The Establishment Of DsRed-labelled Fusarium Proliferatum Genetic Transformation System

Fusarium proliferate is an important soil-borne pathogenic fungi with can cause wheat, barley, corn and other cereal crops infections. F. proliferate can not only infect the roots of wheat and give rise to wheat root rot, but also cause corn ear rot by infecting the ear of corn, which affects the yield and quality of wheat and corn especially in the wheat-maize rotation area like Huang-Huai-Hai region,but there are few researches about the pathogenesis of F. proliferate relatively. In recent years, fluorescent protein was widely used in pathogenic fungi, plants and animal aspects as a marker protein. Labeled by fluorescence protein, the infection and colonization process of fungal pathogens can be observed directly in plant tissues, and the interaction between plant and pathogenic also can be studied. In this study,Ds Red gene was transferred into F. proliferatum strain Fp-1 by means of eletroporation-mediated transformation and the fluorescence protein labeled transformants of F. proliferatum was obtained, the genetic transformation system was established. This finding will provide a theoretical basis for the study of the interactions between pathogens and plants.The main findings are as follows:1. Through gradient screening, we found that the concentration of hygromycin that F.proliferatum could resist was 600μg·ml-1. The optimum conditions for electroporation was 6d for F. proliferatum culture and 10 h for spore’s germination. The voltage parameter range for F. proliferatum spores electroporation was determined as 1400¹800v.2. We constructed the filamentous fungi electroporation expression vector containing of Ds Red, and induced Ds Red-labelled into F. proliferatum strains Fp-1 by the electric conductance method, and then have got about 140 transformants. DNA extraction, PCR,fluorescence microscope detecting for the transformants, the results showed that 84 transformations hyphae and conidia expressed bright red fluorescence, and by using gene specific marker amplification proved the Ds Red was integrated into the genome of these fungi.3. The grown rate and and growth ability of transformation strain were measured in different p H value. Finally we selected the transformation Fp-RFP which is similar to the wildtype strain in all aspect. Analysising Fp-RFP growth characteristics and genic stability, it is shown that the Ds Red in transformants was stable genetic, colony growth speed had no significant difference compared with wild type strains. Though in different p H conditions of growth are slightly different, but the natural p H had no significant difference, does not affect the transformant strains in wheat tissue invasion and amplification. Extracellular enzyme metabolism ability between Transformants and wild type strain was basically consistented,and both of them could produce pectinase and protease, amylase and cellulase.4. Content of H₂O₂ and changes in activity of protective enzymes in wheat Lumai-21 were determined respectively at different times after inoculating with transformant strain Fp-RFP and wild type strain Fp-1. Compared with the wild-type strain, the effects on H₂O₂ content and protective enzymes activity in wheat by inoculating transgenic strain was not significant, indicating that transgenic strains maintained similar pathogenicity to wild type. At the early stage of inoculation, the activity of SOD, CAT, MDA and content of H₂O₂ were significantly increased, and the changing trend of SOD and other enzymes was consistent with that of H₂O₂ content, which matched the theoretical prediction. At the beginning of inoculation did not significantly improve with the activity change of theoretical predictions of POD enzyme, and the enzyme activity caused by the transformant strains decreased, while the enzyme activity have increased in 48 h. It show that POD enzyme infection of Fusarium in the only play a role in the medium and later period of innoculation. Through the analysis of the results shows that in the period before infection, CAT enzyme has the biggest change in the activity, especially in late infection, the activity of POD against the greatest change,prevention, resistance breeding, and provide a theoretical basis for the identification of disease resistance.The establishment of the genetic transformation system offered a theoretical basis for the future study of molecular genetic characteristics and pathogenic mechanism of F. proliferatum,the interactions between F. proliferatum and the host.