Map-based Cloning Of Arabidopsis N Nutrition Genes And Preliminary Functional Verification

Nitrogen (N) is one of the necessarily mineral elements for plant growth anddevelopment as well as a dominical effect factor on crop productivity. As the problemof N resource and environment has been increasingly severe, it is great theoretical andapplicable value for increasing N utilization efficiency through transgenic technique.In this study, two mutants resistant to N deficient, lnt3and lnt5form Arabidopsisthaliana Col-0mutated by EMS were used as materials to identify important genesrelated to N nutrition with map-based cloning technique.TT8gene was cloned with lnt3mutant. The gene translation protein product isTT8, which belongs to basic-helix-loop-helix domain (bHLH) transcription factors. Inthe lnt3, the760thbase of TT8exon was mutated from C to T, which leads tononsense mutation caused by genetic codon “CAA” changing into stop codon. Formutant lnt5, the location of candidate gene was narrowed between SSLP molecularmarker5-5654and5-5443, which was211kb and overlap3BAC Contigs, such asMKP11, F2K13and F5E19. During the cloning,21pairs of SSLP,1pair of CAPs and2pairs of SNP molecular markers were developed and a PCR-based AS-PCR methodwas modified and optimized, these enriched the molecular marker resource formap-based cloning, saved a great deal of time and improved cloning efficiency.The current research about TT8mainly focused on flavonoid biosynthesispathway, while there is little progress on its role in regulation of N metabolism. Inthis study, transcription factor TT8was suggested to be involved in the regulation ofN metabolism through analysis of lnt3mutant’s phenotype under stress of N deficient,N content in root and shoot, and N absorption results by using non-invasivemicro-test technique (NMT). At the same time, considering NMT results and thephenotype under stress of N deficient, the gene lnt5would play a critical role in Nuptake. The functional analysis of both genes should lay a solid foundation for furtherstudies of N utilization mechanism in plant and application of transgenic technique toimprove productivity on crops.