Screening And Functional Verification Of NADP-ME Promoter Binding Protein In Maize

Maize is one of the three major food crops in the world,and it is also the main feed and industrial raw material.It can be an effective way to improve maize yield by improving the photosynthetic efficiency of maize so as to give full play to its high yield potential.In C₄plants,nicotinamide adenine dinucleotide phosphate-dependent malic acid enzyme（NADP-ME）is the most widely used decarboxylase,whose main function is known to release CO₂ from vascular bundle sheath cells to effectively enter Rubisco,thereby improving the catalytic efficiency of substrates.In this study,the differences of nadp-me in related photosynthetic physiological indexes were analyzed to determine that the normal gene functional expression of NADP-ME is indispensable for the normal growth of maize plants.According to the Ch Ip-seq data predict NADP-ME transcription factors function structure domain,using Yeast one Hybrid maize photosynthesis related technical mining ERF transcription factors family,build the ERF-C₄ NADP-ME transcription regulation pathways.Analyze the influence of external light intensity,light quality,and photoperiod on this pathway,and lay a theoretical foundation for revealing the regulation mechanism of corn photosynthesis and achieving C₄ transformation of C₃ plants.The main research results are as follows:（1）The leaves of UFMu5 mutants（5’UTR inserted）seedlings were pale green after unearthed.The illu3 mutant（third exon insertion）has yellow leaves and trefoil death after emergence,suggesting that this gene is necessary for the normal survival of the plant.By measuring the photosynthetic physiological indexes of UFMu5,found that the mutant net photosynthetic rate of about 3μmol m?2s?1,and Fv/Fm（The maximum photosynthetic capacity）decreased significantl.It was proved that the mutant was a typical photosynthetic defective mutant.（2）Two mutants of nadp-me,UFMu5 and illu3,were identified to be inserted into the5’UTR and the third exon of NADP-ME（GRMZM2G085019）.According to the results of Fluorescence Quantitative PCR and Western Blot detection,NADP-ME gene could not be transcribed and translated normally in the mutant.It is speculated that the failure of the gene to perform normal functional expression may lead to phenotypic changes in the mutant and the failure of maize plants to grow and bear fruit normally.（3）Through Yeast one Hybrid technology,Ch IP-Seq data and Dual-Luciferase technology,it was found that ZmERF172 and ZmERF198 could bind to the promoter region of NADP-ME,activating the expression of NADP-ME,and then affecting the photosynthesis of maize by affecting the activity of NADP-ME.（4）Through Yeast two Hybrid technology,we found that ZmERF198 interacts with PIF₃,ZmMYB106,IAA14,etc.（5）MEAG7 software was used to perform cluster analysis of AP2/ERF transcription factors in two species of maize and Setaria viridis,and homologous genes of ZmERF172 and ZmERF198 in Setaria viridis were respectively recorded as Sv ERF172 and Sv ERF198.Through the techniques of CRISPR/Cas9 and plant tissue culture,positive plants of partial stable transformation of Setaria viridis have been obtained.（6）ZmERF172 and ZmERF198 have a high degree of homology in evolution.The sequences of genes ZmERF172 and ZmERF198 were edited by using CRISPR/Cas9technology.At present,we have been obtained zm-erf198-crispr plants.The sequencing results showed that the exon deletion of 1 bp,the base shift,and the amino acid sequence changes,but the mutants showed no significant phenotypic changes.