Establishment of T-DNA Insertion Mutation Library of Fusarium Oxysporum f.sp.cubense Race1 and Location of Insertion Site of Some Related Pathogenicity Mutants
|Keywords||Fusarium oxysporum f.sp. cubense (Foc) racel T-DNA Insertion Mutant Genetic Transformation TAIL-PCR|
Banana wilt (Fusarium oxysporum f.sp. cubense=Foc), also known as banana Panama disease or yellow leaf disease, is caused by a Cuban isolate of Fusarium oxysporum resulting in devastating soil-borne vascular disease. Foc has four physiological races, with races No.1 and No.4 causing serious damage to banana in China. As Foc’s genetic background is complex and its pathogenesis is poorly understood, at present there is no really effective disease control method and there is no banana varieties highly resistant to Fusarium wilt. Therefore, an in-depth understanding of the pathogenicity of Fusarium oxysporum f.sp.cubense and variations of the genetic mechanism will help in the breeding of resistant varieties and development of control strategies.In this study, T-DNA insertion technology was used. The genetic transformation system of Fusarium oxysporum f. sp cubense race 1 was constructed with a mutant library containing 1200 mutants. All mutants were tested in pathogenicity. Those mutants that had lost pathogenicity, had severely reduced pathogenicity or greatly enhanced pathogenicity were selected for further analysis for the T-DNA insertion sites of the right-wing sequence clone. The results are as follows:1. Established T-DNA into the genetic transformation system of Focrl-N2 strain (with strain genome sequencing) and factors affecting transformation efficiency were optimized for the efficient transformation:Agrobacterium OD600:0.15, AS (Acetosyringone) induced concentration of 150μmol/L, induction time:7 hours, Focrl N2 spore concentration:1×106/ml, hygromycin B:150μg/mL, induction medium pH:5.5, temperature for culture:25 C and culture time:48 hours.2. Established a mutant library containing 1200 mutants. There were 20 mutants that had lost their pathogenicity,18 with significantly reduced pathogenicity,53 with a marked increase of pathogenicity,2 with greatly enhanced pathogenicity to Fen Jiao (AAB) and Baxi Jiao(Cavendish,AAA), and 1 with serious pathogenicity to Baxi Jiao(AAA) but less severe virulence to Fen Jiao (AAB).3. The four mutants that had lost their pathogenicity (Focr1-N2-65, Focr1-N2-194, Focrl-N2-273 and Focrl-N2-328), two with greatly reduced pathogenicity (Focr1-N2-71 and Focrl-N2-73), and three with greatly enhanced pathogenicity (Focrl-N2-409, Focrl-N2-418 and Focrl-N2-531) were selected for further studies. TAIL-PCR method was used for PCR amplification of its wing sequences of T-DNA insertion site.15 TAIL-PCR products were amplified from these nine mutants. Except for Focrl-N2-409, the other eight mutants’T-DNA loci can be found in the genome of Focrl-N2.4. This study successfully acquired a number of mutants with total loss, serious decline or greatly enhanced pathogenicity, obtained part of insertion sites from the T-DNA mutants, and found these insertion sites in genome sequence of race 1. Our findings served as a good foundation for the next step in the cloning of related genes.