Identification Of Strawberry SWEET17-like Gene And Preliminary Analysis Of Its Function In Regulating Sugar Metabolism

SWEET proteins（Sugars will eventually be exported transporters,SWEETs）are newly discovered sugar transporters.Its function is mainly involved in the transportation of sugar,and it plays a role in plant growth and development and adversity stress.At present,the research on the function of strawberry SWEET gene family is still in the blank stage.The previous research of our group found that adding an appropriate amount of potassium sulfate can increase the sugar content of strawberry fruit.In order to further clarify which genes play a role in this process,the differentially expressed SWEET17-like gene was screened by transcriptome sequencing technology.The SWEET17-like gene was cloned from’Ruegen’and’Yanli’,and the Fve SWEET17-like gene fusion vector was constructed to analyze its function.The main results are as follows:（1）The fruits with significant differences in soluble solids in the red fruit stage strawberry treated with potassium sulfate(0.0 g·L^-1 as CK,0.4 g·L^-1 as T1)were used as test materials,and mixed samples were sent for transcriptome sequencing analysis.742 differential genes were screened out,and SWEET17-like genes with relatively high differential folds and possibly related to glucose metabolism were screened out by gene function annotation analysis as the research object.（2）Using’Ruegen’and’Yanli’strawberries as test materials,the full-length sequence of the differentially expressed gene SWEET17-like was cloned.The sequencing results showed that the SWEET17-like coding region was 708 bp in length and encoded 235 amino acids.It was compared with the diploid strawberry SWEET17 sequence downloaded from the NCBI website,and it was found that the amino acid sequence identity between them was 100%,and the gene was highly conserved in different strawberry resources.（3）q RT-PCR analysis showed that SWEET17-like was expressed in the roots,stems,leaves,flowers and red fruits of’Ruegen’and’Yanli’strawberries,with the highest expression in flowers and leaves,and a low expression in red fruits.Moreover,the expression of SWEET17-like gradually decreased with the ripening of fruit.Through GUS tissue staining,it was also found that the expression of pro Fve SWEET17-like::GUS was the strongest at the green fruit stage,and the expression level decreased as the fruit matured,indicating that the Fve SWEET17-like gene may have an impact on strawberry fruit development and sugar accumulation.Subcellular localization assay showed that Fve SWEET17-like protein was localized on the tonoplast.（4）Using p RI101-AN as the vector,the Fve SWEET17-like overexpression vector and silencing vector were constructed and named as Fve SWEET17-like-AN and p RNAi-Fve SWEET17-like,respectively.With the constructed vector,the transient transformation method mediated by Agrobacterium was used to obtain the silencing and overexpression of Fve SWEET17-like transiently transformed strawberry fruits using’Yuehua’and’Yanli’strawberry fruits as test materials.Compared with the control,it was found that the sucrose content in the overexpressed fruit was significantly decreased,while the sucrose content in the silent fruit was significantly increased,and there were also certain changes in glucose and fructose.Through q RT-PCR experiment,it was found that Fa SPSA3 and Fa SUT2,which are related to sucrose synthesis,were up-regulated in silenced fruits,while the expression of sucrose hydrolysis-related enzymes,Fa CWINV1,Fa VIN1,and Fa SUS1,were all down-regulated.In the overexpressed fruit,the expression levels of Fa VIN1,Fa SUS1,and Fa CWINV1,which are related to sucrose hydrolysis,increased,and the genes related to sucrose synthesis and fructose and glucose,Fa FRK2-like,Fa HK2,Fa SPSA3,and Fa SUT2 were down-regulated.Overall,the expression of genes related to sucrose synthesis and metabolism changed significantly,while the expression of fructokinase and hexokinase genes did not change significantly,indicating that the significant changes in sucrose content in transiently transformed fruits may be due to the overexpression or silencing of Fve SWEET17-like affecting the differential expression of sucrose synthesis and degradation-related enzyme genes.（5）By constructing yeast functional vectors p DR-Fve SWEET17-like,p DR-At SWEET1,p DR-At SUC2 and transforming them into yeast mutant strains EBY.VW4000 and SUSY7/ura3,it was found that Fve SWEET17-like protein has the ability to transport glucose and sucrose.