山葡萄及其杂交品种间单、双糖苷花色苷组成差异的机制研究

The anthocyanin compositions in red grape berries have an important impact on the qualities of their wines. There are only monoglucosidic anthocyanins found in Vitis vinifera grape berries, while many diglucosidic anthocyanins, which are easy to brown in the aging process of red wines, also exist in the berries of V. amurensis grapes and their hybrids. Furthermore, there are significant differences exsited in the composition of monoglucosidic anthocyanins and diglucosidic anthocyanins among different V. amurensis cultivars and hybrids. The key enzyme of diglucosidic anthocyanin synthesis is anthocyanin 5-O-glucosyltransferases (5GT). A functional allele of 5GT was found in non-V. vinifera grapes, while an allele in V. vinifera grapes was nonfunctional. Then are the composition differences of monoglucosidic and diglucosidic anthocyanins among different grape cultivars controlled by haplotype compositions at the region of 5GT loci? The differences have any relationships with the expressions and activities of 5GT? These are the primary questions in this study. On the basis of preliminary work, the 5GT allels will be identified and isolated from the region of 5GT loci in gDNA of V. amurensis cultivars and hybrids. The recombinant enzyme activities and substrate specificities will be characterized through heterologous expression. And functions of 5GT alleles will be verified by transient gene expression assays in V. vinifera grape berries. Then the functional 5GT alleles will be confirmed. After that, the relationship of phenotype with haplotype compositions, expression and enzyme activities of 5GT will be analyzed in the process of anthocyanin acummlation and also in a group of V. amurensis cultivars and hybrids. The purpose of this study is to clarify the reason of composition differences of monoglucosidic and diglucosidic anthocyanins among V. amurensis cultivars and hybrids, and lay foundation of further revelation of the synthesis and regulation mechanism of diglucosidic anthocyanins in grapes. The results have a significant effect on improving wine quality made from V. amurensis grapes through the proportion decrease of diglucosidic anthocyaninans via molecular methods and directed breeding.

葡萄花色苷组成是影响葡萄酒品质的重要因素。山葡萄及其杂交品种含大量酒陈酿过程中易褐变的双糖苷花色苷，且不同品种间两种花色苷的组成差异显著。双糖苷花色苷合成关键酶为5-O-葡萄糖基转移酶（5GT），非欧亚种存在5GT功能性等位基因，欧亚种存在5GT非功能性等位基因。而山葡萄品种间这种花色苷组成差异是否由5GT基因座单倍型组成控制？又是否与5GT的表达和活性差异有关？这是本项目拟研究的首要问题。本项目在前期工作基础上，鉴定分离山葡萄及其杂种的5GT等位基因，异源表达测定酶活性，瞬时表达验证其功能，从而确定5GT功能性等位基因。然后分别在花色苷积累过程中和17个品种中分析表型与5GT基因座单倍型组成、表达、酶活性的关系，确定山葡萄及其杂种间Atc-3-glc和Atc-3,5-diglc组成差异的机制，为揭示葡萄双糖苷花色苷合成调控机制奠定基础。本研究对通过定向育种提高山葡萄酒品质具有重要意义。

葡萄花色苷组成是影响葡萄酒品质的重要因素。山葡萄及其杂交品种含大量酒陈酿过程中易褐变的花色素双糖苷，严重影响山葡萄酒的陈酿潜力和品质。双糖苷花色苷合成关键酶为5-O-葡萄糖基转移酶（5GT），据报道其合成基因定位于葡萄9号染色体上，非欧亚种存在5GT功能性等位基因，欧亚种存在5GT非功能性等位基因。然而对于山葡萄花色素双糖苷的合成调控机制的研究较少。本研究采集了大量的山葡萄及其杂交品种，分析它们糖基化花色苷（即花色素单糖苷和花色素双糖苷）的含量和组成，并选取代表性的品种克隆9号染色体及其他染色体上已知和未知的5GT等位基因，通过序列分析初步判断基因的功能性，以期从基因型的角度分析山葡萄及其杂交品种间糖基化花色苷组成差异的机制。研究结果：1.不同品种的糖基化花色苷组成差异显著，山葡萄与欧亚种杂交品种的花色素双糖苷比例（65.13%）远远低于山葡萄品种（88.89%）。2. 从代表性品种的9号染色体上克隆出7个5GT等位基因，其中3个5GT等位基因有功能，4个5GT等位基因由于移码突变和提前终止密码子导致C-末端的截断而丧失功能。3.从5号染色体上克隆到5个5GT等位基因，其中3个5GT等位基因有功能，2个5GT等位基因无功能。4.从5号和13号染色体上克隆出未知功能的5GT等位基因26个，由于缺乏功能性等位基因的对比序列，无法判断它们的基因功能，但蛋白质结构预测表明‘哈桑’中克隆到的3个5GT等位基因有明显的结构缺失，可初步推测这3个基因为非功能性的5GT等位基因。5.分析表明代表性品种所含功能性等位基因的数量、每个基因位点的分型与品种糖基化花色苷的组成并没有明显的相关性，而且每个品种中都存在若干数量未知功能性的等位基因。因此，我们仍在进行进一步的分析和研究来阐明不同品种间糖基化花色苷组成差异的机制，这一机制的阐明对通过定向育种提高山葡萄酒品质具有重要意义。