Preliminary Analysis Of DNA Methylation Changes In Banana Induced By TE Fusarium Oxysporum F.Sp. Cubense Race4

Organisms are constantly exposed to environmental stimulated stresses, including biotic-stresses and abiotic-stresses, and they are capable of establishing mechanisms of protection and adaptation. Information content of the genome (DNA sequence) and its expression in response to stress are crucial for the adaptability of a genotype. Fusarium wilt is a destructive fungal disease of banana plants, and there is no effective measure to control either in traditional or genetic breeding. To date, the underlying molecular basis of pathogenesis and resistance remains elusive. To investigate whether DNA methylation plays a role in banana Fusarium wilt molecular mechanism, this paper using methylation-sensitived amplification polymorphism (MSAP) compared the DNA methylomes of adult Brazil banana (Musa spp. AAA) plant artificially inoculated with Fusarium oxysporum. sp. race4and the control group. The results were as following:1. This paper utilized the MSAP method to investigate the Brazil Banana genome CCGG site methylation status under the stress of the Fusarium oxysporum. sp. race4inoculation, and there were25pairs of selective primers using in MSAP analysis during five treated time points(4h,12h,24h,3d and6d), and totaled, in health adult Brazil banana, the CCGG bands of the genome were detected to be methylated on the average of29.23%, of which the fully-methylated CCGG sequences were on the average of19.376%. After artificially inoculated with the pathogen of Fusarium oxysporum. sp. race4, the the total level of genome CCGG methylation increased to34.73%in average during the five treated time points, which implied that DNA methylation might play a role in banana Fusarium wilt molecular mechanism.2. To analysis the polymorphic DNA methylatd fragments, there were66pathogen induced polymorphic DNA methylatd fragments detected and sequenced.19of polymorphic fragments were identified to be homologous to known gene encoding protein such as NBS-type resistance protein in banana, or encoding genes in Oryza sativa and Arabidopsis lyrata, whereas mostly homologous to Musa acuminata genomic clone BAC sequence, and the remaining fragments were no significant similarity.3. The MSAP polymorphic fragment M27hypomethylated under the pathogen stress and it was homologus to the NBS-type resistance protein RGC5encoding gene with79%in Genebank. Inorder to confirm whether its’ expression consistent with the methylation variation pattern, the semi-quantified RT-PCR and Methylated-specific PCR were used to ascertained the expression of the gene was down-regulated. The result showed the genome-wide CCGG sites hypermethylation and the related resistance gene hypomethylation and showed these can be used for the research of the interaction between pathogen Fusarium oxysporum. sp. and the plant banana.4. Subsequently, to identify more information of the resistance protein encoding gene homology, the TAIL-PCR was performed to extent the flanking sequence of the M27, and after twice of PCR, the M27fragment was amplified from171bp to1324bp. Blasted it with the Genebank, the result showed that it was homologued with NBS-RGC5CDS of Musa acuminata subsp. Malaccensis, which implied that the resistance protein encoding gene existed dissimilar with the different varieties and it was demethylated under the Fusarium oxysporum. sp. race4induction.