黄瓜圆叶突变体基因克隆及功能分析

Perfect leaf morphology is the important guarantee for high yield of cucumber. A round leaf mutant with stable inheritance in cucumber was obtained by using EMS mutagenesis technique in previous studies. The leaf shape of mutant plant was round, leaf margin of mutant plant had no crack, the stomata of mutant plant were smaller, and the downy mildew resistance of mutant plant was higher than wild type. Genetic analysis revealed that the round leaf phenotype in cucumber was controlled by a single recessive gene. Considering its abnormal pollination character, the related map-based cloning techniques, which are based on hybridization between mutant and wild type to construct the genetic population, can not be applied to the gene cloning of the round leaf mutant in cucumber. Therefore, this present study intends to identify the candidate causal gene of round leaf mutant in cucumber by using Mutmap+ technique with M3 segregation population. The causal gene will be then verified by gene cloning, gene sequencing and comparison, gene expression analysis, CAPs marker and transgenic techniques. The function of causal gene of round leaf mutant in cucumber will be analyzed further. The results of this study will help people understanding further the molecular regulation mechanism of leaf morphology in plants, and will provide theoretical reference for the improvement of leaf morphology, yield incensement, and cultivation of ideal plant type in cucumber.

良好的叶形是黄瓜高产的重要保障。本项目在前期研究中利用EMS诱变获得了一个能稳定遗传的黄瓜圆叶突变体，其叶片为圆形，叶缘无裂刻，气孔较小，霜霉病抗性强于野生型。遗传分析表明该突变体为单基因隐性遗传。鉴于该黄瓜圆叶突变体不能正常授粉，基于突变体和野生型杂交手段构建遗传群体的相关图位克隆技术无法应用于该突变体基因克隆。因此，本研究拟在前期获得黄瓜圆叶突变体基础上，以黄瓜圆叶突变体M3分离群体为材料，利用MutMap+技术对黄瓜圆叶突变体候选基因进行初步鉴定，进而通过基因序列测定和比较、基因表达模式分析、CAPs标记验证以及转基因等技术对候选基因进行鉴定并分析其功能。本研究结果将会帮助人们进一步了解植物叶形发育分子调控机制，并为黄瓜叶形改良、产量增加和理想株型培育等研究提供理论参考。

黄瓜是世界十大蔬菜之一，良好的叶形是黄瓜高产的重要保障。为了研究黄瓜叶片形态建成的分子机制，本项目对黄瓜EMS诱变圆叶突变体（rl），进行了表型鉴定、遗传分析、基因定位以及分子机制研究，主要研究结果如下：与野生型相比，rl突变体叶片由掌状变为圆形，卷须数量明显减少，胚珠起始发生缺陷，雄花花蕊异常，导致rl突变体雌性器官完全不育以及雄性器官的部分不育性；利用MutMap+和KASP基因分型检测，初步确定候选基因为CsPID，等位性检测结果进一步证明CsPID为调控黄瓜叶片和花器官形态的建成的基因。将CsPID超表达至拟南芥pid-14突变体，转基因株系表型恢复了部分育性，花器官形态也基本上与野生型一致，说明CsPID的功能在拟南芥中具有保守性，参与花器官的发育，同时也确定了CsPID为调控黄瓜rl突变体的目标基因；原位杂交结果表明CsPID主要在叶片、顶端分生组织两侧、花瓣、萼片以及子房的胚珠原基中有表达，另外qRT-PCR的结果也表明CsPID在叶片和子房中表达量较高。IAA定位的结果表明IAA主要在野生型子房的胎座以及胚珠原基中富集，而在rl突变体中的相同部位并没有产生富集，该结果也与CsPID在子房中的表达模式一致；亚细胞定位结果证明了CsPID定位于细胞膜和核膜上，而Cspid定位情况则发生了变化，在细胞膜上呈点状分布，从而推测rl可能通过改变亚细胞定位的模式从而破坏了CsPID的功能；利用转录组测序技术筛选出与生长素极性运输和子房发育相关的基因，CsPID可能通过调控这些基因的表达从而影响了黄瓜侧生器官和胚珠的发育过程；酵母双杂交结果表明CsPID可以与极性基因CsREV在蛋白水平上发生互作，BiFC的结果也进一步证明CsPID和CsREV共定位于细胞膜和核膜。本项目研究结果可为黄瓜叶形改良、产量增加、培育理想株型等研究提供参考。