【摘要】：抗壞血酸(Ascorbate acid,AsA)是果實品質(zhì)的一個重要組成成分,植物的AsA含量主要依賴于生物合成,其中GDP-L-半乳糖磷酸化酶(GGP),是植物體內(nèi)AsA生物合成的主要限制酶。本文首先以AsA含量差異較大的山梨和毛花獼猴桃為研究對象,分析了其GGP基因的序列和活性差異;并從蘋果中克隆了與擬南芥GGP表達負調(diào)控因子AMR1同源的基因MdAMR1.1和MdAMR1.2,初步分析了二者表達與AsA的關系,并利用酵母單雜交技術篩選了與其啟動子相結合的轉錄因子。主要結果如下:1.在獼猴桃兩個GGP基因中,GGP1表達水平與毛花獼猴桃果實高AsA含量相關。在山梨和毛花獼猴桃雜交后代果實AsA含量的測定表明,AsA含量顯著的偏向毛花獼猴桃。在雜交后代中,GGP啟動子遺傳分離分析表明GGP1和GGP2連鎖,且毛花GGP1啟動子可能與AsA含量相關。進一步對山梨和毛花獼猴桃GGP1等位基因啟動子序列和活性分析表明,與山梨獼猴桃相比,毛花獼猴桃AeGGP1啟動子存在217bp和215 bp的缺失,毛花獼猴桃AeGGP1啟動子活性顯著高于山梨獼猴桃的,且毛花獼猴桃AeGGP1等位啟動子均存在高活性,而山梨獼猴桃ArGGP1等位啟動子活性存在差異。這些結果表明,獼猴桃GGP1啟動子序列的遺傳特性在AsA含量決定中起重要作用。同時,通過對擬南芥GGP基因上游的uORF區(qū)與AsA含量的關系研究也表明,不同物種中高度保守的uORF區(qū)序列與AsA有關,該區(qū)域的基因編輯能提高AsA含量。2.通過同源對比,在蘋果基因組中找到了與擬南芥GGP表達負調(diào)控因子AMR1基因同源度最高的MdAMR1.1和MdAMR1.2基因。表達分析表明,在果實發(fā)育過程中MdAMR1.1與AsA含量顯著負相關�？寺×恕吕病O果MdAMR1.1和MdAMR1.2基因全長,分別轉化進野生型和AMR1突變型擬南芥,發(fā)現(xiàn)MdAMR1.1和MdAMR1.2在擬南芥中的過量表達均可以降低野生型和突變型擬南芥AsA含量,且MdAMR1.1在擬南芥互補試驗中AsA下降更明顯。為探明MdAMR1.1轉錄調(diào)控途徑,從‘嘎啦’蘋果基因組中克隆了MdAMR1.1啟動子序列,構建了酵母單雜交重組質(zhì)粒,通過篩選蘋果果實的cDNA文庫,得到了候選相關基因8個,分別命名為AMR1-1、AMR1-5、AMR1-6、AMR1-7、AMR1-14、AMR1-16、AMR1-22和AMR1-27,為MdAMR1.1轉錄調(diào)控的進一步研究奠定了基礎。
[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.
【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】：2017
【分類號】：S66;Q943.2

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