Functional Analysis Of Transaminase Gene ZmLL-DAP-AT In Maize

LL-diaminopimelate aminotransferase is involved in the biosynthesis of lysine and is also related to pathogen resistance.In our previous study,the transaminase gene ZmLL-DAP-AT was cloned in two maize inbred lines N04 and Dan232 with significant differences in grain size.Based on the sequence of two inbred lines,preliminary predictions were performed,including sequence comparison,homology analysis and function prediction.In this study,the promoter of ZmLL-DAP-AT gene was amplified from the dent maize inbred line Dan232 with large-sized grains,the popcorn inbred line N04 with small-sized grains and several other maize inbred lines.The cis-elements were predicted and compared.Overexpression of ZmLL-DAP-AT in Arabidopsis thaliana was analysis functionally.Transcriptome sequencing analysis were done using the siliques of 20 days after pollination in the wild-type and transgenic lines as materials.The main results are as follows:1.The promoter of ZmLL-DAP-AT gene was amplified from the inbred line Dan232,N04 and other inbred lines.The prediction showed that its promoter contained various light-responsive elements,endosperm expression element and elements involved in stress and hormone.The plant expression vector EGFP-ZmLL-DAP-AT was constructed for ZmLL-DAP-AT gene and transiently expressed in Nicotiana benthamiana.The results indicated that the ZmLL-DAP-AT protein was localized to the cell membrane.2.The plant expression vector p CAMBIA3301-ZmLL-DAP-AT was transformed into Arabidopsis thaliana.Expression analysis of ZmLL-DAP-AT in homozygous transgenic lines showed that the expression level of the gene was higher in leaves and in the fruit pods at 20 days after pollination.Analysis of the mature grain phenotypes of T3 plants showed that the leaf size of the transgenic lines increased significantly and the flowering period was significantly delayed;the grain weight per grain increased significantly;grain surface area,grain length,and mature grain cotyledon area all significantly increased,and the mature embryonic cotyledon epidermal cell area increased.ZmLL-DAP-AT might regulate the size of Arabidopsis cells by affecting the expression of related genes.3.Transcriptomic sequencing was performed on the fruit pods of 20 days after pollination for the wild-type and transgenic lines.According to the sequencing results,there were 202 differentially expressed genes,9 up-regulated and 193 down-regulated.GO enrichment indicated that these differentially expressed genes were involved in processes such as cells,cellular components,single biological processes,cellular processes,metabolic processes,organelles,catalytic activity,stimulatory responses,cell membranes,and binding.Pathway enrichment analysis revealed that these differential genes were totally involved in 72 pathways,includingmetabolism,synthesis of secondary metabolites,starch and sucrose metabolism,amino acid biosynthesis,plant hormone signal transduction,tryptophan metabolism,and other.The results preliminarily confirmed the role of ZmLL-DAP-AT in leaf development,flowering regulation and grain development.Its related genes was identified by transcriptome.It was speculated that the overexpression of ZmLL-DAP-AT might affect the Arabidopsis phenotype by regulating the expression of other genes.The function and molecular mechanism of ZmLL-DAP-AT in maize kernel development and lysine biosynthesis need to be studied in further research.