大白菜早抽薹突变基因ebm1的克隆与功能鉴定

The development of early bolting mutants and function analysis of the relevant mutant genes are the effective approach to explore the mechanism that control the switch from the vegetative growth to reproductive growth in Chinese cabbage. In the previous study, the applicant obtained an early bolting mutant in Chinese cabbage by using the microspore culture and EMS treatment. Genetic analysis showed that the early bolting trait was controlled by a single recessive gene, designated as ebm1. ebm1 has been mapped within the 88.2kb region on the chromosome A04. Based on the genome resequencing of the wild-type and mutant, and BAC clone sequencing, the aim of this study is to fine map ebm1 and clone the candidate gene. The real mutant gene will be confirmed by functional complementary verification. The expression characteristic of ebm1 will be studied by subcellular localization analysis, promoter activity analysis and spatiotemporal expression pattern analysis. The interaction protein and the genes that are regulated by ebm1 will be searched by yeast two hybridization, ChIP-seq, EMSA, etc. Transcriptome analysis will be performed on the wild type and mutant. The relation between ebm1 and the known bolting related genes will be analyzed. Furthermore, the signal pathways and metabolic pathways that ebm1 involved in will be screened. The research results will be helpful to reveal the regulatory mechanism of bolting and flowering in cruciferous plants, and lay a certain theoretical basis for the genetic improvement of bolting traits in Chinese cabbage.

创制早抽薹突变体和解析突变基因的功能，是研究大白菜从营养生长向生殖生长转变分子调控机制的重要策略和手段。在前期研究中，申请者以大白菜DH系为试材，通过小孢子培养结合EMS诱变处理，创制出1个早抽薹突变体，证明其早抽薹性状由单隐性基因ebm1控制，已将其定位在A04染色体88.2kb区域内。本研究拟通过对突变体和野生型基因组重测序，以及BAC克隆的筛选测序，进一步精细定位ebm1，预测其候选基因；通过功能互补验证，克隆ebm1基因；借助亚细胞定位、启动子活性和时空表达模式分析，明确其表达特性；通过酵母双杂交、ChIP-seq和EMSA等技术，分离ebm1互作蛋白和受其调控的下游基因；通过转录组测序分析，探讨其与已知抽薹基因间的关系，发掘ebm1促进抽薹所涉及的信号通路和代谢途径。研究结果可以丰富植物抽薹开花分子调控机制方面的知识，并可为白菜类蔬菜抽薹性状的遗传改良提供参考。

创制早抽薹突变体和解析突变基因的功能，是研究大白菜从营养生长向生殖生长转变分子调控机制的重要策略和手段。在前期研究中，申请者以大白菜DH系为试材，通过小孢子培养结合EMS诱变处理，创制出1个早抽薹突变体，证明其早抽薹性状由单隐性基因ebm1控制，已将其定位在A04染色体88.2kb区域内。本研究中，将ebm1重新命名改为ebm3，利用基于改良的MutMap测序、基因分型分析和等位性试验，证明编码组蛋白甲基转移酶CURLY LEAF（CLF）的BraA04g07190.3 C是emb3的强候选基因。BrEBM3第14外显子的C到T碱基替换导致氨基酸变化（S到F）和emb3的早期抽薹表型。突变发生在SET结构域，该结构域催化组蛋白中的位点和状态特异性赖氨酸甲基化。组织特异性表达分析表明（QRT-PCR），BrEBM3在花蕾中优势表达。启动子活性分析，证实BrEBM3启动子在花序中具有活性。亚细胞定位显示BrEBM3定位于细胞核内。转录组分析表明，BrEBM3突变可能抑制H3K27me3的沉积，并激活AGAMOUS（AG）和AGAMOUS-like（AGL）基因座的表达，从而导致早期开花。最终，我们确认EMS诱导的大白菜早抽薹突变体ebm3是由编码组蛋白甲基转移酶CLF的BraA04g017190.3C非同义突变引起的。这些结果提高了我们对白菜类蔬菜抽薹和开花的认识，可能有助于大白菜的遗传改良。同时，我们构建了基于小孢子培养的大白菜基因编辑技术体系，为该类作物的基因功能鉴定和资源改良提供了有效的工具。