Clone And Function Analysis Of Strawberry Fruit-Specific Promoter

Constitutive promoter is one kind of promoters used in plant genetic engineering. This kind of promoter can drive the non-specific,sustained,stable expression of target gene;but not regulate the gene expression with the temporal and spatial control, which may result in the waste of plant nutrition,and even damage to the transgenic plant.Tissue-specific promoters can induce high expression of target gene in one or several specific tissues.As one important kind of tissue-specific promoters, fruit-specific promoter can allow the specific expression of a transgene in fruit tissue(s),which would offer more precise time and spatial control to the target genes. Cloning and studying of fruit-specific promoters can make foundation for improving the quality of fruit.By searching in GenBank database,an EST sequence(DY633382),obtained from a subtractive library of strawberry,was selected for further analysis,which has high similarity(83%)with a grape serine/ammonia acid kinase gene related to fruit ripening.The expression pattern of the gene corresponding gene(DY gene)in strawberry(Fragaria ananassa,Duch.,cv Chandler)was also analyzed by RT-PCR, which showed the presence of DY transcripts only in fruit.The result indicated that the level of transcription in strawberry fruit was the highest in green fruit stage,the lowest in the white fruit stage,and then rose in the turning stage and red fruit stage. The RT-PCR analysis revealed the specific-expression of DY gene in strawberry fruit.According to the sequence of EST DY633382,specific primers were designed for Tail-PCR to obtain upstream sequence of DY gene.A fragment containing 1021 bps of upstream sequence 5' of the DY gene start codon was isolated using the TaiI-PCR.The 1021bps 5' flanking sequence of the DY gene was used to analyze plant cis-regulatory DNA elements using NSITE and PLACE on-line software.The results indicated that TATA box(TATAAT)is located at-32～-27bp of the ATG stat codon.DY gene promoter contains 18 putative cis-elements,such as,CCGTCG motif,GA-1,AT-rich F et al.To analyze the transcriptional regulation pattern of DY gene promoter,three plant expression vector pCAMBIA1305-DI-GUS,pCAMBIA1305-Ⅱ-GUS, pCAMBIA1305-DⅢ-GUS,which contained 1021bp,699bp and 523bp promoter fragment,respectively,were constructed.These vectors were introduced into tobacco via A.tumefaciens-mediated transformation.Transgenic plants were confirmed by PCR and Southern Blotting analysis.The transgenic plants containing a single copy insertion were selected for the analysis of transcriptional regulation pattern of DY gene promoeter.Using histochemical staining method,Gus activity was detected in the fruit(ovary) parts for transgenic tobacco plants carrying the three constructs pCAMBIA1305-DⅠ-GUS,pCAMBIA1305-DⅡ-GUS,pCAMBIA 1305-DⅢ-GUS.In contrast,staining of roots,shoots,leaves and flowers structures were not observed in these lines.Thus,the GUS gene can be specific-induced by the DY promoter,which indicated that the region(-523-ATG)contained some cis-element to regulate the gene fruit-specific expression.Temporal and spatial expression of GUS gene was studied at different development stages of fruit(ovary)in transgenic lines.The GUS activity is higher in development stageⅠcompared with in the other development stages.During the fruit stageⅠ,GUS activity in the transgenic plants containing pCAMBIA1305-DⅡ-GUS and pCAMBIA1305-DⅡwere higher than that in the transgenic lines containing pCAMBIA1305-DⅢ.The results indicated that the cis-element AT-rich F located in the-699～-523 of DY promoter might enhance expression of genes.HyPRR is specific-expressed gene in strawberry fruit,and its product HyPRP is a hybrid proline-rich structure protein in cell wall,which plays a role in the anchoring of polyphenols in the strawberry fruit during the growth and ripening.We isolated a 778bp fragment of the HyPRR promoter using PCR.The sequence of the HyPRR promoter was used to analyze plant cis-regulatory DNA elements using NSITE and PLACE database on line.TATA-box(TATAAT)is located at-59～-56bp of the ATG start codon,and HyPRR gene promoter contains 23 putative cis-elements,such as, ABRE,ACGT box,GT motif et al.Two fusion vectors containing the GUS gene under the control of promoter fragments of different length(581bp and 778bp)was constructed,named pCAMBIA1305-HⅠ-GUS and pCAMBIA1305-HⅡ-GUS,and transformed separately into tobacco.Transgenic plants were used to analyze the expression of GUS gene through Histochemical analysis.The GUS signal was only seen in fruit(ovary)tissue,indicating that the promoter can specifically regulate the gene expression in fruit,and the region(-581-ATG)contained the cis-element to regulate the gene fruit-specific expression.The activity of GUS in transgenic lines transformed with CAMBIA1305-HⅠ-GUS was considerably higher than that in lines with CAMBIA1305-HⅡ-GUS construct.These results showed there might be some cis-elements to increase the gene expression level in the-778—581 region of promoter.