| Regulation of nitrogen metabolism in yeast is complicated under different nitrogen condition.In this thesis,the genome of Saccharomyces cerevisiae N85 and XZ-11 were firstly sequenced and annotated.Comparative genomic analysis was conducted to explore the key genomic variants involved in nitrogen metabolism.Besides,the expression pattern of genes related to nitrogen metabolism under different nitrogen conditions and in different strains were investigated through comparative transcriptomic analysis.Finally,a rapamycin-mediated protein anchoring system was established and used to regulate the intracellular localization of nitrogen catabolism repression(NCR)regulators.The main content are as follows:(1)Whole genome sequencing of S.cerevisiae N85 and XZ-11.The genomes of strain N85 and XZ-11 were sequenced by both next-and third-generation sequencing technologies.The genomes of S.cerevisiae N85 and XZ-11 were assemblied through reference assembly and de novo assembly methods,respectively.The complete genome sequence of strain N85 and XZ-11 were obtained through integrating the results of these two assembly methods.The genome size of S.cerevisiae N85 and XZ-11 is 12.10 Mbp and 12.17 Mbp,and the GC content is 38.28% and 38.32%,respectively.Gene features were annotated through the combination of three different approaches: ab initio,evidence-,and homology-based predictions.6,635 and 6,648 protein coding regions were identified in the genome of strain N85 and XZ-11,respectively.In addition,275 tRNA were annotated using tRNAscan-SE in both genome of strain N85 and XZ-11.(2)Comparative genomics analysis to identify genomic variants associated with nitrogen metabolism.Totally 12,309 genomic variants were identified between genomes of strain N85 and XZ-11.Besides,6,564 genes were found on both genomes of strain N85 and XZ-11.71 and 84 genes were unique in the genome of strain N85 and XZ-11,respectively,in which ARG2 is an important gene involved in urea metabolism.ARG2 may guide the carbon and nitrogen metabolic fluxes flowing through glutamate into the urea cycle.Replenishing ARG2 in strain XZ-11 decreased the urea utilization by about 25.04%.Comparative genomic analysis of the genome of evolved strain 4B and the wild type strain XZ-11 revealed that AAT2 and PRO1 evolved rapidly during adaptive evolution.Heterologous expression of the AAT2 and PRO1 of strain 4B in strain XZ-11 decreased the urea amount by 34.29% and 23.96%,respectively.(3)Genome-wide association study(GWAS)to identify genomic variants associated with nitrogen metabolism.Firstly,a flow cytometry-based high-throughput screening technology was established.The haploid N85 and XZ-11 strain were crossed to generate diploid heterozygote.After inducing the sporation of heterozygote,9600 haploid segregants were screened.Among them,96 segregants with the highest and 96 segregants with the lowest urea accumulation were selected and named as H and L group,respectively.The mixed genomes of group H and L were sequenced.Three genes,PUT3,VBA5 and VBA3 were identified to be associated with nitrogen metabolism via GWAS.Replenishing the variant sites of the three genes in strain N85 decreased the urea accumulation by 16.53%,20.17% and 10.24%,respectively.(4)Comparative transcriptomic analysis to interpret the regulation mechanism of yeast nitrogen metabolism.The transcriptomes of strain N85 and XZ-11 under different nitrogen conditions were sequenced.After analyzing the different expression genes,it was found that yeast cells promoted the interaction between intracellular amino acids and free tRNAs by upregulating the expression of amino acid-tRNA ligase under the prefered nitrogen condition.As a result,the level of intracellular free tRNA decreased,which leads to activate the TOR pathway and inhibit the utilization of non-preferred nitrogen sources.In addition,in strain XZ-11,the nitrogen metabolic flux from glutamate into urea cycle is smaller than that in strain N85,but the activity of urea degradation pathway is higher than that in strain N85.These two patterns result that the urea accumulation in strain XZ-11 is lower than that in strain N85.Furthermore,the activation function of Gln3 for the transcription of nitrogen metabolism-related genes in strain XZ-11 is stronger than that in N85 strain.The inhibition function of Dal80 for the transcription of genes involved in nitrogen metabolism is weaker than that in the strain N85.(5)Regulating the subcellular localization of NCR transcription factors to release NCR.The protein anchoring system was constructed by knocking out FPR1,which encodes the rapamycin binding subunit and mutating TOR1.On the basis of this system,the transcriptional activators Gln3 and Gat1 were anchored in the nucleus and the transcriptional repressors Gzf3 and Dal80 were anchored in the cytoplasm after induced by rapamycin.It was found that,in strain XZ-11,the prefered nitrogen sources induced inhibition of non-preferred nitrogen sources utilization was mainly due to the sequestration of Gln3 in the cytoplasm and the translocation of Dal80 into the nuclear.Furthermore,anchoring Gln3 and Gat1 in the nucleus can reduce the urea amount by 49.45% and 38.99%,respectively.And anchoring Dal80 and Gzf3 in the cytoplasm can reduce the urea amount by 42.44% and 21.52%,respectively. |
