Identification And Characterization Of The Transcription Factors Regulating Banana Fruit Starch Synthesis Related Gene MaSBE4

As an important fruit and food crop,banana has a higher content of resistant starch(RS)in its pulp than other fruits and conventional crops.There are many health efficacies of resistant starch in banana,including regulation of digestive tract function,blood cholesterol level,microbial flora and blood glucose.There is a high content of RS in unripe banana fruit--about 15%(DW).In banana,the RS content is reported to be positively correlated with amylose content.Starch branching enzyme(SBE)is a crucial enzyme involved in determining the ratio of amylose/amylopectin.In developmental stage of banana fruit,the expression level of Ma SBE4 is significantly higher than other homologs.In order to understand the molecular mechanism of RS accumulation during developmental stage,we perform experiments to investigate the key regulator of Ma SBE4.A series of proteins are screened out though the yeast one-hybrid library screening assay,using Ma SBE4 promoter as a bait.After clustering and conserved domain analysis,five putative transcription factors are selected,namely,Ma C3H24,Ma HOX32,Ma SPL15,Ma EID1 and Ma MBD13.Their function of DNA binding is further confirmed by yeast one-hybrid assay.The result of subcellular localization in rice and banana protoplasts reveals that all five proteins except for Ma EID1 are nucleus-located,which is an essential feature of transcription factors.To identify the transcriptional regulation ability of Ma C3H24,Ma HOX32,Ma SPL15 and Ma MBD13,we performed a dual-luciferase transient expression assay.Ma SBE4 promoter and transcription factors are co-transformed into rice and banana protoplasts.We found that the set of Ma C3H24 showed a result of three times up-regulated reporter gene compared to the control,while reporter gene in sets of Ma SPL15 and Ma MBD13 are significantly down-regulated,respectively,45.2% and 38.4%.Meanwhile,the transcriptional activation capability of Ma C3H24 is further confirmed by examination based on yeast two-hybrid system.Transformation of Ma C3H24-GAL4 BD fusion protein in Y2 Hgold yeast can turn the yeast into blue on selective medium containing X-α-gal.In conclusion,Ma C3H24,Ma SPL15 and Ma MBD13 can regulate the transcription of Ma SBE4 as transcription factors.Among these proteins,Ma SPL15 and Ma MBD13 are negative regulatory factors while Ma C3H24 can activate the expression of down-stream gene.In order to investigate the influence of these factors exerted on phenotype,banana overexpression transgenic lines of Ma C3H24,Ma SPL15 and Ma MBD13 are created,using agrobacterium infection.