Promoter Activity Analysis And Subcellular Localization Of Four SlSWEETs Genes In Tomato

Sugar provides plants with important sources of carbon and energy.During the growth and development of plants,sugar transporters play a crucial role in regulating the movement and distribution of sugars.The SWEETs gene family as a sugar transporter discovered in recent years can be involved in the transport of various monosaccharides and polysaccharides,but there are still many problems to be solved for the physiological functions and mechanisms of the SWEET family members.Such as SWEET family members subcellular localization and spatiotemporal expression characteristics.Previous studies showed that Sl SWEET7 a,-11 b,-12 c and-14 genes were different in the green ripe stage and the red ripe stage of tomato fruit ripening.It was speculated that SWEETs gene may participate in the sugar transport and distribution in tomato fruit ripening.Promoters that drive gene expression are important elements that regulate gene expression and are particularly important in the functional regulation of gene expression.In this study,four genes and promoters of Sl SWEET7 a,-11 b,-12 c,and-14 were analyzed for their tissue distribution and subcellular localization.The SlSWEETn::GUS and SlSWEETn::GFP vectors were constructed by cloning the full-length gene and the promoter by polymerase chain reaction.The histochemical analysis of GUS activity was performed by transient analysis of tomato fruit and genetic transformation of Arabidopsis to illustrate the promoter expression pattern.Subcellular localization of four SWEET genes was performed by transient expression of onion and tobacco leaves.The main research results obtained are as follows:1.The SlSWEETn::GUS vectors containing four SWEETs were constructed using Gateway technology.Transient expression of tomato fruits using Agrobacterium-mediated method GUS activity analysis was performed after 3 days.The results showed that Sl SWEET7 a,-11 b,-12 c,and-14 promoters all have activity.Among them,SlSWEET7 a has strong activity in the vascular bundle,gelatinous placenta and seed coat of fruit;The distribution of GUS activity of-11 b is similar to that of-7a.The activity of SlSWEET12 c was mainly distributed in the pulp,vascular bundle,ventricular wall and seed coat of fruit.The activity of SlSWEET14 is mainly distributed in the seed coat and the flesh part of the fruit.The above results suggest that Sl SWEET7 a,-11 b,-12 c and-14 may play an important role in the sugar transport of tomato fruits.2.The truncated cis-acting elements of the two SlSWEET promoters were analyzed and further transformed into Arabidopsis thaliana.The T2 transgenic pure lines obtained were subjected to GUS histochemical staining.The results showed that the promoter of SlSWEETgene had almost no detectable GUS activity in Arabidopsis thaliana,but the GUS activity was significantly increased after the promoter was truncated,and the GUS activity of the promoter sequence that does not contain HSE elements after truncation was enhanced.These results suggest that HSE elements negatively regulate the expression of SWEET7 a and SWEET14 in trophic tissues.3.Four genes of SlSWEETn::GFP green fluorescent protein fusion expression vector were constructed and used Agrobacterium-mediated transient expression of onion epidermal cells and tobacco leaves.The results showed that the SlSWEET genes differentially expressed in the four fruits were located on the plasma membrane.