Analysis On Genetic Diversity Of Populations Of Sporisorium Reilianum In Northern China And The Use Of The Green Fluorescent Protein In Pathology Research

Based on random amplified polymorphic DNA(RAPD) technology,genetic diversity and differentiation of Sporisorium reilianum populations were analyzed. Agrobacterium tumefaciens-mediated transformation was successfully applied to Sporisorium reilianum.The green fluorescent protein(GFP) was expressed in Sporisorium reilianum,and the Sporisorium reilianum in the maize tissue can be observed through detecting GFP.The main results were outlined as follows:1.The genetic diversity of 68 isolates of Sporisorium reilianum in northern China was analyzed using 26(RAPD) markers.The database calculated by Popgene software showed that the polymorphism was 98.33%.It indicated an abundant genetic diversity and a distinct differentiation among the populations of Sporisorium reilianum,and the genetic difference in populations was smaller than that among populations.The genetic differentiation was affected not only by geographical regions,but also by gene transference through pathogens carried by seeds.2.A dendrogram of genetic relationship was constructed using UPGMA method.All the 68 populations were classified into 13 cluster groups with a similar coefficient of 0.76. This result had perfected and enriched the researches on genetic diversity evaluation of Sporisorium reilianum in China.It was valuable for both disease-resistance resources selection and disease-resistance breeding engineering.3.We constructed a plasmid vector pCAMBIA0611,containing gfp gene as a reporter.The plasmid was transformed to Agrobacterium tumefaciens by heat shock.Then the Agrobacterium tumefaciens with gfp was used to transfer genes into Sporisorium reilianum.And transformants were obtained.4.A novel transformation system mediated by Agrobacterium tumefaciens was firstly established in Sporisorium reilianum.In addition,different factors which influenced transformation were optimized including Agrobacterium strains,cultivation time for Sporisorium reilianum,Agrobacterium concentrations and so on.The results signified this fit together can obtain the highest transformation efficiency(280 transformants/10⁶ spores) when the strain is GV3101,the culture time is 3d in CM,the Agrobacterium concentrations was OD₆₀₀ 0.4.5.When transformants and wild were co-cultiavted in PD,they can combine with each other and the conjugation hyphae can be observed by microscope.It indicated that the insertion of foreign gene did not change matching-related genes.And the transformants exhibited normal physiological functions.6.The maize mescotyl was injected with mated basidiospores suspensions when the seeds began to germinate.The genome DNA of maize in two-leaf and one-leafiest stage was detected to habor gfp gene fragment by PCR.Disease symptoms became apparent after the onset of flower development when the fungal sori replaced male or female inflorescences.7.Through microscope,some transgenic hyphae can be observed in stamen flowers without any symptom observed by nake eyes.To observe the transgenic fungi inside the tissue,laser scanning confocal microscope(LSCM) was more effective attributing to the reduced interference from plant cells auto-fluorescence.Green fluorescence was visible in stamen tissues using LSCM which signified an infection by fungi before symptom exhibited exteriorly.