated transformation system of Botrytis cinerea was established and a T-DNA insertion mutants library was constructed. TAIL-PCR was an effective method for isolating the flanking sequence of T-DNA inserted in genome.