Cloning,Expression And Functional Analysis Of Amino Acid Transporter ZmAAP Gene In Maize

The amino acid transporter AAPs gene was first discovered in Arabidopsis thaliana.A large number of experimental results demonstrated that the AAPs gene in Arabidopsis thaliana regulates the type and number of amino acids in the plant.More and more scientists have demonstrated that AAPs are actively involved in the absorption of nutrients and transport of metabolites in some plants.However,the study on AAP gene in maize is rarely reported.Therefore,this study aims to clone the AAP gene from maize and explore its function.In this study,maize ZmAAP gene was cloned and its function was analyzed by background expression and eukaryotic expression.The main results were as follows:1.By designing degenerate primers based on the AAP1 gene sequence of Arabidopsis thaliana and barley,a partial sequence of the maize AAP gene was cloned using the synthetic maize cDNA as a template.Sequencing of the AAP gene partial sequence in Arabidopsis and Arabidopsis was confirmed by sequencing.The similarity of barley AAP gene sequence reached 87.08%.A fragment of maize gene with unknown function was obtained by BLAST of maize AAP gene partial sequence in NCBI.The unknown functional gene fragment 1396 bp was cloned by RT-PCR.Using bioinformatics analysis of this sequence,we know that the nucleotide sequence of the unknown functional gene coding region has the highest similarity with the barley AAP1 gene,and the similarity is 87%.It is initially proved that the gene cloned from corn may be the nucleic acid of AAP gene.The sequence,named ZmAAP.The ZmAAP gene full-length cDNA sequence includes a 1212 bp open reading frame,the ZmAAP gene encodes an amino acid sequence of 403 amino acids,a relative molecular weight of 42882.13,a theoretical isoelectric point of 9.24,and the total number of positive and negative charge residues is 18 and 30,respectively.Among the 20 amino acids encoded by ZmAAP protein,serine accounted for 13.4%,while histidine accounted for only 1.0%,and the total average hydrophilicity was 0.220.The ZmAAP protein contains a signal peptide with cleavage sites at 38,39 residues;there are 5 transmembrane regions.2.Fluorescent quantitative PCR was used to determine the different growth stages(emergence stage,jointing stage,big bellmouth stage,tassel phase,silking stage,milk ripening stage,and ripening stage)of maize plants and different maturity(10 days,20 days,30 days).Days,40 days,50 days,60 days)Tissue expression of ZmAAP gene in maize seeds.At the same time,the three physiological and biochemical indexes of total free amino acids,soluble protein content and 20 kinds of amino acid components were determined at different growth stages of maize plants and seeds of different maturity.The correlation between physiological and biochemical indexes and ZmAAP gene expression was analyzed.The change of total free amino acid content in maize roots,leaves and seeds was consistent with the change of ZmAAP gene expression.The change of soluble protein content in maize leaves and seeds was consistent with the change of ZmAAP gene expression.3.After the plant expression vector pCAMBIA3301-ZmAAP was constructed,the ZmAAP gene was introduced into Arabidopsis thaliana by Agrobacterium-mediated method,and then the ZmAAP gene Arabidopsis thaliana was obtained by screening with herbicides to obtain T2 transgenic Arabidopsis thaliana 25 strains.The T2 transgenic Arabidopsis thaliana was used as experimental material.Phenotypic analysis of Arabidopsis thaliana at different growth stages showed that the root length,number of lateral roots,leaf surface area,and number of branching stems of the transgenic Arabidopsis thaliana were significantly higher than those of Arabidopsis thaliana.Wild type Arabidopsis thaliana.4.Through determination of total free amino acid content and soluble protein content in transgenic and wild Arabidopsis thaliana,it was found that total free amino acid content and soluble protein content in roots and leaves of transgenic Arabidopsis thaliana were significantly higher than in wild type during the whole growth period.Arabidopsis thaliana.The tissue expression of the ZmAAP gene in transgenic Arabidopsis thaliana plants at different growth stages was determined by real-time fluorescence quantitative PCR.The correlation analysis of ZmAAP gene expression levels with total free amino acid content and soluble protein content showed that the ZmAAP gene expression level was determined.The changes were consistent with changes in total free amino acid content.Therefore,expression of the ZmAAP gene in Arabidopsis can increase the amino acid content in Arabidopsis thaliana.