GGP基因在果实Vc含量调控中的分子机理研究

发布时间：2018-08-13 20:06

【摘要】：抗坏血酸(Ascorbate acid,AsA)是果实品质的一个重要组成成分,植物的AsA含量主要依赖于生物合成,其中GDP-L-半乳糖磷酸化酶(GGP),是植物体内AsA生物合成的主要限制酶。本文首先以AsA含量差异较大的山梨和毛花猕猴桃为研究对象,分析了其GGP基因的序列和活性差异;并从苹果中克隆了与拟南芥GGP表达负调控因子AMR1同源的基因MdAMR1.1和MdAMR1.2,初步分析了二者表达与AsA的关系,并利用酵母单杂交技术筛选了与其启动子相结合的转录因子。主要结果如下:1.在猕猴桃两个GGP基因中,GGP1表达水平与毛花猕猴桃果实高AsA含量相关。在山梨和毛花猕猴桃杂交后代果实AsA含量的测定表明,AsA含量显著的偏向毛花猕猴桃。在杂交后代中,GGP启动子遗传分离分析表明GGP1和GGP2连锁,且毛花GGP1启动子可能与AsA含量相关。进一步对山梨和毛花猕猴桃GGP1等位基因启动子序列和活性分析表明,与山梨猕猴桃相比,毛花猕猴桃AeGGP1启动子存在217bp和215 bp的缺失,毛花猕猴桃AeGGP1启动子活性显著高于山梨猕猴桃的,且毛花猕猴桃AeGGP1等位启动子均存在高活性,而山梨猕猴桃ArGGP1等位启动子活性存在差异。这些结果表明,猕猴桃GGP1启动子序列的遗传特性在AsA含量决定中起重要作用。同时,通过对拟南芥GGP基因上游的uORF区与AsA含量的关系研究也表明,不同物种中高度保守的uORF区序列与AsA有关,该区域的基因编辑能提高AsA含量。2.通过同源对比,在苹果基因组中找到了与拟南芥GGP表达负调控因子AMR1基因同源度最高的MdAMR1.1和MdAMR1.2基因。表达分析表明,在果实发育过程中MdAMR1.1与AsA含量显著负相关。克隆了‘嘎啦’苹果MdAMR1.1和MdAMR1.2基因全长,分别转化进野生型和AMR1突变型拟南芥,发现MdAMR1.1和MdAMR1.2在拟南芥中的过量表达均可以降低野生型和突变型拟南芥AsA含量,且MdAMR1.1在拟南芥互补试验中AsA下降更明显。为探明MdAMR1.1转录调控途径,从‘嘎啦’苹果基因组中克隆了MdAMR1.1启动子序列,构建了酵母单杂交重组质粒,通过筛选苹果果实的cDNA文库,得到了候选相关基因8个,分别命名为AMR1-1、AMR1-5、AMR1-6、AMR1-7、AMR1-14、AMR1-16、AMR1-22和AMR1-27,为MdAMR1.1转录调控的进一步研究奠定了基础。
[Abstract]:Ascorbic acid (Ascorbate acididae ASA) is an important component of fruit quality. The content of AsA in plants is mainly dependent on biosynthesis, and GDP-L- galactose phosphorylase (GGP), is the main limiting enzyme of AsA biosynthesis in plants. In this paper, the sequence and activity of GGP gene were analyzed based on the difference of AsA content in wild pear and Actinidia chinensis (Actinidia deliciosa). The genes MdAMR1.1 and MdAMR1.2 homologous to Arabidopsis thaliana GGP expression negative regulatory factor AMR1 were cloned from apple. The relationship between the expression of MdAMR1.1 and AsA was preliminarily analyzed, and the transcription factors combined with the promoter were screened by yeast single hybrid technique. The main results are as follows: 1. The expression of GGP1 in two GGP genes of Actinidia deliciosa was correlated with the high AsA content in the fruit of Actinidia deliciosa. The determination of AsA content in the fruit of the hybrid progenies of Pyrus sorghum and Actinidia chinensis showed that the content of AsA was significantly skewed to kiwifruit (Actinidia angustifolia). Genetic segregation analysis of GGP promoter in hybrid progenies showed that GGP1 and GGP2 were linked, and the GGP1 promoter of hairy flower might be related to AsA content. Further analysis of the GGP1 promoter sequence and activity of Actinidia chinensis showed that there was a deletion of 217bp and 215bp in the AeGGP1 promoter of Actinidia chinensis compared with Actinidia chinensis. The activity of AeGGP1 promoter of Actinidia deliciosa was significantly higher than that of Actinidia chinensis, and the activity of AeGGP1 allele of Actinidia chinensis was higher than that of Actinidia chinensis, but the activity of ArGGP1 allelic promoter of Actinidia chinensis was different. These results suggest that the genetic characteristics of GGP1 promoter sequence play an important role in the determination of AsA content in kiwifruit. At the same time, the relationship between the uORF region upstream of Arabidopsis GGP gene and the content of AsA also showed that the highly conserved uORF region sequence in different species was related to AsA, and the gene editing in this region could increase AsA content. By homology comparison, MdAMR1.1 and MdAMR1.2 genes, which have the highest homology with Arabidopsis GGP, expressed the negative regulatory factor AMR1 gene, were found in the apple genome. Expression analysis showed that MdAMR1.1 and AsA content were negatively correlated during fruit development. The full-length MdAMR1.1 and MdAMR1.2 genes of 'Gara' apple were cloned and transformed into wild-type and AMR1 mutant Arabidopsis thaliana respectively. It was found that overexpression of MdAMR1.1 and MdAMR1.2 in Arabidopsis could reduce AsA content of wild type and mutant type of Arabidopsis. MdAMR1.1 decreased more obviously in Arabidopsis complementation test. In order to investigate the transcriptional regulation of MdAMR1.1, the MdAMR1.1 promoter sequence was cloned from the genome of 'Gara' apple, and the recombinant plasmid of yeast single hybrid was constructed. By screening the cDNA library of apple fruit, 8 candidate genes were obtained. They were named AMR1-1, AMR1-5, AMR1-6, AMR1-7, AMR1-14, AMR1-16, AMR1-22 and AMR1-27, respectively, which laid a foundation for the further study of MdAMR1.1 transcriptional regulation.
【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】：2017
【分类号】：S66;Q943.2

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