Construction And Function Research Of The Fpd1Gene Deletion Mutants Of Fusarium Oxysporum F. Sp. Cubense

Fusarium wilt of banana is a disease that destroys vascular Systemly. The pathogen is Fusarium oxysporum f.sp.cubense, mainly including racl?rac2and rac4, and its cause plants death by destroying the vascular. Banana wilt lack of effective prevention and control method. At present, disease-resistant variety and biological control are applied to prevention and cure of banana wilt. However, People understand the molecular mechanism of pathogenic bacteria banana is very limited, which to some extent hindered the conduct breeding for disease resistance. Therefore, it is necessary to strengthen study forpathogenic mechanism of Foc, which will provide theoretical guidance for resistance breeding. In this study, function of Foc4fpdl gene encoded chloride conductance protein was research and analysis by reverse genetics mainly.By bioinformatics analysis for Fusarium oxysporum fpdl gene, we found genes encoding proteins FPD1chloride conductance and has44%protein ICln partial similarity, may be involved in the process of cell swelling from the cytoplasm to the cell membrane transpose. we found that the proteins encoded fpd1genes has44%similarity with chloride conductance protein ICln and may be involved in the process of cell swelling from the cytoplasm to the cell membrane transpose. The putative FPD1protein have multiple sites of phosphorylation and is a unstable hydrophilic protein instability.According to Fusarium oxysporum fpdl gene (Genbank No. EU334871.1), the primers for PCR amplification of upstream and downstream homology arms were designed and, knockout vector was constructed. Using PEG-mediated protoplast transformation method transformed wild-type strain and identifying transformants by PCR specific primers, two knockout mutants were useful, namely Foc4-?fpd1-1/2. These mutant carries a hygromycin resistance gene (hph)and green fluorescent protein (GFP).The fpdl gene sequences was obtained on Genbank number (EU334871.1), and primers of the downstream homology arms were designed to build knockout vectors according the sequences. Protoplast transformation use PEG-mediated protoplast transformation method, and specific PCR primers were use to identificat transformants so that two transformants, Foc4-?fpd1-1/2, were right. They carries hph and gfp gene as double label.Two mutants phenotype (Foc4-?fpd1-1/2) were observed and compared with the wild type. Mutant colony have obvious reduction in growth rate. Spore production rate is reduce. Mutant colony only product a small number of aerial hyphae and snapping medium. Furthermore, compared to wild type, the mutant did not significantly change sensitivity of hydrogen peroxide, but sensitivity increased to sodium chloride. Bacillus resistant result showed increased sensitivity, and cell wall of treated mutant has lysing, protoplasts has leaking. It did not change significantly for pesticides (carbendazim and prochloraz) sensitivity, the control without transparency. Mutant and wild-type grew in media containing various factors, and the growth rate in the PDA and mutant MM medium was significantly slower than the wild type. In the MM+Congo red medium, mutants grow more slowly. In MM+Sorbitol medium, the growth rate of mutant have not been restored. These results indicate that knockout of fpdl gene in Foc lead more sensitive to the cell wall interference and suggesting that the gene may be involved in maintaining the cell wall integrity and stress response regulation.In pathogenicity experiments of potted plants banana seedlings, pathogenesis index of two mutations was14.4%and16.2%, but pathogenesis index of wild-type strain was62.4%and water control index is only4.1%. This suggests that the mutant significantly reduced virulence. Toxin crude extract of mutant and wild type was used to soak roots of banana seedlings. Result showed that they both can lead to banana seedlings browned and suggesting that the toxin synthesis wasnot be affected by fpdl gene.Analysis of secreted proteins, cytoplasmic proteins and cell wall protein content on mutant and wild-type, result show that mutants secreted protein (0.78mg/mL and0.83mg/mL) is lower about30%than wild-type (1.17mg/mL). The wild-type cytoplasmic protein content was only1.43mg/mL, lower about40%than the mutant. Analysis about Cell wall protein binded by Coomassie blue show when the cell wall content was50mg, the OD465absorbance of two mutant and wild-type was difference up to48%and46%, respectively. The absorbance values of mutants was significantly lower than the wild-type. So it suggests that mutant cell wall protein content was lower than the wild type. Both SDS-PAGE of wild type and mutant proteins did not have difference band.Through analysis of two mutants functional recovery experiments, colony morphology of reply complementary mutant Re-Foc4-?fpd1was similar to wild-type colonies, and also can generate a lot of aerial hyphae. The growth rate had increases, far different from the gene deletion mutants, Foc4-?fpdl-1/2colonies. In addition, pathogenesis index was about58.3%, and similar to the wild type62.4%.In summary, the fpdl gene of Fusarium oxysporum.cubane involved in growth and development, stress response, pathogenicity and cell wall integrity maintain. These findings have laid a good foundation for the Foc pathogenic mechanism.