Indentification And Functional Analysis Of Malate Regulatory Gene SLALMT9 Interaction Proteins CESA2,TP And TEP In Tomato

Tomato（Solanum lycopersium）is a kind of vegetable which is widely grown in the world.Its distinctive taste and abundant nutrition make the tomato popular among people.Malate is one of the most important organic acids in tomato fruit,and its content significantly affects the taste and flavor of tomato fruit.Sl ALMT9 is a key gene regulating the accumulation of malic acid in tomato fruit,and its expression in the root enables tomato adapt to Al³⁺ toxicity.In this study,three proteins interacting with Sl ALMT9 were screened by yeast two-hybrid.The main results are as follows:1.The p GBKT7 bait vector containing the complete CDS fragment of Sl ALMT9 was constructed,we obtained 28 proteins that may interact with Sl ALMT9 gene through yeast two-hybrid library screening and sequencing.Combining yeast two-hybrid point-to-point validation and RNA-seq in Sl ALMT9-OE fruit,the results indicated that three genes,CESA2（Solyc01g087210.2.1）,TEP（Solyc03g005270.2.1）and TP（Solyc05g026540.2.1）,encode proteins that interact with Sl ALMT9.2.We obtained CESA2,TEP and TP c DNA in AC fruit by reverse transcription.Construction of CESA2,TEP,TP truncated prey vectors and Sl ALMT9-p GBKT7 truncated bait vector were performed by structure prediction basd on TMHMM and Inter Pro Scan prediction.Yeast two-hybrid results indicated that complete fragment of TP and TEP interact with SLALMT9,CESA2150-350 aa area interact with SLALMT9-N.The results of bimolecular fluorescence complementation（Bi FC）further verified the interaction between CESA2、TEP、TP and Sl ALMT9.3.The subcellular localization vectors of CESA2::YFP,TEP::YFP and TP::YFP were constructed,results of subcellular localization indicated that the three proteins were located in plasma membrane,vacuolar membrane and vacuolar membrane respectively.Therefore,there is a spatial possibility of interaction between CESA2,TEP,TP and Sl ALMT9.4.GUS staining of p CESA2::GUS,p TEP::GUS,p TP ::GUS and expression profiles of three genes in different tissues of AC showed that the three genes were involved in the life activities of AC fruits and roots.In addition,CESA2,TEP,TP have the same or opposite expression patterns of Sl ALMT9 in different development stages of AC fruits,which indicated that the binding of CESA2,TEP and Sl ALMT9 is a positive regulation pattern,while the binding of TP and Sl ALMT9 is a negative regulation pattern.p TP::GUS and p TEP::GUS showed staining differences after Al³⁺ treatment,suggested that expression of TP and TEP may be induced by Al³⁺,root growth differences of CESA2,TEP,TP transgenic materials before and after Al³⁺ treatment indicated that they may also participate in the mechanism of Sl ALMT9 response to aluminum toxicity.5.Over expressed CESA2,TEP and knocked out TP in AC led to a significantly increase of malate content in fruits,indicating that the binding of three genes with Sl ALMT9 affected their transport function and further affected the accumulation of malic acid in fruits.The expression results of each genes in fruits showed that the binding of CESA2,TEP and Sl ALMT9 positively regulated the content of malic acid in fruits,while the binding of TP and Sl ALMT9 negatively regulated the content of malic acid in fruits.6.In the fruits of CESA2-OE and TEP-OE lines,the expression of TP was significantly reduced,indicated that there might be an inner link between CESA,TEP and TP.7.CESA2,TP and TEP can partially restore their growth in E.coli malate transporter mutant,indicated that they also have malate transport capacity.8.Over expressed CESA2 and TEP increased the stomatal number and malic acid content of tomato cotyledon,and the knockout CESA2 and TEP reduced the stomatal number and malic acid content of tomato cotyledon,indicating that the combination of CESA2,TEP and Sl ALMT9 positively regulated the stomatal number of tomato cotyledon.