Construction Of T-DNA Insertion Mutation Library Of Fusarium Oxysporum And Analysis For Growth And Development Related Genes

Agrobacterium tumefaciens-mediated transformation(ATMT),a natural transformation tool with high transformation efficiency,was widely applied in transgenic manipulation for plant and fungi.Fusarium oxysporum,the infectious agent of Panax notoginseng Root Rot,caused the loss of production and quality for P.notoginseng can not be estimated.To study the molecular mechanisms in growth,development and pathogenicity of F.oxysporum,firstly,we optimized a range of conditions to improve the ATMT transformation efficiency.Then a T-DNA insertion mutant library of F.oxysporum was established.Meanwhile,several characters including stresses tolerance,sporulation and germination has been measured.Finally,the flanking sequences of inserted site has been confirmed using thermal asymmetric interlaced PCR(TAIL-PCR).The main results are as follows:1.Plasmid p Camhyggfp which containing hygromycin resistance and kanamycin resistance gene was transformed to A.tumefaciens LBA4404 and established a transformation system for F.oxysporum.The cultivation conditions affecting the transformation efficiency were optimized,like LBA4404 concentration,co-cultivation time,co-cultivation temprature and spore concentration.600 mutants were abtainted using above optimized protocol.60 transformants were randomly selected and determined by hygromycin resistance and PCR amplification,all of transformants can grow in PDA containing with hygromycin B and the PCR production included targeted fragments,suggested that the T-DNA has been inserted into the F.oxysporum genome.2.Analyses were conducted in morphology,stress response,sporulation and germination for mutants.Under osmotic stress,the growth of mutant Δ 11 was significantly inhibited compared with wild type.Under cell wall stress,the growth of mutantΔ3,Δ7,Δ11 andΔ12 was significantly inhibited compared with wild type.Under cell membrane stress,the growth of mutant Δ11,Δ12,Δ320 andΔ336 was significantly inhibited didn’t even grow compared with the wild type.Under oxidative stress,the growth of mutant Δ3,Δ320 and Δ336 was significantly inhibited didn’t even grow compared with wild type.The spore production of mutant Δ7 and Δ336decreased significantly compared with wild type.The spore germination rate of mutantΔ3,Δ7,Δ320 and Δ336 decreased compared with the wild type.3.Genome DNA of six mutants was extracted by CTAB,and flanking sequences were amplified by TAIL-PCR.In all,6 flanking sequences were obtained successfully.After sequencing and BLAST analysis,flanking sequence of Δ320 located in F.oxysporum f.sp.cubense strain race 4 chromosome 1 but no more information was obtained.Flanking sequence of Δ12 showed 99% similarity to Rho2,which encoding GTP binding protein of Fusarium oxysporum 4287.Flanking sequence of Δ 336 showed 99% similarity to the LST8,which encoding the targeted of rapaxin(TOR)subunit of Fusarium oxysporum 4287.Flanking sequence of Δ 3,Δ 7 and Δ 11 encoding putative protein with unknown functions.In summary,T-DNA was successfully inserted into the genome of FO by ATMT in this study,a batch of mutants were obtained and the locations of disrupted genes successfully found by TAIL-PCR.All of genes were unreported in FO,which are of great research value.ATMT as a transformation technology opens a new perspective on the molecular level of F.oxysporum.The successful construction of F.oxysporum mutant library is conducive to the exploitation of unknow functional genes,which lays a foundation for the study of F.oxysporum growth,development and pathogenic mechanism.