| Ustilago esculenta can infect the plant Zizania latifolia and stimulate stem swollen at the base of the plant to form edible galls called 'Jiaobai'. In our study, we found that there are two types of U.esculenta: yeast form and mycelium form; as for Ustilago maydis, yeast form has no pathogenicity, but mycelium form could infect the corns. It was accepted that dimorphism of U.esculenta which played important roles in the edible galls's formation was regulated by a mating type loci containing functional genes which encoded lipopeptide pheromone precursors (mfa) and receptors (pra). The genome sequencing data showed the existence of mfa, pra homologous genes in U.esculenta, but there is no report about their structures and molecular mechanisms in dimorphism. In this study, we isolated the affinity haploid strains of U.esculenta (UeT14, UeT55, UeMT10, UeMT46) as experiment materials to study the structure and function of a loci at molecular biology level, and found out the mfa, pra genes'regulation mechanism of U.esculenta dimorphism.First, using the UeT55 haploid strain as experiment material, the conditions of enzyme hydrolysis, protoplast regeneration medium, osmotic pressure stabilizers, vectors, resistance marker were explored to optimize protoplast transformation system of U.esculenta. The protoplast transformation system provides experimental basis for U.esculenta gene function research. The UeT55 cells were almost completely broken after treated with 15 mg/mL Lywallzyme enzyme solution at 30? for 3 h when sorbitol or sucrose were used as osmotic stabilizer. The conversion rate was higher when the UeT55 strain was transformed with a linear plasmid with carboxin or hygromycin resistance gene and regenerated on a sucrose or sorbitol-containing regeneration medium.At the same time, the specific primers were designed and used for long fragment cloning according to the whole genome sequence of U.esculenta. The a locus of four strains (UeMT10, UeMT46, UeT14, UeT55) were cloned and analyzed, respectively. The results showed that there were three alleles al, a2, a3 in U.esculenta. Besides, each site had two pheromone genes (mfa) and a pheromone receptor gene (pra), which were analyzed by bioinformatics tools. The al locus was cloned from UeT14 strain, including mfal.2, mfal.3, pral genes; a2 locus was cloned from UeT55 and UeMT46 strains, including mfa2.1, mfa2.3, pra2 genes; a3 locus was cloned from UeMT10 strain, including mfa3.1, mfa3.2, pra3 genes. The high homology of mfa and pra genes was found among the U.esculenta, U.maydis and Sporisorium reilianum, suggesting that they might have similar functions that the a genes were related to cell fusion and filamentous growth in U.esculenta dimorphic conversion pathway.Then the ersonality affinity haploid strains UeT14, UeT55 were used as experiment materials, the mfa or pra gene deletion strains were obtained by PEG mediated protoplast transformation method, and the regulation mechanism was studied. The UeT14-? mfa1.2, UeT14-? mfa1.3, UeT14-? pral, UeT55-? mfa2.1, UeT55-? mfa2.3, UeT55-? pra2 gene deleted strains were used in fusion experiments, the results showed that:the growth of germ tube, cell fusion and mycelium growth could be performed in UeT14 and UeT55 strain when the two pairs of mfa1.2-pra2, mfa2.1-pral genes existed at the same time (wild strain or mfal.3/mfa2.3 gene deletion strain in fusion culture); the growth of germ tube could be performed in UeT14 and UeT55 strains when one pair of mfa1.2-pra2 or mfa2.1-pral genes existed at the same time; when mfa1.2-pra2 and mfa2.1-pral two pairs of each genes deletion were deleted, the UeT14 and UeT55 strains neither growth, nor for cell fusion and mycelial growth.Using Quantitative Real-time PCR (qRT-PCR) to analyze the expression patterns of a mating-type locus and its downstream pathway genes (Uegpa3, Ueubc2 genes) by the fusion experiments to study the signal regulation mechanism of dimorphic transition. First, the Uegpa3 and Ueubc2 genes in U. esculenta were cloned according to the transcriptome information database, the connections between Uegpa3, Ueubc2 genes and U.esculenta mycelium growth were studied by cell fuse process. Further qRT-PCR results showed that:the mfa, pra genes'expressions were significantly affected when any of mfal.2-pra2, mfa2.1-pral genes were missing; when lack of mfa 1.3, mfa2.3 genes, the other mfa, pra genes expression were almost not affected. The studies suggested that UeT14 and UeT55 compatible haploid cell fusion and mycelium growth signaling pathways were regulated by mfal.2-pra2, mfa2.1-pral genes. At the same time, the expression levels of Uegpa3 and Ueubc2 genes were detected, and the results showed that:the expression of Uegpa3 and Ueubc2 genes were relatively high at the germ tube initial growth stage and hyphal growth anaphase, and the trend was basically the same; when the colony of yeast type and no germ tube growth, Ueubc2 and Uegpa3 genes expression changes failed to show obvious regularity. That means there may be an interaction involved in U.esculenta dimorphic transition amony Ueubc2, Uegpa3 genes and mfa, pra genes, and need more experiments to further test. |
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