Screening And Functional Analysis Of SWEET Family Genes Regulating Fructose Accumulation In Strawberry Fruit

Carbohydrates such as sucrose,glucose,and fructose are the major products of photosynthesis in higher plants,Because it cannot be transported independently across the plant cell membrane system,it requires the assistance of the corresponding sugar transporter,such as the monosaccharide transporter,the sucrose transporter,and the sugar efflux transporter.As a novel sugar transporter,SWEET protein plays an important role in sugar export and transport,including plant growth,development and response to environmental stimuli.Existing results have shown that they regulate carbohydrates in plants.Transportation,distribution and storage are involved in important physiological processes of plant growth and development.Fructose is mainly stored in the vacuoles of plants.Most of the SWEET family members are located on the plasma membrane,but some members also act on the vacuolar membrane,which is responsible for transporting vacuolar fructose.SWEET1 plays an important role in regulating transmembrane of glucose.Therefore,the major goal of this paper is to find out the way of fructose transport in cells,to explain the expression of SWEET gene,artificially control the flow of plant carbohydrates,the accumulation process in fruit and the molecular mechanism of fruit quality formation,and improve crop yield and quality.There are very important theoretical and practical application values for breeding disease-resistant and stress-resistant varieties.In this experiment,the fruit of the red-colored strawberry coloring period was used as a test material,and two SWEET family transporter gene FaSWEET1 were cloned and analyzed by bioinformatics analysis.The bioinformatics analysis was carried out by using real-time quantitative PCR technology on FaSWEET1 in strawberry.The expression patterns of seven different developmental stages,starvation days,tissue culture seedlings,and different sugar starvation periods were analyzed.At the same time,the expression pattern of FaSWEET family related functional genes in different sugar concentrations of strawberry was compared by real-time PCR.The overexpression vector pCXSN-Flag-FaSWEET1 was constructed for gene transformation and functional verification studies.The main resμLts of this experiment are as follows:1.The strawberry system was optimized,and the Rugen tissue culture seedlings of octoploid strawberry with 45-day cycle were used as test materials,which were screened from different media,different plant hormones,sucrose concentration ratio and callus induction.The best generation formula.The experimental results showed that MS+0.5 mg/L 6-BA+0.1 mg/L IAA+0.1 mg/L GA3 was the best subculture formul a in the screening process,and the number of buds on the differentiation side was 45 cycles in light culture.5-6,and grow robust.From the sucrose concentration ratio,the tissue culture seedlings were green and the lateral buds were completely differentiated at the concentration of sucrose 2.5%,followed by 1.5%concentration,1%growth,leaf yellowing and lateral bud differentiation.From callus induction screening results,MS+0.5 mg/L IAA+2.0 mg/L ZT induced callus as the best medium,sucrose 3%,agar 7g/L,which provided better transgenes.Test materials.The results of antibiotic concentration screening showed that the differentiation buds were the most in 25 mg/L Kan,followed by 50 mg/L Kan,and 300 mg/LKan did not differentiate.Two fructose transporter genes FaSWEET1 gene were cloned from the red strawberry fruit of 30 days after flowering.The ORF of FaSWEET1.1 was composed of 1073 bases,encoding a total of 244 amino acids.The ORF of FaSWEET1.2 was 1196 bases.The composition,encoding a total of 236 amino acids,showed that it has the highest homology with Arabidopsis thaliana ATSWEET16 and ATSWEET2,and ATSWEET16 and ATSWEET2 belong to the SWEET family,indicating that FaSWEET1.1 and FaSWEET1.2 are also possible.A member of the SWEET family.The subcellular localization resμLts of tobacco showed that the FaSWEET-GFP fusion protein was localized to the cell membrane.The expression analysis of fruit in seven different developmental stages of strawberry showed that the expression of FaSWEET was significantly up-regulated,indicating that this gene may be related to fruit ripening.The overexpression vector pCXSN-Flag-FaSWEET1 was constructed and transformed into Arabidopsis thaliana and tomato.Three transgenic Arabidopsis plants were obtained,and two tomato transgenic plants were obtained.Real-time quantitative PCR analysis of SWEET family-related functional genes in different sugar concentrations showed that the expression of mannitol and sucrose was significantly up-regμLated at 3%and then decreased at 6%sucrose concentration,while fructose and glucose concentrations were at 1%,3%and 6%are raised in turn.Real-time quantitative PCR analysis was carried out by treatment of tissue-free seedlings with sugar-free starvation days and SWEET family-related genes in the starvation period of different sugars.The resμLts showed that the expression was significantly up-regulated on the 6th day after glucose-free starvation treatment,in fructose and glucose.The expression of 6h after starvation treatment was significantly up-regulated,while the expression of mannitol and sucrose was up-regμLated after starvation treatment for 3h.