| Melon is an important economic crop in Xinjiang.Wilt diseases had caused heavy losses to the melon production.Now many studies on molecular resistance mechanisms of melon were focused on resistance genes cloning,but the pathways of their signal transduction are unknow.The large chloroplast genome,complex genetic background,long reproduction cycle and genetic operation difficulties are main obstacles to the comprehensive study on melon resistance mechanisms at the molecular level.Arabidopsis thaliana is a model plant which has many advantages that have been brought out great breakthroughs in molecular mechanism research of plant-microbe interaction.In this dissertation,the pathogenic fungi Fusarium oxysporium and Arabidopsis thaliana are used as a plant-microb interaction system to identify and isolate the effective wilting factor from Fusarium oxysporium.We preliminary separated the cultured Fusarium oxysporium into different components and respectively treated melon and Arabidopsis thaliana seedings and seeds.The promoter of pathogenesis-related gene was cloned from Arabidopsis genome and the plant expression vector in which the reporter gene GUS is droved by PR1 promoter was constructed.The vector named pBI-prp1 was transformed into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated vacuum filtration.We used the transgenic plants to identify the probable elictor components of culture medium of Fusarium oxysporium,which will provid a reliable basis for the further study of host-pathogen interaction.The main contents and results of this thesis are as following.1.Using PCR amplification,the 910bp promoter of PR-1 gene was cloned from Arabidopsis to and plant expression vector was constructed to build inducible expression system.DNA sequence analysis show that the transcriptional initiation site,TATA box and high-degree conserved sequence TGAC was in coincidence with the sequence registered in GenBank.The vector named pBI-prp1 in which the reporter gene GUS is droved by the cloned PR-1 promoter was constructed and transferred into Agrobacterium tumefaciens.2.The transgenic Arabidopsis plants was obtained through Agrobacterium-mediated transformation and identified by antibiotics screening.Gene integration and expression were verified by PCR and GUS staining method.3.Six compounds separated from Fusarium wilt pathogen culture medium were tested for elicitor activity using transgenic plants.Histochemical analysis indicated that the component of crude toxin and mycelium both have elicitor activity to inducing the expression of PR-1.In addition,the sterilized crude toxin still has its activity showed it is heat stable.4.The pathogenic secreted crude toxin had the strongest inhibition effect on seedling growth and seeds germination.The crude toxin lost its activity partially after high temperature sterilization,thus we infer there are heat-stable and heat-unstable substances existing in crude toxin. |
