Cloning Of Soybean Amino Acid Transporter Gene UmamiT25 And Its Functional Study In Arabidopsis Thaliana

Soybean(Glycine max)is a native crop in China and also an important economic crop in the world.It can supply human beings with vegetable oil accounting for nearly60% of the total output of oil crops every year,and it is also the main source of human edible-plant protein.China is the world’s largest soybean consumer,with an average annual consumption of more than 100 million tons in recent years,accounting for about one-third of the total global soybean consumption.However,due to limited per capita arable land and yield per unit area,our country total annual soybean output is less than 20 million tons,and it is very dependent on imported soybeans.Therefore,it is particularly important to combine modern biotechnology with traditional breeding to cultivate new soybean varieties with stable,high-yield,high-quality,and independent intellectual property rights.Nitrogen is the main factor limiting crop yield.Nitrogen absorbed by higher plants from the outside is mainly assimilated into amino acids in the body,and then transported and distributed by amino acid transporters to various tissues and organs for their use.It has been found in Arabidopsis and rice that amino acid transporters can participate in plant growth and development and affect seed size and yield,but there are currently few reports in soybeans.This study analyzed a type of soybean amino acid transporter UmamiTs mainly expressed in reproductive organs through bioinformatics methods,and finally cloned the GmUmamiT25,which is only expressed in large quantities in seeds.Then the gene was constructed into plant expression vector and yeast expression vector,and its biological function was deeply studied through genetic transformation,subcellular localization analysis,transgenic plant phenotype analysis,substrate transport experiment,amino acid uptake experiment,etc.The main results as follows:1.The GmUmamiT25 with a total length of 1104 bp in CDS was cloned using the c DNA of the high protein soybean variety Zhechun-3 as a template.Analysis of its tissue expression pattern and subcellular localization showed that the GmUmamiT25 was localized to the cell membrane,mainly during the filling stage of seed development have a lot of expression.2.The GmUmamiT25 was overexpressed in Arabidopsis,analyzed and compared the phenotypes of overexpressed Arabidopsis and wild type,and found that the seeds of overexpressed Arabidopsis were significantly larger,protein content and yield per plant increased.Further analysis of the expression of genes related to the regulation of seed size,nitrogen transport and storage metabolism,we found that the expression of multiple positive regulatory genes of seed size and the expression of genes related to nitrogen transport and storage metabolism in overexpressed Arabidopsis were up regulated.3.We expressed GmUmamiT25 in yeast,and then through yeast secretion experiments,we found that transgenic yeast secreted more 14 kinds of amino acids such as Ala,Gly,Cln,and Val into the medium,indicating that soybean UmamiT25 is an amino acid exporter with a wide range of substrates.4.Adding different kinds of amino acids to the 1/2 MS medium to treated Arabidopsis seedlings,we found that the overexpressed Arabidopsis grows better than the wild type,indicating that GmUmamiT25 can export the excess amino acids accumulated in the cell to the outside of cell,thus reduce the toxic effect of excessive amino acid on plants.Prove that it also has the ability to export a variety of amino acids in plants.5.The amino acid content of siliques of Arabidopsis 12 days after flowering was determined,the results showed that GmUmamiT25 transgenic Arabidopsis increased significantly compared with the wild type,but the amino acid content in the pericarp of8 days after flowering was significantly reduced,indicating that GmUmamiT25 was involved in the amino acid transport process form pericarp to seed.6.We sequenced the transcriptome of the siliques 12 days after flowering and found that overexpressed Arabidopsis had 3513 differentially expressed genes compared with the wild type,of which 2312 genes were up-regulated and 1201 genes were down-regulated.GO enrichment analysis and KEGG pathway annotations show that most differentially expressed genes are enriched in different metabolic processes of plants,and their main molecular functions may be are enzymes with catalytic activity and binding proteins.In summary,this study cloned the GmUmamiT25,which is highly expressed in soybean seeds,and proved that it encodes the protein is an amino acid exporter in yeast and plants,located at the plasma membrane,which can mediate a variety of amino acids.At the same time,this gene was overexpression in Arabidopsis can significantly increase the size of seeds,increase protein content and yield per plant.This provides a theoretical reference for the follow-up research of soybean amino acid transporter,and also provides a new idea for cultivating new soybean varieties with stable,high-yield,high-quality and independent intellectual property rights.