本文選題：蘋果　切入點：液泡糖轉運蛋白　出處：《西北農(nóng)林科技大學》2016年碩士論文

【摘要】：糖是果實品質的重要組成,其中可溶性糖的種類及組成影響著果實的甜度和風味。果糖作為蘋果果實中糖的主要成分,是甜度最高的可溶性糖,其在果實中的積累受液泡膜糖轉運蛋白(TST)和果糖激酶(FK)的高度調控。本試驗以‘嘎啦’蘋果為試驗材料,克隆了蘋果中液泡膜糖轉運蛋白基因MdTST1和果糖激酶MdFK2基因的啟動子片段并分析了其表達特異性和啟動子活性,利用酵母單雜技術篩選了MdTST1和MdFK2啟動子結合的轉錄因子,獲得以下結果:1.從‘嘎啦’蘋果基因組中克隆了MdTST1和MdFK2兩個基因的啟動子序列,其長度分別為1595bp和1780bp。生物信息學分析順式作用元件發(fā)現(xiàn),兩個基因的啟動子中均含有響應植物激素、逆境脅迫誘導的相關元件,其中,MdFK2啟動子核心區(qū)域有多個響應干旱脅迫的元件。在煙草中的瞬時表達試驗表明,MdTST1和MdFK2基因啟動子活性均受到外源糖的高度誘導,其中葡萄糖(Glu)和果糖(Fru)對MdTST1基因啟動子活性誘導效應較蔗糖(Suc)明顯;MdFK2基因啟動子活性在果糖(Fru)誘導下遠遠高于葡萄糖(Glu)和蔗糖(Suc)誘導。此外,干旱條件處理下,MdFK2基因啟動子活性增強。2.在分別對MdTST1和MdFK2兩個基因啟動子5’缺失體的活性分析中發(fā)現(xiàn),不同長度的啟動子片段均可以啟動GUS報告基因的表達。其中,MdTST1基因啟動子Th2缺失體活性最高,表明其核心區(qū)域位于轉錄起始位點至上游-900bp之間;MdFK2基因啟動子中Th4缺失體活性最高,表明轉錄起始位點至上游-600bp是其最大啟動子活性區(qū)域。3.構建MdTST1和MdFK2基因啟動子與植物表達載體PBI121-GUS的融合載體,轉化擬南芥研究其組織表達特異性。結果表明,MdTST1主要在糖形成與積累相關的器官中有較高的表達,如成熟葉片和花器官,而MdFK2主要在葉片、花和莖尖中表達。4.構建了用于酵母單雜交篩選的蘋果果實cDNA文庫——pGADT7-REC2-DEST酵母單雜交文庫,其庫容量CFU為1.44×107,重組率達到了100%,插入片段大小在500bp-2500bp之間。綜合這3個衡量文庫質量的重要指標,表明此cDNA文庫代表性可以得到保證。通過酵母單雜交,我們篩選到與MdTST1基因相關候選轉錄因子18個,分別命名TST1-m1、TST1-m2,……,TST1-m18,,與MdFK2基因相關候選轉錄因子6個,分別命名FK2-m1、FK2-m2,……,FK2-m6。qRT-PCR分析發(fā)現(xiàn),在植物生長發(fā)育期及果實形成的各個階段中,編號為TST1-m5、TST1-m6、TST1-m15的這三個候選基因與MdTST1的表達量呈正相關,而TST1-m4、TST1-m18則與呈負相關;編號為FK2-m1的轉錄因子無論在果實發(fā)育過程還是整個植株的生長過程中,表達量均和MdFK2表達量呈正相關,而FK2-m4則呈負相關。
[Abstract]:Sugar is an important component of fruit quality, in which the species and composition of soluble sugar affect the sweetness and flavor of fruit. Fructose, as the main component of sugar in apple fruit, is the most sweet soluble sugar. Its accumulation in fruit was highly regulated by vacuolar sugar transport protein (TST) and fructose kinase (FK). The promoter fragments of vacuolar glucose transporter gene (MdTST1) and fructokinase (FK) MdFK2 gene in apple were cloned and their expression specificity and promoter activity were analyzed. The transcriptional factors binding to MdTST1 and MdFK2 promoter were screened by yeast hybridization technique. The following results were obtained: 1.The promoter sequences of MdTST1 and MdFK2 genes were cloned from the genome of 'Gara' apple, with the lengths of 1595bp and 1780bp.Bioinformatics analysis showed that the cis-acting elements, The promoters of both genes contain plant hormone responsive elements, which are induced by stress. The transient expression test of MdFK2 promoter in tobacco indicated that the promoter activity of MdTST1 and MdFK2 gene was highly induced by exogenous sugar. The activity of MdTST1 gene promoter induced by Gluand fructose (Glu) and fructose fruit was much higher than that induced by fructose fruit (Glu2) and sucrose (Sucu). In addition, the activity of MdFK2 gene promoter induced by fructose fruit was much higher than that induced by fructose fruit. Under drought condition, the promoter activity of MdFK2 gene was enhanced. 2. The activity of 5 'deletions of MdTST1 and MdFK2 gene promoter were analyzed, respectively. The GUS reporter gene expression was initiated by different length promoter fragments, and the activity of Th2 deletion of MdTST1 gene promoter was the highest. It was suggested that the core region of MdFK2 gene was the most active in the promoter of MdFK2 gene, which was located between the transcription initiation site and the upstream region of MdFK2 gene. The results showed that the transcriptional initiation site to the upstream of -600bp was the most active region of the promoter. The fusion vector of MdTST1 and MdFK2 gene promoter and plant expression vector PBI121-GUS was constructed. The specific expression of MdTST1 in Arabidopsis thaliana was studied. The results showed that MdTST1 was highly expressed in organs related to sugar formation and accumulation, such as mature leaves and floral organs, while MdFK2 was mainly expressed in leaves. The cDNA library of apple fruit, pGADT7-REC2-DEST, was constructed for single hybrid screening. The library capacity CFU is 1.44 脳 107, the recombination rate is 100 and the inserted fragment size is between 500bp-2500bp. By synthesizing these three important indexes to measure the library quality, the representativeness of the cDNA library can be guaranteed. We screened 18 candidate transcription factors related to MdTST1 gene and named TST1-m1TST1-m2O.TST1-m18G respectively. We named FK2-m1FK2-m2O.FK2-m6.qRT-PCR analysis showed that, during the growth and development stage and fruit formation stage of plant, FK2-m1FK2-m2-m6.qRT-PCR analysis showed that TST1-m1TST1-m18 and FK2-m1FK2-m2FK2-m2-m6.qRT-PCR analysis showed that, The three candidate genes numbered TST1-m5, TST1-m6 and TST1-m15 were positively correlated with the expression of MdTST1, while TST1-m4 and TST1-m18 were negatively correlated with the expression of TST1-m18. The transcription factors numbered FK2-m1 were positively correlated with the expression of MdFK2 during fruit development and the whole plant growth. FK2-m4 was negatively correlated.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S661.1

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