Preliminary Functional Study Of Transaminase Gene ZmDAP And Its Related Genes In Maize

High and stable yield of maize is very important for food security.Mining related genes which controlling grain weight is beneficial to achieve high and stable yield of maize.Diaminopimelate aminotransferase is involved in the biosynthetic pathway of lysine and participates in the regulation of pathogen resistance.On the basis of previous study,the open reading frame（ORF）and promoter sequence of the transaminase gene ZmDAP of the corn inbred line Dan232 and N04 were successfully cloned,and the sequence alignment and function prediction were carried out.And,overexpression transgenic plants were obtained through the genetic transformation of Arabidopsis thaliana,and transcriptome sequencing was performed on the pods of the overexpression lines 20 days after pollination.In this study,based on the transcriptome results of Arabidopsis lines overexpressing the ZmDAP gene,we selected 6 genes（ethylene biosynthesis,nitrogen cycle,circadian rhythm,TCA cycle,and photosynthesis）related to ZmDAP gene function.The ORF and promoter sequences were amplified in maize inbred lines N04 and Dan232,and their sequences were analyzed and compared.The functions of ZmDAP and its related genes were studied by bioinformatics,Arabidopsis genetic transformation,subcellular localization and maize transgenic methods.The main results are as follows:1.Bioinformatics analysis showed that the DAP gene belongs to the fifth family Aminotran₁₂（PF00155）in the AAT-I superfamily（aspartate aminotransferase superfamily）.The protein sequence analysis of DAP family genes in Arabidopsis（31）,rice（32）,and maize（50）showed that the sequence of this gene is not conservative in different plants,indicating that the DAP gene has functional specificity.Expression analysis indicated that this gene may have developmental temporal and spatial specificity.In addition,the plant height of the Mu mutant of ZmACS6 in this gene family was significantly reduced.2.Using the ninhydrin colorimetric method,the lysine content in Arabidopsis thaliana was reduced when the ZmDAP gene was overexpressed.In addition,the leaves of At DAP mutants became smaller,the number of rosette leaves decreased,and the thousand-grain weight decreased.3.Analyzing the transcriptome data of Arabidopsis plants overexpressing the ZmDAP gene,we found out 6 genes（ZmACO1,ZmSAM1,ZmMYBE1,ZmLHCb7,ZmNIT2,and ZmFH1）related to the function of the gene,and the full length of these genes was amplified in Dan 232 and N04.The ZmMYBE1 gene has 2 amino acid differences between Dan232 and N04,and the amino acid sequences of the remaining5 genes are the same.Bioinformatics predictions show that ZmACO1 protein belongs to the 20G-Fe_Oxy superfamily;ZmMYBE1 belongs to the Myb_dna binding protein;ZmLHCb7 contains the Chloroa_b-bind superfamily domain;ZmSAM1 belongs to the S-adenosylmethionine synthase superfamily;ZmNIT2 contains carbon Nitrogen hydrolase（CN_hydrolase）domain;and ZmFH1 belongs to the lyase family.To predict its subcellular location,only ZmMYBE1 is located in the nucleus,and the rest are located in the cytoplasm,mitochondria,etc.In different plants,phylogenetic tree analysis and sequence alignment of the protein sequences of these six genes showed that they are relatively conserved among different species.The plant expression vectors were constructed and transfected into Arabidopsis.Positive plants have been obtained.4.Using Dan 232 and N04 corn inbred lines as materials,this study cloned the promoter sequences of the above 6 genes.The results showed that the promoter sequences of these six genes in Dan232 and N04 were not significantly different.Among them,the promoter sequence of ZmMYBE1 in N04 has a relatively long sequence insertion compared with Dan232 and B73.The cis-acting elements of the promoter sequence were predicted,and the results showed that the elements related to light response,hormones,and stress were predicted.5.The two promoter sequences of the ZmDAP gene that have been obtained were constructed on the p GreenⅡ0800-LUC vector,and their activity was detected by a dual luciferase reporter gene detection kit.The results showed that the promoter activity of the ZmDAP gene was in higher Dan232.Next,we constructed the differential sequence of the promoter into the p ABAi vector,and analyzed its upstream transcriptional regulatory factors through the yeast one-hybrid technique.6.GFP-ZmDAP vector was constructed and transformed into maize protoplast for subcellular localization and observation under laser confocal microscope.At the same time,ZmDAP corn overexpression and CRISPR vector were constructed to transform B104 corn inbred line.Currently,over-expression and CRISPR-positive strains have been obtained.