| The banana fusarium wilt is a kind of fascicular systemic disease caused by Fusarium oxysporum f. sp. cubense, which caused destructive destruction in the production of banana and brought serious economic loss. The Foc1 that widely distributed in the world mainly infests “Gros Michel” and the Foc4 that were most dangerous infests all cavendish cultivars. In the interaction with host, the Fusarium oxysporum usually produce some kinds of cell wall degradation enzymes that closely related to pathogenicity, which can degrade the defense and protection organization in the cell wall of the host. Among the cell wall degradation enzymes, pectinase plays an important role in the pathogenicity of Fusarium oxysporum.Based on the comparative transcriptomic analysis of Foc1 and Foc4 of Fusarium oxysporum f. sp. cubense induced with three carbon sources, The BLAST results of comp12226_c0 and comp14619_c0 that were pectin esterase gene in the analysis of the transcriptome showed that they belonged to pectinesterase family genes, whose homologies were respectively 92%, 93% with Fusarium moniliforme and which were respectively named pme1 and pme2. Induced under different carbon source, the results of the transcriptome analysis indicated the pme1 and pme2 were obviously different in the expression level with the expression amount of Foc4 higher than the Foc1. Verified by q PCR, the expression of pme1 in Foc4 is 300-500 times of Foc1 and the expression of pme2 in Foc4 is 100-700 times of Foc1, which were in conformity with the results of transcriptome analysis.The DNA length of both Foc1-pme1 and Foc4-pme1 consisting of 2 introns and 3exons were 1141 bp and their coding regions were 990 bp with the homology of 98.7%.The DNA length of both Foc1-pme2 and Foc4-pme2 consisting of 2 introns and 3 exonswere 1331 bp and their coding regions were 1221 bp with the homology of 98.5%. The proteins encoded pme1 and pme2 belonged to the family of Pectinesterase, located outside the cell. The conserved domains of pme1 and pme2 were respectively 26-315 aa and 57-381 aa and the function sites of amino acid were both IQGATDFIFG, which having glycosylation sites and phosphorylation sites.Pichia pastoris GS115 was used to expressed pme1 and pme2. p PICZ?A was choosed as expression vector. Through efficient electroporation and identification, recombinant Pichia pastoris strains were obtained. Aim proterins were induced by 1% methanol and havrested the supernate. SDS-PAGE showed that aim proteins had produced at 24 h.Western blot proved purpose proteins have expressed again. The crude protein activity was determinated by continuous spectrophotometer method and agarose diffusion assay, the results show that, the highly esterification of pectin was degradated with recombinant protein, the medium of pectin can detected transparent circle, show that had isolated the activity of recombinant proteins.The flanking sequences of Foc1-pme1, Foc4-pme1, Foc1-pme2 and Foc4-pme2 were obtained by hi TAIL-PCR. The method of homologous recombination was used to construct the knockout vector. Using ATMT method transformation, successfully obtained the gene deletion mutant. Using root dip inoculation method, knockout mutant and wild-type Foc were inoculated host, observation results in 45 days. The results showed that, these host inoculated knockout mutant and wild-type all have the typical symptoms of banana wilt.The disease index of the knockout mutant were lower than the wild-type, but statistical analysis showed that the disease index has no significant difference between knockout mutant and wild-type. The results suggest the impact of knockouting pme1 and pme2 gene is small to pathogenicity of Foc in host, then PME1 and PME2 are not the key pathogenic factors of host, but it may be related to the severity of the banana fusarium wilt. |
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