Question

Plant physiology

You have done a genetic screen for a mutant plant line (Arabidopsis of course!) that can not use glutamate as a nitrogen source. Describe one experiment you will do to provide proof that your mutant is defective in nitrogen assimilation. Be specific and thorough with your answer.

Answer 1

Mutant plant ability to sense the nitrogen -Internal N status can be determined by the existence mechanisms which are affects key effector molecules which can provide reliable information in mutant plant tissue which are specific.Other words , finding molecules (proteins) which can act as sensors to specific N compounds by binding and transferring information to downstream compounds as a signal transduction pathway.

Genetic Screening of a mutant plant line can be obtained by following steps-

Arabidopsis contain nitrate regulatory mutants(nrg) which can be used as genetic screening to determine presence of N, followed by employing them as nitrate inducer promoter which are fused to yellow flurescent protein marker gene (YEP).Thus, identification of mutation was determined by impaired nitrate induction and localization to nitrate regulatory gene(NLP7) which demostrate the validation of screening.

By identifying two nitrate- nonresponding mutants, Fusion of nitrate inducible promoter(NRP) to DNA encoding YEP was transformed to arabidopsis. Homozygous arabidopsis transgenic plant line was obtained and screened for presence of nitrate responsive for YFP expression by studying plant tissue under flurescence microscope.After that ,Control Seeds were grown with 2.5mM ammonium succinate and grown on agarose plates containning no nitrate for 4 days and later treated with 20 mm KNO3 or 20 mm KCl. Test seeds were obtained from transgenic plant and grown with 2.5mM ammonium succinate and grown on agarose plates containning nitrate for 16 hours, and were treated with 20 mm KNO3 or 20 mm KCl.

Expression was analysed under fluroscence microscope which determined that test seed contain strong fluorescence than Control treated seeds with chloride,YFP expression succesfully induced by nitrate in nirogem assimilated plants.

Validation of mutant plant - Quantitaive PCR was performed by using genomic DNA obtained from transgenic plant which was amplified and sequenced.,which shoen the presence of codon q301 was succesfully converted to stop codon.