苹果资源果实糖酸性状评估及酸度性状的候选基因关联分析

发布时间：2018-03-27 15:02

本文选题：苹果　切入点：HPLC　出处：《中国科学院研究生院(武汉植物园)》2016年博士论文

【摘要】：苹果(Malus domestica Borkh.)是世界上广泛种植的经济果树之一。可溶性糖和有机酸的组分与含量是决定果实风味品质的重要因素。本论文中利用高效液相色谱法(HPLC)对苹果资源成熟果实中的可溶性糖组分及苹果酸的含量进行测定,并开展了酸度性状的候选基因关联分析研究,主要结果如下：1.构建了一个包含321份栽培种和43份野生种成熟果实的糖酸含量数据库。苹果果实中可溶性糖组分包括蔗糖、果糖和葡萄糖,其中,栽培种可溶性糖组分以蔗糖和果糖为主,而野生种可溶性糖组分以果糖和葡萄糖为主。苹果有机酸组分以苹果酸为主,野生种果实中苹果酸的含量显著高于栽培种,但总糖含量及总甜度在野生种与栽培种之间非常相似。可见,苹果风味品质驯化过程中酸度选择起着关键作用。2.苹果成熟果实中,苹果酸含量与葡萄糖含量呈正相关,但与蔗糖含量呈负相关,这表明苹果驯化过程中酸度选择能够引起果实可溶性糖组分含量的变化。3.控制苹果果实酸度的一个主效QTL位于第16号连锁群的Ma(Malic acid)区间,通过区间关联分析研究发现一个编码铝诱导的苹果酸转运体(ALMT)的Ma1基因是Ma位点候选基因。Ma位点包含两个等位基因：Ma1和ma1。Ma1定位于液泡膜上,其在酵母中异源表达能显著提高细胞中苹果酸的积累,这表明Ma1可作为液泡膜上的苹果酸盐通道而促进果实有机酸积累。但ma1基因由于最后一个外显子发生了G→A的单碱基突变,造成终止密码子提前(少了84个氨基酸),编码的不完整蛋白定位于细胞质膜上,不能行使液泡膜上苹果酸盐的转运功能,不利于有机酸的积累,导致低酸性状的形成4.为分析Ma1基因同源基因对果实酸度存在的潜在影响,对苹果铝诱导的苹果酸盐转运体基因家族成员进行了分析,发现该基因可分为三个亚家族,其中Ma1基因属于ALMTII亚家族成员。开发了所有ALMTII亚家族成员的分子标记,并开展了候选基因关联分析,结果表明除Ma1基因与苹果成熟果实中苹果酸含量具有显著相关性外,其他同源基因与果实酸度均不存在显著相关性。5.对苹果资源Ma位点基因型进行了分析,结果表明,栽培苹果Ma1Ma1基因型频率较低,而野生苹果该基因型频率较高,这表明苹果果实酸度驯化可能与Mal基因的选择有关。此外,研究还发现Ma位点基因型相同的不同资源成熟果实中苹果酸含量仍有较大的变异,这表明Mal基因并不是决定苹果果实酸度的唯一遗传因子。上述研究不仅为苹果果实风味品质遗传改良提供了理论依据和材料保障,而且也为果树等作物数量性状基因的发掘提供了新的思路。
[Abstract]:Malus domestica Borkh. is one of the most widely planted fruit trees in the world. The composition and content of soluble sugar and organic acid are important factors to determine the quality of fruit flavor. The contents of soluble sugar and malic acid in ripe fruits were determined. The main results are as follows: 1. A database of sugar and acid contents of 321 cultivated species and 43 wild mature fruits was constructed. The soluble sugar components in apple fruits include sucrose. Fructose and glucose, the soluble sugar components of cultivated species were mainly sucrose and fructose, while those of wild species were fructose and glucose. The content of malic acid in wild fruit was significantly higher than that in cultivated species, but the total sugar content and total sweetness were very similar between wild and cultivated species. It can be seen that acidity selection plays a key role in the process of apple flavor quality acclimation. Malic acid content was positively correlated with glucose content, but negatively correlated with sucrose content. This indicated that the selection of acidity during apple acclimation could result in the change of soluble sugar content. 3. A major QTL controlling apple fruit acidity was located in the Ma(Malic acidregion of the 16th linkage group. By interval correlation analysis, it was found that the Ma1 gene encoding an aluminum-induced malate transporter was a candidate gene at Ma locus .Ma locus contained two alleles: Ma1 and ma1.Ma1, which were located on the vacuolar membrane. Its heterologous expression in yeast significantly increased the accumulation of malic acid in the cells, which indicated that Ma1 could promote the accumulation of organic acids in fruit as a channel of malate in vacuolar membrane. However, the ma1 gene had G due to the last exon. 鈫扵he single base mutation of A caused the termination codon to be prematurely (84 amino acids missing, the encoded incomplete protein was located on the cytoplasm membrane, and could not perform the function of transporting malate on the vacuolar membrane, which was not conducive to the accumulation of organic acids. In order to analyze the potential effect of the homologous gene of Ma1 gene on fruit acidity, the members of the malate transporter gene family induced by apple aluminum were analyzed, and the results showed that the gene could be divided into three subfamilies. Ma1 gene belongs to the ALMTII subfamily. Molecular markers of all ALMTII subfamily members were developed and candidate gene association analysis was carried out. The results showed that the Ma1 gene was significantly correlated with malic acid content in apple fruit. There was no significant correlation between other homologous genes and fruit acidity. 5. The Ma locus genotypes of apple resources were analyzed. The results showed that the frequency of Ma1Ma1 genotype in cultivated apple was lower than that in wild apple. The results suggested that the acidity acclimation of apple fruit might be related to the selection of Mal gene. In addition, the malic acid content in mature fruits with the same Ma locus genotype was still varied greatly. These results indicate that Mal gene is not the only genetic factor to determine the acidity of apple fruit, and this study not only provides theoretical basis and material guarantee for genetic improvement of apple fruit flavor quality. It also provides a new idea for the discovery of quantitative trait genes in fruit trees and other crops.
【学位授予单位】：中国科学院研究生院(武汉植物园)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S661.1

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