苹果果糖积累相关基因MdTSTl和MdFK2启动子功能鉴定及其转录因子筛选

发布时间：2018-03-24 08:01

  本文选题：苹果　切入点：液泡糖转运蛋白　出处：《西北农林科技大学》2016年硕士论文

【摘要】：糖是果实品质的重要组成,其中可溶性糖的种类及组成影响着果实的甜度和风味。果糖作为苹果果实中糖的主要成分,是甜度最高的可溶性糖,其在果实中的积累受液泡膜糖转运蛋白(TST)和果糖激酶(FK)的高度调控。本试验以‘嘎啦’苹果为试验材料,克隆了苹果中液泡膜糖转运蛋白基因MdTST1和果糖激酶MdFK2基因的启动子片段并分析了其表达特异性和启动子活性,利用酵母单杂技术筛选了MdTST1和MdFK2启动子结合的转录因子,获得以下结果:1.从‘嘎啦’苹果基因组中克隆了MdTST1和MdFK2两个基因的启动子序列,其长度分别为1595bp和1780bp。生物信息学分析顺式作用元件发现,两个基因的启动子中均含有响应植物激素、逆境胁迫诱导的相关元件,其中,MdFK2启动子核心区域有多个响应干旱胁迫的元件。在烟草中的瞬时表达试验表明,MdTST1和MdFK2基因启动子活性均受到外源糖的高度诱导,其中葡萄糖(Glu)和果糖(Fru)对MdTST1基因启动子活性诱导效应较蔗糖(Suc)明显;MdFK2基因启动子活性在果糖(Fru)诱导下远远高于葡萄糖(Glu)和蔗糖(Suc)诱导。此外,干旱条件处理下,MdFK2基因启动子活性增强。2.在分别对MdTST1和MdFK2两个基因启动子5’缺失体的活性分析中发现,不同长度的启动子片段均可以启动GUS报告基因的表达。其中,MdTST1基因启动子Th2缺失体活性最高,表明其核心区域位于转录起始位点至上游-900bp之间;MdFK2基因启动子中Th4缺失体活性最高,表明转录起始位点至上游-600bp是其最大启动子活性区域。3.构建MdTST1和MdFK2基因启动子与植物表达载体PBI121-GUS的融合载体,转化拟南芥研究其组织表达特异性。结果表明,MdTST1主要在糖形成与积累相关的器官中有较高的表达,如成熟叶片和花器官,而MdFK2主要在叶片、花和茎尖中表达。4.构建了用于酵母单杂交筛选的苹果果实cDNA文库——pGADT7-REC2-DEST酵母单杂交文库,其库容量CFU为1.44×107,重组率达到了100%,插入片段大小在500bp-2500bp之间。综合这3个衡量文库质量的重要指标,表明此cDNA文库代表性可以得到保证。通过酵母单杂交,我们筛选到与MdTST1基因相关候选转录因子18个,分别命名TST1-m1、TST1-m2,……,TST1-m18,,与MdFK2基因相关候选转录因子6个,分别命名FK2-m1、FK2-m2,……,FK2-m6。qRT-PCR分析发现,在植物生长发育期及果实形成的各个阶段中,编号为TST1-m5、TST1-m6、TST1-m15的这三个候选基因与MdTST1的表达量呈正相关,而TST1-m4、TST1-m18则与呈负相关;编号为FK2-m1的转录因子无论在果实发育过程还是整个植株的生长过程中,表达量均和MdFK2表达量呈正相关,而FK2-m4则呈负相关。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S661.1

【相似文献】

相关期刊论文 前10条

1 郝迪，

本文编号：1657404