Studies On Fruit-specific Regulation Mechanisms Of AGPL1 Promoter From Watermelon (Citrullus Vulgaris S.) And Transgenic Research In Tomato

The ADP-glucose pyrophosphprylase is a heterotetramer consisting of two small and two large subunits in plants.The small subunit has one or two isoforms that highly conserve among the plant species and express constitutively in all tissues,while the large subunit is present as multiple isoforms and shows tissue-specific expression.The large subunit gene was expressed specifically in fruits in watermelon(Citrullus vulgaris S.).A 1.8kb 5' flanking region of the large subunit of the AGPase was isolated from watermelon,which was proved to driving report genes expression specifically in transient transformed watermelon fruits and stable transformed tomato fruits.In this work,the fruit-specific regulation of AGPL1 promoter was intensively studied.The transcription start site(TSS)of watermelon AGPL1 promoter was mapped to an A residue 33-bp downstream of the TATA-box by primer extension methods. Consistently,in order to separate the specific-related cis elements of A GPL1 promoter,a series of deletion from 5'- and 3'- were generated by PCR and fused with gus (β-glucuronidase).All of the fragments were transferred into fruits of watermelon by particle delivery system.GUS activity assays revealed that the 1301 bp fragment(-868 to +433 from TSS)was sufficient for the maximal and fruit-specific promoter activity.With the deletion to 3'-end of the promoter,driving activity was decreased and drop to the undetectable level.The results also showed that an AT-rich(about 65%)intron,found after TSS(+136 to+459)in the 5'-untranslational region of the gene,could enhance the promoter activity in fruit but have not effect on the fruit-specific promoting manners.All of the AGPL1 promoter deletions were fused with GFP/RFP double markers transient expression vectors and were transformed into watermelon leaves.Two negative regulatory regions,27bp from -986 to -959 and 41bp from -464 to -424,were identified in the further deletion analysis.Deletion of both regions led to an activity of constitutive-like expression in leaves epidermal cells,which were named putatively negative region 1 and 2 respectively(PN1 and PN2).Two positive regulatory regions (PP1 and PP2),24bp from -892 to -868 and 56bp from -424 to -368,were coupled with both negative regulators respectively,which deletion led to the constitutive-like activity disrupted.Gain-of-function experiments showed that the two negative regulatory regions were sufficient to inhibit expression of RFP in transformed epidermal cells when fused to CaMV 35S minimal promoter,which gave reason of why the promoter has not activity in leaves.Gel mobility shift experiments showed strong binding of PN1 and PN2 with watermelon leaf nuclear proteins.Those results demonstrated the presence of leaf nuclear factors that interact with the two regions.PP1 and PP2 also could bind to the leaf nuclear proteins respectively.Consistently,we draw a conclusion of thoroughly different regulation mechanisms with other specific promoters.That is,the cis element in AGPL1 promoter inhabits driving activity in tissues out of fruits.To evaluate the fine nucleotides of the element,4 nucleotides of internal deletion and single nucleotide mutation was carried in competition assays.The nucleotides important for the binding in PN1 are TC/TCAAAA,of which A¹²and A¹³are most important.The cis was proved to be present in several monocot and dicot plants.Moreover,AGPL1 promoter was fused to lycopeneβ-cyclase RNAi vector and transformed into tomato.The fruit-specific regulation of AGPL1 promoter was analyzed in stable transgenic plants,and produced lycopene-rich tomato fruits.