生菜叶缘卷曲控制基因的遗传克隆及作用机理研究

Lettuce is one of the most important leafy vegetables worldwide. There are considerable phenotypic variations among different horticultural types. Leaf flatness is an important sensory trait for lettuce, and curved leaf margin is preferred sometimes. Leaf curvature is caused by imbalance development of leaf lamina, with increased cell division and/or cell expansion at the leaf margins, and therefore serves as a good material for studies on leaf development. Our lab has constructed a GWAS population for lettuce, and obtained at least 5 Gb RNA-seq data for each genotype. In this proposed study, we will perform GWAS analysis for leaf curvature. At the same time, biparental segregating populations are constructed by crossing cultivars with flat leaves and cultivars with curved leaves. BSA in combination with RNA-seq will be used to dissect the genetics of leaf curvature. At least two genes controlling leaf curvature will be fine mapped and at least one of them will be cloned using map-based cloning method. Then, genes associated with leaf curvature will be identified through combined analysis of eQTL, differentially expressed genes and gene expression network. This study will provide insight on leaf development, the mechanism for leaf curvature in particular. The results will also be useful for the breeding of lettuce cultivars with ideal leaf shape.

莴苣（Lactuca sativa）是世界上最重要的蔬菜之一，包含叶用的生菜和茎用的莴笋。作为叶用类型，生菜叶色和叶型是其重要的园艺性状。散叶生菜（尤其是色拉生菜）的叶缘卷曲是育种家和消费者关心的感官品质。叶缘卷曲由叶发育不平衡所致。本实验室已构建了一个莴苣关联分析群体，对240份材料进行了RNA-seq测序，本研究拟利用该群体对叶缘卷曲进行关联分析，鉴定控制叶缘卷曲的位点。同时，我们还构建了多个叶缘卷曲双亲分离群体，利用BSA与RNA-seq相结合的方法快速定位了叶缘卷曲控制位点，成功对3个位点进行了遗传定位。本研究拟精细定位这3个基因并克隆至少其中2个，研究其控制叶缘卷曲的作用机理。我们还将利用eQTL、差异表达分析以及基因表达网络分析挑选2-3个可能参与叶缘卷曲形成的基因，分析其功能，进而深入了解叶缘卷曲形成的分子机理。本研究的结果将丰富植物叶发育理论，并对生菜育种具有指导意义。

生菜叶缘卷曲性状是重要的农艺性状，影响着生菜的感官品质。我们通过对两个杂交分离群体进行QTL-seq分析，获得两个控制生菜叶缘卷曲的基因。第一个分离群体是叶卷曲生菜与油麦菜杂交的分离群体。QTL-seq分析发现，该群体中叶缘卷曲由三个位点控制。我们对5号染色体的QTL进行了深入研究，构建了该位点的单基因分离群体，最终将该控制叶缘卷曲基因定位在252.34 Mb - 253.35Mb区域范围内。该区域内的LsTCP4基因为控制叶缘卷曲的候选基因。该基因在卷叶生菜中存在一个约5 kb的反转录转座子插入，使其表达量下降。通过超量表达和敲除LsTCP4基因，验证了该基因对叶缘卷曲的作用。反转座子的插入未导致甲基化程度的变化，也未检测到小RNA。我们的结果表明反转座子的插入可能是通过破坏下游调控基因表达的序列（如增强子）进而改变基因的表达。我们通过对一个叶缘卷曲和一个叶平整的生菜进行杂交构建了第二个控制生菜叶缘卷曲的分离群体。BSA分析发现，该分离群体中的叶缘卷曲由单基因控制。通过对2000多株的群体进行遗传分析，最终将该基因定位在7号染色体，候选基因是LsKN1。该基因的第一个外显子插入了一个CACTA转座子，导致基因表达量提高。转基因互补实验、超量表达和敲除实验都证明了LsKN1为调控叶缘卷曲的基因。LsKN1蛋白结合CK合成途径基因并促进了细胞分裂素的提高，进而导致叶缘卷曲。LsKN1蛋白同时通过抑制GA的合成导致叶缘卷曲。叶面喷施CK可促进生菜叶缘卷曲，而喷施GA则一直叶缘卷曲，进一步证明了LsKN1调控生菜叶缘卷曲的作用机理。本研究结果可用于今后生菜的叶缘卷曲性状育种。