生菜4个叶色调控基因（PLL1-4）的遗传克隆及其作用机理分析

Lettuce is one of the most important vegetables worldwide. Purple/red lettuce is welcome by consumers due to its color appearance and health benefits. Purple/red color of lettuce leaf is attributed to anthocyanin, accumulated in the epidermal cells of leaf. Anthocyanin is shown to be a strong antioxidant, and high concentration of anthocyanin in food has efficient anticancer effects.The research on leaf colour is still rare, though leaf, the harvest organ, is one of important horticultural traits. Green and purple are the two main colors of lettuce leaf, though different cultivars may vary slightly. In this proposed study, we will map and clone genes controlling purple leaf of lettuce. Primary study, which combined traditions genetics approach and the second generation sequencing technology, successfully mapped three loci (PLL1-3) controlling leaf color. At least one additional locus (PLL4) controlling purple leaf exists in this segregating population. We are going to fine map and clone at least two of them, and then analyze their molecular mechanisms. These four genes (or loci) controlling purple leaf will be used to screen a collection of 950 lettuce cultivars. If a purple cultivar does not have these four genes, it will be considered to harbor new sources of genes controlling purple lettuce leaf. Highly linked markers will be developed for these new genes. The cloned genes and markers highly linked with new source of genes controlling purple leaf will be helpful for the development of cultivars with high concentration of anthocyanin in lettuce.

生菜和莴苣是世界上最重要的蔬菜之一。由于有色生菜/莴苣中的花青素具有抗氧化、抗癌和抗衰老作用，紫色和红色生菜/莴苣越来越受到消费者的青睐。叶色是生菜重要农艺性状之一，但生菜叶色相关研究仍较少。本项目前期工作利用紫色生菜和绿色油麦菜（叶用莴苣）进行杂交，构建F2叶色分离群体,通过传统遗传学和第二代测序技术相结合的方法已快速定位了3个控制紫色生菜的位点（PLL1-3），发现该分离群体中还有至少1个（PLL4）未定位的紫色调控基因。本研究拟精细定位这4个基因，克隆至少其中2个并分析其作用的分子机理。根据这些基因扫描本实验室收集的950份生菜材料，找到含有其它紫色调控基因的生菜/莴苣品种，建立新的分离群体，利用上述方法快速定位叶色调控基因。本研究将系统分析生菜中紫色调控基因以及作用机理，为一步了解花青素的合成与调控以及培育高含量花青素的生菜品种奠定基础。

生菜是世界上最重要的蔬菜之一，而叶色是生菜重要品质。生菜叶片颜色通常是紫色和绿色，其紫色叶片又存在一系列的深紫和浅紫，紫色主要是由花青素类物质在叶表皮细胞积累形成。本项目利用紫色生菜和绿色油麦菜进行杂交，构建叶色分离群体，通过传统遗传学和第二代测序技术相结合的方法，精细定位克隆了4个控制生菜紫色叶片性状的基因（Purple Lettuce Leaves 1-4, PLL1-4），并系统分析它们的作用机理。PLL1编码一个bHLH转录因子，该基因在绿色亲本中第七个外显子起始位置存在5 bp的缺失，导致功能丧失。PLL2编码一个R2R3-MYB转录因子，该基因存在于紫色亲本中并高量表达；该基因在绿色亲本中缺失，呈现有无多态性。PLL1和PLL2蛋白都能结合花青素合成途径结构基因DFR和ANS的启动子顺式元件。PLL3是花青素合成的负调控因子，它编码一个R3-MYB转录因子，PLL3是通过与R2R3-MYB（PLL2）竞争结合bHLH（PLL1）转录因子来抑制花青素的合成。利用上述基因上标记，扫描了本实验收集的生菜种质资源，发现在PI491070× PI536760的F2群体中存在新的位点PLL4，在该区间内发现花青素合成酶基因ANS在编码区发生点突变造成提前终止。从栽培和野生生菜中PCR扩增PLL1-4基因序列并进行进化分析，发现PLL-4基因的功能变化都发生在驯化后。其中PLL1或PLL4的突变使植株失去合成花青素的能力，植株变成绿色；PLL2突变则是植株颜色变淡， PLL3的突变使植株表现的红色更深。在驯化过程中，生菜叶片色泽向两个极端方向（更紫或者纯绿）进行，呈现典型的歧化选择模式。. 通过本项目的研究克隆了4个生菜中紫色控制基因，明确了它们的作用机理，为一步了解花青素的合成与调控以及培育高含量花青素的生菜品种奠定分子基础。