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


CONCLUSIONS
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.
Feng
Juan
J
College of Biological & Environmental Engineering, Zhejiang University of Technology, Hangzhou 310014, China.
Zhu
Tingheng
T
Cui
Zhifeng
Z
Wang
Kun
K
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001483:Base Sequence; D020171:Botrytis / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D004269:DNA, Bacterial / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D018551:Solanum lycopersicum / Q000382:microbiology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010935:Plant Diseases / Q000382:microbiology; D014170:Transformation, Genetic
D004269:DNA, Bacterial; C036483:T-DNA