Construction Of T-DNA Insertion Mutant Collections In Verticillium Dahliae

Cotton is the most important fiber crops in our country, and is closely related to the development of national economy. Verticillium wilt disease of cotton, caused by Verticillium dahliae, might result in destructive economic damage. Verticillium wilt fungi are notoriously difficult to combat due to extremely persistent resting structures, residing in the soil, are difficult to eradicate since the only effective control measure, soil fumigation, is expensive and has harmful environmental effects. Furthermore, crop cultivation is difficult to find the ideal of source material, producing high resistance species to Verticillium wilt is impossible. Furthermore, the disease control methods and reagents also are difficult to reach ideal control result. Furthermore, the broad host ranges of V. dahliae make crop rotation ineffective, and fungicides to cure infected plants are not available. Now, genetic engineering resistance is the preferred method to control Verticillium wilt diseases, and Verticillium resistance has been described in some plant species. Therefore, understanding the molecular mechanism of the fungus is particularly important because we can develop the new strategies, new methods and new fungicides to control Verticillium wilt.In order to further understand the molecular mechanism of the interaction between pathogenic fungi and cotton, it will help realize the effective prevention and control. Though screening and cloning a number of genes related to Veticillium pathogenicity, will help understand the pathogenitic mechanism of cotton Verticillium disease. In this thesis, I found a transformation system of V. dahliae mediated by Agrobacterium tumefaciens, and constructed a V. dahliae T-DNA random insertion mutant library. After that, through phenotypic variation and pathogenicity of functional identification and screening out associated with pathogenicity mutants, further cloning the mutants of the flanking sequences, confirming the missing gene. These are useful for cloning the virulence associated genes and studying their function, and also help to elucidate the pathogenic molecular mechanism of V. dahliae.1 Construction of T-DNA insertion mutant collection in V. dahliaeI firstly optimized the A. tumefaciens-mediated transformation (ATMT) system by several factors increasing the transformation efficiency.I established optimal conditions for transformation of V. dahliae as follows:A. tumefaciens LBA4404 (contained the pPK2 vector) cultured to OD660 0.5-0.6 and diluted to OD660 0.15 in induced medium (IM) with 200 μM Acetosyringone(AS) for 6 h; mixing the A. tumefaciens with same volume V. dahliae conidia suspension which concentration was 107 conidia per milliliter; spreading 100 μL mixture on the co-cultivation medium (IM medium blending 200 μM AS, pH 5.3) with a synthetic cellulose membranous on it; incubating 48 h at 25 ℃. Under these conditions, the transformation efficiency of ATMT on V. dahliae is 100～300 mutants per 107 conidia. Then I constructed a T-DNA insertion mutant library of V. dahliae with 600 mutants.2 Evaluated the quality of T-DNA insertion mutant library in V. dahliaePCR analysis of randomly selected seven transformants showed that Hygromycin B was transformed into the V. dahliae genome. All the randomly selected seven resistant colonies were proved to be genetically stable through mitotic cell division.3 Screening phenotype mutants from T-DNA insertional mutagenesis collections and analysis of T-DNA flanking sequencesA total of 60 transformants were screened for phenotype mutants. Thirty one mutants were selected from transformants cultured on PDA medium. Compared with the wild type V. dahliae Vd991, these mutants, to some degree, showed difference in growth rate, colony phenotype and pigment aggradations. I also analyzed the T-DNA flanking sequence from 31 V. dahliae mutants. The T-DNA flanking sequences of insertion site were isolated using hiTAIL-PCR method followed by sequencing. We successfully obtained 31 T-DNA left border flanking sequences.4 Isolated and analysis the T-DNA tagging gene from V. dahliae mutantsDNA sequences flanking to the T-DNA were isolated and sequence comparison was performed using BLAST available on the Verticillium group database web site, I have got a number of reference data about mutant T-DNA insertional locus upstream or downstream of gene. Combined with the phenotypic characteristics of mutants, the important candidate genes can be screened. By comparing, I found that the T-DNA sequences flanking insertion the mutant D5 coding trehalose phosphorylase, the mutant N4 T-DNA flanking insertion coding chloride channel protein and other mutants T-DNA sequences flanking insertion coding hypothetical proteins.