Expression Pattern And Regulation Of Fructose 1,6-Diphosphate Aldolase Gene On Photosynthesis In Tomato

Tomato?Solanum lycopersicum L.?is one of the most important protected vegetables in the world,and it is a cold-sensitive and light-loving plant.In the north of China,tomatoes were mainly cultivated through the winter in solar-greenhouses,where low temperature and weak light stresses are frequent,and their growth and development were frequently impacted.Fructose 1,6-bisphosphate aldolase?FBA?is a key enzyme in the Calvin Cycle.It exerts flux control of carbon by limiting the Ru BP regeneration steps.In order to elucidate the regulating mechanism of FBA on photosynthesis in tomato,genome wide identification,phylogenetic analysis and expression pattern of SlFBA gene family were performed.In addition,SlFBA4and SlFBA7 were cloned and the over-expression and RNAi expression constructs were constructed,which was genetic transformed into tomato through Agrobacterium-mediated method,respectively.The transgenic lines were obtained and the effects of FBA changes on the growth and photosynthesis in transgenic tomato plants was analyzed for studying the regulating mechnism of FBAs.The main results are as follows:1.Eight Arabidopsis FBA proteins were used as query sequences to search the genome database of tomato with the BlastP program.Through BlastP and Pfam,we identified eight FBA genes in tomato which contain the glycolytic domain and named SlFBA1^SlFBA8.Among these members,SlFBA1^SlFBA5 were located in chloroplastid and SlFBA6^SlFBA8 were located in cytoplasm.2.Phylogenetic tree of SlFBAs was structed through MEGA 4.0,and the subfamily of SlFBAs was analyzed based on the phylogenetic relationship.SlFBAs gene family can be classified into two subgroups.SlFBA1^SlFBA5 were belongs to the first subgroup,and SlFBA6^SlFBA8 were in the second subgroup.The phylogenetic analysis of FBAs from tomato,Arabidopsis,rice,and other organisms suggested that SlFBAs shared the highest protein homology with FBAs from other plants.Synteny analysis indicated that segmental duplication events contributed to the expansion of the tomato FBA family.3.Different SlFBAs showed different spatial expression.The mRNA of SlFBA1,2,3,5and 6 were abundant in leaves and that of SlFBA4,7 and 8 were abundant in fruits.SlFBAs expression at the mRNA and protein levels was higher in mid-position leaves(5^th apical expanded leaves)than in upper position leaves(1^st)and base position leaves(10^th and 15^th).A similar difference in FBA activity was found among different position leaves.In a sunny day,the diurnal variation of photosynthetic rate?Pn?of tomato functional leaves showed a single-peak pattern curve,and the peak appeared at 12:00.The tomato leaves revealed similar diurnal variation of SlFBAs expression and FBA activity to Pn,except that they were increase again in 16:00¹8:00.These data indicate that FBA participate in the diurnal variation of Pn in tomato leaves.4.The gene expression of SlFBAs were induced by stress of low or high temperature.However,the expression level were decreased after 48 h of stress.FBA activity declined gradually with the prolonging of tress time.These results suggest that the decrease in SlFBAs expression and FBA activity caused by low or high temperature is one of the main reasons of the decline in Pn of tomato plants.5.The full-length SlFBA4 and SlFBA7 cDNA were cloned and their over-expression and RNAi silence expression constructs were constructed,which were transformed into tomato cotyledon through Agrobacterium mediated method,respectively.The successful transformants were screened with kanamycin and confirmed by qPCR and Western blot.The results showed that the expression level of SlFBA4 and SlFBA7 increased in over-expression transgenic lines and decreased in RNAi transgenic lines.6.Over-expressed transgenic tomato showed an increase in seed weight and germination rate under suboptimal temperature,compared with the WT plants.However,the seeds of RNAi transgenic plants were withered and the germination rate were significantly lower than that of the WT plants.Over-expression of SlFBA4 and SlFBA7 increased the FBA activity,mRNA abundance and activities of other main enzymes in Calvin cycle,Ru BP generation rate and Pn.Whereas,the RNAi transgenic plants revealed the contrary results to the over-expression plants.7.Under low temperature and low light intensity,the seed germination rate and Pn of over-expressed transgenic seedlings were obviously higher than those of the WT.over-expressed transgenic seedlings showed a decreased in the accumulation of MDA and active oxygen?ROS?under low temperature and low light intensity,compared with the WT seedlings.However the RNAi transgenic seedlings revealed the contrary results to the over-expression ones.This data suggested that over-expression of SlFBA4 and SlFBA7increased the tolerance of tomato to low temperature and weak light stress,while it led to just the opposite when the expression of SlFBA4 and SlFBA7 were interfered.