| Soybean(Glycine max)is an important grain,oil and feed crop in China,and the fourth largest crop after rice,wheat and corn.China’s soybean yield is seriously insufficient,the relationship between supply and demand is tense,and the increasingly harsh natural conditions restrict its yield.Sucrose,a source of carbon and energy,is produced during plant photosynthesis.However,salt,saline-alkali and drought stress can inhibit photosynthesis,thus affecting sucrose supply and reducing plant tolerance to environmental stress.Therefore,sucrose transport and allcation are critical steps in plant stress resistance.Sucrose transporters,the key role of sucrose transporters,is named of SUC or SUT.There are 16 members in soybean,which are divided into 3 subfamilies,and 9 members have been cloned,but their functions and mechanisms have not been studied in detail.This study first through bioinformatics analysis and fluorescence quantitative PCR method,found GmSUT4 under salt,saline-alkali and drought stress,which is significantly up-regulated expression,and high expression in different organizations.Then,the subcellular localization vector was constructed,and the subcellular localization results of GmSUT4 were analyzed.In addition,the function of GmSUT4 was preliminarily identified in yeast cells and transgenic Arabidopsis thaliana by heterologous expression system.Further,the predicted interacting proteins were identified by yeast BiFC and BiFC experiments.Finally,GmSUT4 was overexpressed in soybean,and phenotypes of T2 generation transgenic soybean were preliminarily identified,which laid a theoretical foundation for the subsequent study on the molecular mechanism of GmSUT4 gene’s involvement in stress response in soybean.The main results are as follows:1.Through tissue-specific expression analysis of soybean sucrose transporter family members and abiotic stress expression analysis,it was found that GmSUT4 gene was highly expressed in some parts of soybean tissues and significantly up-regulated under stress.2.The plant expression vector of GmSUT4::GFP fusion protein,PGDG-GmSUT4 was constructed.Transient expression of GmSUT4::GFP fusion protein was carried out by Nicotiana benthamiana,and fluorescence signal was detected under laser confocal microscope,and the results showed that GmSUT4 protein was located on the cell membrane.3.Saccharomyces cerevisiae SUSY7/ura3 was used to verify the sucrose transporter function of GmSUT4 protein.At the same time,the phenotype of yeast was observed on the medium under drought,salt and saline-alkali stress,which preliminarily indicated that the overexpression of GmSUT4 gene had the function of improving stress resistance.4.Through website prediction and related literature review,it was found that GmSUT4 and Gm Cyt b5-4 may interact with each other,and then the interaction relationship between GmSUT4 and Gm Cyt b5-4 was identified by yeast two-hybrid and BiFC method.Further,the possible interactive motifs were verified by yeast twohybrid by cutting.In addition,combined with the potential binding sites predicted by Pi SITE,the 82 Leu was proved to be the key amino acid site influencing the interaction of two genes by point mutation and yeast double hybrid methods.5.The results showed that the germination rate of transgenic Arabidopsis was significantly better than that of sut4 and wild-type material by analyzing the salt,saline-alkali and drought phenotypes of sut4,wild-type and overexpressed GmSUT4.However,there was no significant difference in root length between wild-type and overexpressed lines,while the root length of sut4 was significantly shorter.In addition,under salt,saline-alkali and drought stress,the transgenic lines were also significantly better than sut4 and wild-type materials,among which the sut4 had the most severe chlorosis.The contents of sucrose,glucose,fructose and starch in the above-mentioned Arabidopsis materials were further detected,the results found that the over-expression plant as salt and saline-alkali stress after compared with other materials,The high concentration of soluble sugar increases and accumulates,which improves the stress resistance of soybean.6.GmSUT4 was overexpressed in soybean by agrobacterium tumefaciens mediated stable genetic transformation of soybean cotyledon nodes,and 5 transgenic soybean lines were obtained.Furthermore,preliminary phenotypic identification was conducted on the T2 generation transgenic soybean with high expression,and the results showed that compared with the wild type,the plant height was significantly increased,which provided material basis for the subsequent study of its stress-related functions. |
