萝卜晚花及大根天然变异新种质的分子解析

Six years ago, we identified a unique spontaneously mutated radish (Raphanus sativus L.) germplasm with later flowering time and bigger taproot compared with the original germplasm. It showed that the mutated phenotype is recessive and controlled by a single locus using the F2 population from the cross between the mutated germplasm and the original germplasm. Using the Second-Generation Sequencing technology, we obtained draft sequences of the genomes of the mutated germplasm and another radish line from the UK. Plenty of polymorphisms were identified between these two lines. Then, a number of SSLP (simple sequence length polymorphism), CAPS (cleaved amplified polymorphic sequences) and dCAPS (derived CAPS) markers were developed. Using these molecular markers and a large F2 segregating population composed of 28000 individual plants from the cross between the mutated germplasm and the line from the UK, the controlling locus was defined into a 10kb DNA region. The corresponding genomic region from the original germplasm were cloned and sequenced. After DNA alignment with the original germplasm, a short DNA deletion in the coding region of a gene was identified from the corresponding region in the mutated germplasm. Furthermore, transgenic function complementation test revealed the causative gene RsFTRD-1 (FTRD is the abbreviation for flowering time and root development) controlling flowering time and taproot development. .. In the proposed project, we are going to illustrate the novel molecular mechanism of flowering time and taproot development regulated by the RsFTRD-1 gene. We will study the spatial and temporal expression of the RsFTRD-1 gene using histochemical GUS (β-glucuronidase) staining technique. Chromatin immunoprecipitation followed by sequencing (ChIP-Sequencing) assay will be performed to investigate the target genes of the RsFTRD-1 protein. The candidate target genes will then be tested by ChIP-qPCR and qRT-PCR. Previous study has isolated some candidate proteins interacting with the RsFTRD-1 protein using the yeast two-hybrid system. In this work, proteins interacting with the RsFTRD-1 protein will be screened using HA and FLAG Tag purification system and LC-MS/MS (liquid chromatography-tandem mass spectrometry) analysis followed by confirmation with the bimolecular fluorescence complementation (BiFC) technique. .. The elucidation of molecular mechanism of the RsFTRD-1 gene involving flowering time and taproot development will provide novel insights into the transition from the vegetative to the reproductive phase and taproot development in plants. Furthermore, these cloned gene resources would play an important role in plant genetic improvement.

申请者六年前于试验田中观察到一份较亲本晚花及大根的萝卜种质，并持续研究至今。通过基因组测序技术开发了高密度分子标记和构建了超大分离群体，精细定位了该性状的控制基因，并经转基因功能验证，获得了调控植物开花时间及根发育新基因RsFTRD-1的序列信息。本项目将深入阐明该重要性状的分子机制。拟应用GUS（β-葡萄糖醛酸酶）组织染色技术明确基因RsFTRD-1的表达特征；应用ChIP（染色质免疫共沉淀）结合第二代测序技术解析蛋白RsFTRD-1的靶标基因，并利用ChIP-qPCR和qRT-PCR技术验证；通过蛋白标签技术纯化蛋白并结合液相色谱-串联质谱技术筛选蛋白RsFTRD-1的互作蛋白，并利用双分子荧光互补技术验证。植物开花时间和肉质直根膨大双调控分子机制的深入剖析，将深化植物开花时间分子机理的认识，也为深度解析植物肉质直根的发育提供重要信息，并为植物遗传育种提供重大价值基因。

发现两份种质直根的真根部和根颈部的中柱组织呈左右对称生长，而非辐射对称生长。应用GUS组织染色技术明确了花期及直根发育调控基因RsFTRD-1的表达特征；应用ChIP结合第二代测序技术解析了蛋白RsFTRD -1的靶标基因，并利用ChIP-qPCR和qRT-PCR技术进行了验证；通过蛋白标签技术纯化蛋白并结合液相色谱-串联质谱技术筛选了蛋白RsFTRD-1的互作蛋白。低温显著促进了开花但抑制了直根发育。根部组织的转录组分析表明，低温影响了植物激素信号转导和植物昼夜节律通路基因的表达。而高温也发现影响植物昼夜节律通路基因的表达。.植物开花时间和肉质直根发育的深入剖析，深化了植物开花时间分子机理的认识，也为深度解析植物肉质直根的发育提供了重要信息，并为植物遗传育种提供重大价值基因。