| Soybean is one of important industrial crops in China,and it is also the main source of vegetable oil and vegetable protein.Soy protein is as high as 35%-40%.Various soy products based on soybeans have become the first choice for people to take protein.while the national consumption of soybean has increased dramatically,making domestic soybeans far from meeting the domestic market demand.Therefore,using modern biotechnology combined with traditional breeding methods to discover key genes that regulate amino acid transport in soybean,and clarify the mechanism of protein synthesis in seeds will provide new ideas for breeding high-yield and high-quality new soybean varieties.Amino acids are called "the currency of nitrogen exchange" in plants,and it is the basic unit of protein.Amino acids can function only when they enter plant cells,and the corresponding amino acid transmembrane transporters play an important role in them.Amino acid transporters have been found in many organisms.At present,amino acid permeases(AAPs)are a relatively clear class of amino acid transporters studied,and they are widely involved in the absorption and transport of amino acids in plants.There are eight members of amino acid transporters in Arabidopsis,followed by At AAP 1-8.Existing research results have found that they directly or indirectly affect seed development,quality and yield-related traits.In this study,a bioinformatics method was used to perform analysis of soybean Williams 82 transcriptome data and sequence alignment of Arabidopsis AAP family proteins that have been reported.The Gm AAP3-like2 gene was identified as the research object and cloned.Through quantitative PCR analysis,it was found that Gm AAP3-like2 gene was expressed in all tissues of soybean,especially in flowers,apical meristems and fruit pods,which was consistent with the transcriptome data published online.At the same time,the gene was ligated into the p CAMBIA1300-GFP vector,and the successfully constructed vector was transferred into tobacco and Arabidopsis thaliana,and the subcellular localization and physiological function analysis of soybean Gm AAP3-like2 were performed.The main results are as follows:1.Bioinformatics analysis showed that soybean Gm AAP3-like2 gene is 1464 bp in length and encodes 487 amino acids.At the same time,the similarity between the protein sequence of Arabidopsis At AAP3 and soybean Gm AAP3-like2 was found to be 65.51%.2.Localization analysis results: Transient expression of soybean Gm AAP3-like2 in tobacco leaf epidermal cells revealed that the protein may be localized in the endoplasmic reticulum and plasma membrane;meanwhile,the obtained transgenic Arabidopsis seedlings were observed under a fluorescence confocal microscope.It was found that it has obvious plasma membrane signals.The results of GUS histochemical staining showed that the gene was mainly expressed in vascular tissues of various organs of plants,followed by higher expression on the stigma of the flowers and the pod shells 12 days after flowering.3.Overexpression and wild-type Arabidopsis seedlings were grown on 1/2MS medium containing different concentrations of amino acids,and it was found that Arabidopsis was overexpressed on medium supplemented with 100 m M alanine(Ala)and 4 m M glycine(Gly).Mustard seedlings exhibited stunted growth,short dwarf roots,some even died,and their growth was significantly inhibited compared to wild type.This phenomenon indicated that Gm AAP3-like2 promoted the uptake and transport of alanine(Ala)and glycine(Gly)by root cells.4.Gm AAP3-like2 overexpressed Arabidopsis seeds related agronomic traits were analyzed.Compared with WT,Gm AAP3-like2 overexpressed Arabidopsis strains OE8,OE19,and OE24 increased their thousand-grain weight by 14.02%,15.87%,12.15%,respectively;at the same time,observation and length/width measurement of mature Arabidopsis seeds under a stereo microscope,it is showed that the seed width of the three overexpressing lines had significant changes,which increased by 5.48%,9.46%,8.14%.Then counted the number of siliques per plant,and found that compared with WT,the overexpressed lines increased by 16.38%,12.25%,and 13.58%,respectively.Further analysis of the single plant yield of seeds,compared with WT,found that the yield of overexpressed plants also increased significantly,increasing by 17.93%,17.84%,and 13.97%,respectively.The above results indicated that soybean Gm AAP3-like2 was involved in the regulation of Arabidopsis seed development.In summary,soybean Gm AAP3-like2 participated in the uptake and transport of amino acids by cells and affects the development of Arabidopsis seeds.This provided a theoretical basis for further research on soybean amino acid transporter Gm AAP3-like2 participating in the regulation of protein synthesis.Thus sheding light on breeding new soybean varieties with high protein content and high yield. |
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