The Metabolic Regulatory Network Of The Uracil Salvage Pathway Was Analyzed Using Forward Genetics

Biomacromolecules,including nucleic acids,proteins,lipids,and polysaccharides,are the basic building block for living organisms.Nucleotide is not only the basic substance of metabolism,but also carries the genetic information of the organism.According to the central dogma,the primary metabolism such as the synthesis and metabolism of nucleotide,directly affects the secondary metabolism and the corresponding regulatory network.This study investigated the regulation network of pyrimidine salvage pathway by using forward genetics,molecular biology and bioinformatics.The results are as follows:1.Firstly,in order to identify the genes involved in the regulation network of pyrimidine salvage pathway,we constructed a mutant library generated by Ethyl methane sulfonate（EMS）mutagenesis.We selected wild-type Ler of Arabidopsis thaliana as the M₀generation,the seeds were soaked with Ethyl methane sulfonate（EMS）overnight,the treated seeds are sown into soil,after self-fertilization M₁ seeds were harvested.EMS mutagenesis will lead to base changes on the chromosomes.As a result,different types of mutants could be generated.2.After obtaining the M₁ mutant seed pools,we performed screening of mutants from M₁ seeds.As we reported before,UKL1 and UKL2 are the key players for pyrimidine salvage pathway.When the UKL1 and UKL2 genes are knocked out,corresponding uridine and its fluoro-derivatives cannot be channeled into pyrimidine salvage pathway for nucleotide biosynthesis.Thus,the ukl1 and ukl2 mutants can grow normally on MS medium containing 5-fluorouridine（5-FD）and5-fluorouracil（5-FU）.Based on this phenomenon,we screened mutants that are resistant to 5-FU or 5-FD,and the recovered mutants are more likely involved in the regulation of pyrimidine salvage pathway.3.After four generations of screening,24 stable mutant lines were obtained.Then a genetic analysis was conducted.The mutants were crossed with Ler wild-type and obtained F₁ seeds,then the F₁ seeds were tested for their resistance to 5-FD or 5-FU.Partial of the F₁ seeds were propagated to get F₂seeds,and the separation ratio of resistant to sensitive seedling on 5-FD or 5-FU containing plates were counted to determine if the mutant showed single gene Mendel inheritance.4.We selected 16 single gene recessive mutants and conducted Bulked Segregate Analysis（BSA）.About 150 resistant seedling segregated from F₂ population were mixed to make one“gene pool”,a“gene pool”from WT also was made in the same way as control,this ensures sequencing results highly reliable.5.Based on BSA sequencing results,one mutant（148D-1）was selected for further characterization.This mutant has a base change which change of the serine（S1030）into phenylalanine（F1030）of the encoded protein,which is predicted to be involved in across membrane transport.