Studies On The Physiological Differentiation And Differential Expression Of FochsV Gene From Three Melons Fusarium Wilt

Fusarium oxysporum Schl is a worldwide soil-borne plant pathogenic fungus,which caused enormous loss on economy in China and other countries. In recent years, Fusarium wilt has become a severe disease on cucurbitaceous crops as the enlargement of the cultivated area. At present, the Fusarium wilt was controlled mainly by chemical methods and breeding resisitant cultivars. However, Fusarium wilt important pathogenic factors of Fusarium wilt are investigated.In the thesis,36strains of Fusarium oxysporum Schl. isolated from cucumber, melon and watermelon wilt at melon were studied as materials. Studies on physiological differentiation of their characteristics by combining the methods of pathogenic differentiation and genetic diversity analysis of research. And construct the eukaryotic expression vector of pBC-hygro-GFP, by using the method of PEG-CaCl2mediated successfully marked F. oxysporum strain FSY0957, and observed characteristics of F. oxysporum infection in cucumber, melon, watermelon plant roots use marked strain. The full-length cDNA of FochsV was obtained using from the genome of F.oxysporum f.sp.cucumerium strains FSY0957. And make its bioinformatics analysis. The expression levels of Fochs V on the three host plant root surface was investigated by Real-Time QPCR technology, Main researchs were as follows:1. The research progress of Fusarium wilt forma specialis and pathogenic factorA total of36F.oxysporum isolates were obtained from cucumber, muskmelon, and watermelon at Liaoning province, were selected to make the classification into formae speciales.The genetic relationship of36F.oxysporum isolates was determined using EF-1α, β-tubulin DNA sequence and UP-PCR analyses. The results show that all36isolates were divided into three formae speciales:F.oxysporum f.sp.cucumerium, F.oxysporum f.sp.melon and F.oxysporum f.sp.niveum.On basis of the combined sequence and UP-PCR data, the36isolates were classified into three major groups, but FSY951and FSY0950isolates were divided with other isolates in UP-PCR data, however they were divided in lineage2in DNA sequence data.In addition, there was not opposite ralationship between pathotype and genetype. This study confirmed that phylogenetic groups have little connection with virulence groups.2. The research of Fusarium oxysporum green fluorescent protein as a markerIn the thesis, for the study on colonization of F. oxysporum in plant root, the gf p fragment as a reporter gene had integrated into the form plasmid vector pBC-hygro which containing a expressive promoter of the fungus to facilitate transformation of F. oxysporum. The results show that the gfp fragment integrated into pBC-hygro success fully. Eukaryotic expr-ession recombinant vector pBC-hygro-GFP was constructed corre ctly. The pBC-hygro-GFP was transformed into F.oxysporum by using PEG-CaCl2med iated transformation technique. The infection characteristics of the strain in the cucumb er, melon, watermelon seedling was observed at48h after inoculated. The colonization ability of tagged strain on cucumber plants was obviously higher than that on the m elon and watermelon host. Genetic transformation system established in this experiment provided a basis for the exploration on F.oxysporum pathogen infection mechanism a nd the colonization on plant rhizosphere.3. cDNA clone and sequence analyses of Fusarium oxysporum Fochs V geneThe RNA of F. oxysporum. f.sp. cucumerium isolate FSY0957was extracted. The full-length cDNA of FochsV was obtained using RT-PCR and gene clone methods. It consisted of5589bp nucleotides, include a complete ORF5588bp. and encoded5588amino acids. The protein contains6transmembrane domain and no signal peptide sequence, which belongs to the membrane binding protein. And the results of gene homology comparising with other fungal chitin synthase show that the Gibberella moniliformis and Trichoderma have highest homology with F. oxysporum FochsV gene. The cDNA has been registered to the GenBank and No.KC840941.5Expression analysis of the FochsV gene in F.oxysporum Schl. f.sp. cucumerinum by real-time PCRIn this paper, in order to prove that the differentially expression of Fochs V gene in different periods after infected three host, expression level analysis of the previous proven Fusarium oxysporum pathogenic gene FochsV at6-144h that inoculated on cucumber, melon and watermelon seedlings using SYBR Green I real-time PCR technology. The results show that:the gene were tested in the three host intermittent movement up or down-regulated, infection of cucumber seedlings infection expression levels are high, only infect melon and watermelon seedlings2-3the expression amount higher than that of the control point. The results unless proved once again that of Fochs V genes involved on the F. oxysporum infection on the three melons host, also reveal the changes in gene expression amount of the different infection period.The real-time fluorescence detection method established in this study has laid a theoretical foundation for the rapid prediction of the bacteria and prevent..