| Ziziphus jujuba Mill.is a traditional economic forest fruit tree in China,which is domesticated from Ziziphus jujuba Mill.var.spinosa,the sugar content of fruit is significantly increased in the process of domestication.Sugar transporters play an important role in the distribution of sugar from source organs to sink organs.Therefore,it is of great significance to explore the transport and regulation mechanism of sugar transporters in source and sink organs for the unloading of soluble sugar and sugar accumulation in jujube fruit.Based on the sequencing data of different fruits and tissues of jujube and sour jujube,the spatio-temporal expression and tissue expression patterns of sugar transporters were analyzed,and the sugar transporters with different expression levels in leaves and fruit organs were screened.It was preliminarily identified that the expression level of Zj SWEET2.2 in leaves was significantly higher than that in other tissues,while the expression level of Zj SWEET15 in jujube fruit was significantly higher than that in sour jujube.On this basis,the transport and regulatory functions in leaves and fruits were analyzed by subcellular localization,transient expression,promoter sequencing analysis and yeast single hybrid molecular biology techniques.The specific results are as follows:1.Sequence analysis showed that Zj SWEET2.2 contained 7 transmembrane domains with typical SWEET characteristics;subcellular localization showed that Zj SWEET2.2 was a plasma membrane-located sugar transporter.2.Gene expression analysis of different tissues showed that Zj SWEET2.2 was highly expressed in chloroplast-rich tissues.The analysis of transient overexpression of Zj SWEET2.2in leaves showed that Zj SWEET2.2 promoted the expression of genes related to photosynthetic system,and it was speculated that Zj SWEET2.2 could promote photosynthesis by reducing the sugar content in leaves.3.Transcriptional regulation analysis based on exogenous sugar spray experiments showed that Zj SWEET2.2 was inhibited by high concentration of exogenous sugar and promoted by low concentration of exogenous sugar,and its response to glucose was more sensitive than that to sucrose.DNA sequence analysis of Zj SWEET2.2 promoter region showed that there were cis elements related to glucose signal in the promoter,which suggested that the protein was regulated by glucose signal.4.Gene expression analysis of different tissues showed that Zj SWEET15 was highly expressed in storehouse organs,sequence analysis showed that Zj SWEET15 contained seven transmembrane domains with typical SWEET characteristics,and subcellular localization showed that Zj SWEET15 was a plasma membrane-located sugar transporter.5.The fluorescence quantitative PCR determination of Zj SWEET15 in jujube and sour jujube population samples was carried out,combined with the correlation analysis of sucrose content.The results showed that the expression level of Zj SWEET15 in cultivated jujube was significantly higher than that of sour jujube,and positively correlated with sucrose content.The results of transient over expression and exogenous sugar spraying further showed that Zj SWEET15 promoted sucrose transport and accumulation.6.Sequencing of the Zj SWEET15 promoter regions of 11 high-sugar-content cultivated jujubes and 11 low-sugar-content jujubes showed that there were SNP variant regions（TG/CTTTAAAAAGC/AAAGAAAAATG/AA）of jujube and jujube at the upstream of the promoter region from-4330 bp to-4228 bp,and the variation site is linked with sucrose content.7.Yeast one-hybrid screening experiment was carried out on the variant region of Zj SWEET15 promoter,and the expression of Zj SWEET15 regulated by transcription factor Zj AFP3 was screened,and the spatio-temporal expression analysis of Zj AFP3 showed that the expression level of Zj AFP3 increased with the ripening of fruit,and the expression level of cultivated jujube was higher than that of sour jujube,which was consistent with the trend of fruit sugar accumulation. |
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