| Tomato(Solanum lycopersicum)is a widely cultivated vegetable crop.The sugar content and the ratio of sugar to acid in tomato fruit affect its taste and flavor,and thus affect the economic value of tomato.Therefore,it is of great significance to deeply study the regulation mechanism and influencing factors of sugar metabolism of tomato fruit in order to improve the quality of tomato fruit.Sucrose phosphate synthase(SlSPS)is the key rate-limiting enzyme in sugar metabolism of tomato fruit,while the bridge protein 14-3-3 is an important internal factor that regulates the activity of SlSPS.Studies of previous researchers found that SlTFT1 and SlTFT10 in tomato 14-3-3 protein family are most likely to interact with sucrose phosphate synthase(SlSPS),and regulate sugar metabolism in tomato.However,there is no exact test evidence for the function of SlTFT1 and SlTFT10.Therefore,this research used bioinformatics methods to analyze the promoter sequences of tomato SlTFT1 and SlTFT10 genes.Combined with its cis-acting elements,to preliminary explore the ABA、SA、MeJA and GA hormones treatment on tomato SlTFT1 and SlTFT10 gene expression regulation and its relationship with sugar metabolism.The subcellular localization of SlTFT1 and SlTFT10 proteins were further studied using the GFP fusion expression mapping method.Yeast Two-Hybrid bait expression vectors of the SlTFT1 and SlTFT10 genes were constructed,and self-activation and toxicity assays were performed,which laid the foundation for the screening of the 14-3-3 protein that interacted with SlSPS.The main results of this study were as follows:(1)The promoter sequences of the cloned tomato SlTFT1 and SlTFT10 genes were predicted and analyzed.The promoter of the SlTFT1 gene contained four plant hormone response elements: ABA,SA,GA and MeJA.The promoter of the SlTFT10 gene had ABA and SA response elements.Presumably the tomato SlTFT1 and SlTFT10 genes might respond to hormone signals.(2)Based on the predicted results of cis-acting elements of the promoters,tomato seedlings were treated with four plant hormones: ABA,SA,GA,and MeJA,respectively.The expressions of the SlTFT1 and SlTFT10 genes and enzyme genes related to sugar metabolism were measured,so as to the enzyme activities and the soluble sugar content in tomato leaves.The results showed that the SlTFT1 and SlTFT10 genes negatively regulated the sugar metabolism,and SlTFT10 played a major role.It might respond to the effects of the two hormones through the ABA and SA cis-acting elements on its promoter,thereby regulating sugar metabolism in tomato leaves.MeJA and GA might regulate SlTFT10 and affected sugar metabolism through other channels.(3)Subcellular localization prediction of SlTFT1 and SlTFT10 revealed that the SlTFT1 might be located in the cytoplasm and SlTFT10 might be localized in the cytoplasm and nucleus.With GFP fusion expression localization technology,SlTFT1 and SlTFT10 proteins were fused with GFP and injected intotobacco leaves.The results showed that the SlTFT1 protein was localized in the nucleus and cytoplasm,and the SlTFT10 protein was localized in the cytoplasm.(4)The yeast bait expression vectors of SlTFT1 and SlTFT10 genes were constructed with TA clone technology,which were co-transformed into Y2 HGold with the prey expression vector.And then the auto-activation ability and cytotoxicity of the bait fusion protein were detected.It has been verified that the pGBKT7-SlTFT1 and pGBKT7-SlTFT10 yeast fusion proteins had no auto-activation ability and no cytotoxicity against yeast,which could be used in the subsequent study of yeast two-hybrid. |
