Cloning,Expression Analysis And Functional Study Of GmAAP Gene In Glycine Max

Amino acid permease?AAP?plays a crucial role in the process of absorbing and transporting amino acids in plants.AtAAP1 is the first amino acid transporter found in plants,and has been reported in crops such as rice,corn,potato,pea,broad bean,and kidney bean.At present,research on GmAAP in soybean is rare.AAP can increase the absorption and utilization of exogenous nitrogen in legumes,thereby increasing the storage protein content in seeds and improving soybean quality.In this study,the GmAAP gene was successfully cloned from soybean,and bioinformatics analysis and subcellular localization were performed to identify the region where the gene functions.At the same time,the expression pattern of this gene was studied in soybean and transgenic Arabidopsis,respectively.The amino acid absorption characteristics of GmAAP gene were clarified by 20 kinds of amino acid affinity experiments and the content of 20 encoded amino acids in Arabidopsis thaliana seeds transfected with GmAAP gene.Through a single amino acid culture experiment,the GmAAP gene expression and key enzyme activities of nitrogen metabolism in transgenic soybean hairy roots were determined,aiming to analyze the relationship between GmAAP gene and nitrogen metabolism.The experimental results can lay a foundation for the study of GmAAP function,and also provide candidate genes for the efficient utilization of soybean nitrogen.The main results are as follows:1.The GmAAP gene was homologously cloned by RT-PCR using soybean Williams 82as experimental material.Sequence analysis showed that the fragment length was 1440 bp encoding a complete open reading frame?ORF?of 479 amino acid residues.Bioinformatics analysis showed that the molecular mass of GmAAP protein was 53.286 kD,the theoretical isoelectric point was 8.93,and the instability index was 34.04,which was a stable protein.The?-helix,extended strand,?-turn,and random coil in the secondary structure account for47.18%,15.66%,2.71%,and 34.45%,respectively.The transmembrane region and hydrophobicity analysis showed that it contained 10 transmembrane regions,and the amino acids constituting the transmembrane region were mostly hydrophobic amino acids.GmAAP is most closely related to wild soybean GsAAP,and the consistency of sequence alignment is100%.The consistency of GmAAP with mung bean,pigeon pea and alfalfa was 91.93%,90.55%and 82.78%.The sequence of 10 transmembrane regions is more conservative.In order to investigate the subcellular localization of GmAAP,the fusion expression vector was constructed and then transformed into Arabidopsis protoplasts by the method of PEG-Ca²⁺mediated transformation.The results showed that GmAAP was localized on the plasma membrane.2.The expression level of GmAAP gene in different stages and different parts of soybean was determined by real-time PCR.It was found to be mainly expressed in soybean roots and leaves,followed by expression in pods and seeds,and the lowest expression was in stems and flowers.T3 generation Arabidopsis transgenic with GmAAP gene were obtained,and the expression of GmAAP in transgenic Arabidopsis thaliana was determined by qPCR.It was found to be expressed in Arabidopsis whole growth period and all tissues,and the highest expression in leaves and roots,followed by fruit pods,stems and seeds,and the level of expression in flowers is very low.The relative expression of GmAAP in leaves was significantly higher than that in roots by transgenic Arabidopsis under nitrogen stress.3.GmAAP has a certain affinity for most amino acids,but it is mainly responsible for the absorption and transport of neutral amino acids,especially for aromatic amino acids,amides,prolines and leucine.At the same time,it also has a certain affinity for basic amino acids,but does not participate in the uptake of acidic amino acids.Compared with wild-type Arabidopsis thaliana,the content of 20 amino acids in Arabidopsis thaliana seeds increased to different degrees,and the increase of alanine,asparagine,proline and aromatic amino acids was the most obvious.The content of aspartic acid,lysine,glycine,and cysteine was decreased.The hairy roots of soybeans overexpressing GmAAP were cultured by using aromatic amino acids as a nitrogen source.The activities of nitrate reductase and glutamate dehydrogenase as well as total free amino acid content were enhanced to varying degrees compared to the control group.Correlation analysis showed that the relative expression of GmAAP was significantly correlated with the changes of glutamate dehydrogenase activity and total free amino acid content.This indicates that the GmAAP can absorb specific amino acids and can be used for soybean nitrogen utilization breeding research.