菰黑粉菌T-DNA突变体库的构建及分析

通过建立适用于菰黑粉菌Ustilago esculenta的农杆菌介导遗传转化(Agrobacterium tumefaciens-mediated transformation,ATMT)体系，构建菰黑粉菌T-DNA插入突变体库。针对性地筛选双核菌丝形成缺陷型转化子，并对T-DNA插入位点进行分析，为研究菰黑粉菌二态型转换的分子调控机理打下基础。以构建的菰黑粉菌自融合菌株TSP为出发菌株，以含有遗传霉素(G418)抗性基因(neo)的质粒为载体，通过ATMT构建菰黑粉菌T-DNA突变体库，并对诱导剂乙酰丁香酮(AS)浓度、转化的共培养时间、农杆菌浓度和菰黑粉菌芽孢子浓度等建库影响因素进行单因素条件试验，筛选最优条件；对继代培养的转化子基因组中的遗传霉素抗性基因进行PCR检测，验证转化子遗传稳定性；对突变体库中的转化子双核菌丝生长情况进行观察，测定其双核菌丝形成能力；对上述双核菌丝形成缺陷型转化子进行基因组重测序，分析其T-DNA插入位点。当遗传霉素浓度为75μg/mL时，菰黑粉菌的生长被完全抑制。当AS浓度为100μg/mL、共培养时间为24 h、孢子浓度为1×10⁵

Construction and analysis of the T-DNA insertion mutant library of Ustilago esculenta

Agrobacterium tumefaciens-mediated transformation (ATMT) system was optimized and T-DNA insertion mutant library of Ustilago esculenta was constructed. Mutants defective in fusion and dikaryotic hyphal formation were screened and the T-DNA insertion sites were analyzed for further research on molecular mechanism of dikaryotic hyphal formation of U. esculenta. An artificial modified strain (TSP) with the ability of self-mating and a vector containing resistance gene (neo) of G418 were used for library construction. Optimal conditions of ATMT, including concentration of acetosyringone (AS), co-cultivation time, concentration of A. tumefaciens, and concentration of U. esculenta spores, were screened based on single factor conditional test. Genetic stability of T-DNA in mutant was tested by PCR with the detection of neo target. Mating assay was performed on mutants to test the ability of fusion and dikaryotic hyphal formation. Genome of defective mutants formed in fusion and dikaryotic hyphal formation was sequenced for T-DNA insertion site analysis. When the concentration of G418 was 75 μg/mL, the growth of TSP was completely repressed. AS concentration of 100 μg/mL, co-cultivation time of 24 h, spore concentration of 1×10⁵ spores/mL, and A. tumefaciens concentration of OD₆₀₀=0.3, were the optimal ATMT conditions for U. esculenta. Seven mutants were randomly selected and subcultured for ten times on YEPS solid medium. Result of neo target detection of the seven mutants showed T-DNA insertion were stably inherited. Observations showed five mutants had defects in dikaryotic hyphal formation. Whole genome resequencing of two of them (TSP-1 and TSP-23) showed T-DNA inserted in the exon of mfa2.1 a loci (GenBank: MK097140.1) (TSP-1) and intergenic regions of two hypothetical proteins (TSP-23) respectively. In summary, this study optimized the ATMT system and constructed ATMT mutant library of U. esculenta, and dikaryotic hyphal formation defective mutants were screened and the T-DNA insertion sites were analyzed by genome resequencing. Further study on the regulation mechanism of the dimorphism transition of U. esculenta needs to be carried out.