| Sugar is an important element in fruit quality. The kind and the composition of the soluble sugar affect the sweetness and flavor of fruits. As a main sugar in apple fruit, fructose is the sweetest soluble sugar whereas its accumulation is regulated obviously by TST and FK.In this thesis, promoters of MdTST1 and MdFK2 were cloned in ‘GaLa’ apple, and were usedto analyzeboth expression specificity in different tissue organs and promoter activities,then yeast one-hybrid technology was used to screen out transcription factors combined with promoter of MdTST1 and MdFK2, the results as following:1. The length of the two gene promoter sequences of MdTST1 and MdFK2 cloned from‘GaLa’ apple genome was 1595 bp and 1780 bp respectively. Bioinformatics analysis of cis-acting elements showed that both the promoters of MdTST1 and MdFK2 had elements which respond to plant hormone induction and stress conditions. The centre area of MdFK2 promoter had some elements responding to drought stress.The transient expression experiment in tobacco leaves showed that both of the gene promoter activities of MdTST1 and MdFK2 were induced obviously by three exogenous sugars. IncreasedMdTST1 promoter activity could be significally inducedby Glu and Fru, while Suchas slight increase. The activity of MdFK2 promoter induced by Fru was much higher than that induced by Glu and Suc. Furthermore, in drought condition, the activity of MdFK2 promoter was enhanced.2. It was discovered when analyzing the activity of the two promoter 5’deletants of MdTST1 and MdFK2 that all different lengths of promoter fragments can activate the expression of GUS reporter gene. Th2 deletant of MdTST1 promoter had the highest activity,which showed that its centre area was located between transcription start site and up-stream area of-900 bp. Th4 deletant of MdFK2 promoter had the highest activity, which showed that its maximum promoter active area is located from transcription start site to up-stream area of-600 bp.3. With constructing the fusion expression vector PBI121-TST1, we transformed it into Arabidopsis to analyze tissue expression specificity of MdTST1 gene promoter. The results suggested that MdTST1 had higher expression in organs involving forming and accumulating of sugar, especially in floral organs.4. With constructing the cDNA library for yeast one-hybrid screening——pGADT7-REC2-DEST yeast one-hybrid library, the storage capacity of it was CFU=1.44×107and the recombination rate reached to 100 %. The length of cDNA fragments inserted in vector were 500bp-2500 bp.Considering all these three important targets of the cDNA library,it showed that the representativeness of pGADT7-REC2-DEST yeast one-hybrid library can be ensured.Through yeast one-hybrid, we screened out 18 candidate transcription factors related to MdTST1, named TST1-m1, TST1-m2, …, TST1-m18, respectively and 6 candidate transcription factors related to MdFK2, named FK2-m1,FK2-m2, …, FK2-m6 respectively.Result of qRT-PCR showed that in various phases of plants growth period as well as fruit-formation period, TST1-m5, TST1-m6 and TST1-m15 had positive correlation with the expression quantity of MdTST1 while TST1-m4 and TST1-m18 had negative correlation with that. The expression quantity of transcription factor numbered FK2-m1 had positive correlation with the expression quantity of MdFK2 regardless whether during the development period of fruit or during the whole growth period of the plant while FK2-m4 had the negative correlation. |
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