辣椒黄色成熟果色新型突变体YRF基因的克隆及其调控机理的研究

Pepper fruit contains a variety of pigments with important health and economic value. Capsanthin/capsorubin synthase gene (Ccs) plays an important role in the regulation of red ripe-fruit color formation, which will be changed to yellow ripe-fruit color with deletion or mutation of this gene. Our research group has found a novel mutant of pepper yellow ripe-fruit color which carries the complete Ccs gene sequence with no mutation. However, our knowledge on its regulatory genes and their regulatory mechanisms is still unclear in pepper. Therefore, we have constructed a new mutant MutMap population of yellow color ripen fruit. Basis on this, our research project aims to clone the yellow ripe-fruit color(YRF) gene with different techniques including MutMap, VIGS, qRT-PCR, genomics, and develop its early selection markers. In addition, we are planning to clarify its function with skills of genetic transformation and identification of transgenic lines. Also, we will screen its upstream and downstream target genes and interacting proteins using the techniques of yeast one hybrid library screening, ChIP-seq, yeast two hybrid and affinity purification mass spectrometry. Moreover, we will also analyse the network relationship regulated by YRF gene in the transgenic lines with technique of RNA-seq. The goal of this project is to clarify the role of YRF gene in transcriptional and post transcriptional regulation of yellow color ripen fruit, and to provide theoretical basis for the breeding of yellow color ripen fruit.

辣椒果实含有的各种色素具有重要的保健和经济价值。辣椒红素/玉红素合成酶基因（Ccs）在调控辣椒红色成熟果色形成中发挥着重要作用，该基因缺失或突变后果实呈黄色。然而，前期项目组发现了一个黄色成熟果色新型突变体，其携带完整Ccs基因序列且无突变，尚不清楚其调控基因及其调控机理。为此，在构建黄色成熟果色新型突变体突变位点图谱（MutMap）群体的基础上，本项目拟采用MutMap方法，结合VIGS、qRT-PCR和基因组学方法克隆黄色成熟果色YRF基因，开发其早期选择标记；利用遗传转化和转基因株系鉴定明确其功能；利用酵母单杂交文库筛选和染色质免疫共沉淀测序，以及酵母双杂交和亲和纯化-质谱技术筛选其上下游靶基因和互作蛋白；利用转基因系的转录组测序技术分析YRF基因调控的网络关系，揭示YRF基因在转录调控和转录后调控黄色成熟果色的作用，为辣椒黄色成熟果色育种提供理论依据。

辣椒成熟果色是重要的商品性状和营养性状。项目前期在循化线辣椒（成熟果色红色）田间发现一株黄橙色成熟果色植株，经多代自交选育为黄橙色成熟果色突变体。项目对突变体果实类胡萝卜素合成途径的关键基因，如辣椒红素/玉红素合成酶（Ccs）、八氢番茄红素合成酶（Psy）、番茄红素环化酶（Lcyb）和β-胡萝卜素羟化酶（Crtz）等基因序列及其启动子序列分析表明，与其野生型相比较无突变发生。为此，开展了辣椒黄橙色成熟果色性状的遗传规律、BSA群体构建、基因定位、转录组差异分析，以及辣椒类胡萝卜素代谢、花青苷代谢机理的研究。明确了辣椒突变体黄橙色成熟果色符合单一隐性基因控制的遗传规律。然而其复杂性是，其调控基因以及调控机理并非如常规单一隐性基因控制性状遗传一样。通过BSA群体构建、基因定位、转录组差异基因筛选，以及辣椒类胡萝卜素代谢、花青苷代谢机理的关联性分析，项目组筛选、鉴定出辣椒黄橙色成熟果色形成与发展的关键基因CaMYB306和CaATHB-12。再通过VIGS技术、qRT-PCR和基因组学方法克隆了辣椒黄橙色成熟果色突变体性状形成关键基因CaMYB306和CaATHB-12，并对其进行了生物学信息分析与表达特性分析；利用超量表达、RNAi基因敲除技术和转基因株系鉴定确定其对辣椒黄橙色成熟果色的形成与发展起关键作用，是调控辣椒突变体黄橙色成熟果色形成与发展的关键基因。进一步利用转录组测序（RNA-seq）技术、基因启动子序列分析与鉴定，以及酵母杂交等技术筛选其靶基因和互作蛋白，揭示了CaMYB306蛋白通过对CaCIPK13启动子的负调控作用调控植株的抗寒性。反过来，低温又诱导CaMYB306的表达，从而促进辣椒果色的形成与发展。这些结果对于揭示辣椒成熟果色的遗传与其调控机理，加速我国辣椒品质分子设计水平具有重要理论与应用意义。