Cloning Of NIA Promoter Of Nitrate Reductase Gene And Screening Of Its Interacting Protein In Nicotiana Tabacum

In order to regulate the nitrate assimilation and the response of nitric acid signal well,the transcriptional factors of the nitric acid reductase promoter gene NIA1,NIA2 and the hypothetical cis element NRE2 were screened and identified.In this experiment,tobacco genome was used as template to select approximately 1 kb upstream of NIA gene,primers were designed to clone the promoter area of NIA gene,and the GUS plasmids were constructed to identify the promoter function.The cDNA Library of K326 was used as the material.The construction of K326 yeast one-hybrid system and the screening of transcription factors of gene NIA1,NIA2 and NRE2 cis elements were completed,according to Matchmaker(?)Gold Yeast One-Hybrid Library Screening System from Clontech.As results,the cloned upstream segment of the NIA was confirmed to has the promoter function after bioinformatics analysis and identification of GUS,and the cDNA library was well constructed.14 cDNA sequences were screened out via sequencing and bioinformatics analysis,8 for NIA1,3 for NIA2 and 3 for NRE2.Bioinformatics analysis showed that 12 of the 14 protein gene sequences were tobacco homologous.Analysis of positive cloned amino acid sequence were conducted by PredictProtein online tool.The prediction results of binding sites showed that the five positive clone protein sequences screened by NiA1 promoter bait fragment,two positive clone protein sequences screened by nia2 promoter bait fragment and two positive clone protein sequences screened by 4 × NRE2 bait fragment contain DNA/RNA binding regions and protein binding regions,which have the function of binding with nucleic acid sequence and protein,and can satisfy the structure of transcription factor DNA binding domain requirements.There were many protein binding sites in other positive clones,which may be involved in the regulation of some life processes after forming ligands with other proteins,but its specific functions need to be further verified.