乌菜BcFLC1基因点突变改变抽薹时间的分子机理

Wucai is the special vegetable in the Yangtze-Huaihe River Basin of China. Most of the Wucai varieties are early-bolting, which affects the yields and qualities, and limits the development of this industry. In our previous study, a special material W13-2 was obtained during the creation of germplasm resources, which the bolting time delayed more than 100 days compared with its homologous material W13-1. The genetic and molecular analysis shown that the A base (520th of CDS) and Threonine (174th of amino acid) of BcFLC1 gene from W13-2 were mutated into G base and Alanine compared with W13-1, and the mutated BcFLC1 could delay bolting in the flc mutant of Arabidopsis thaliana. However, the regulation mechanism of this mutated BcFLC1 on bolting time changing is still unclear. In this study, the fluorescence quantitative PCR (qRT-PCR) and Chromatin immunoprecipitation assay (ChIP-PCR) will be used to analysis the splicing and function of COOLAIR, the antisense long non-coded RNA of BcFLC1; the yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) will be employed to evaluate the affect of the mutated BcFLC1 on the polymerization of FLC-FLC and FLC-SVP, and the electrophoretic mobility shift assay (EMSA) and luciferase in vivo expression technology will be adopted to determine the interaction between the promoters of FT, SOC1 and the mutated FLC two-polymer. Finally, the Wucai transgeneic plants will be generated through the gene editing technique CRISPR/Cas9, and the function of the point mutation gene BcFLC1 will be further verified by the qRT-PCR, western blot and ChIP-PCR using the of Arabidopsis thaliana mutant flc and Wucai transgeneic plants. This project will be of great significance in exploring the molecular mechanism of changing bolting time by the point mutation BcFLC1 gene in Wucai, and guiding the reasonable application in the breeding of wucai for enhancing the resistance to bolt.

乌菜是我国江淮流域特色蔬菜，但先期抽薹严重影响产量与品质。课题组前期研究发现，抽薹时间延迟3-4个月的特异材料W13-2，其BcFLC1基因发生突变（CDS第520位碱基A→G；AA第174位亲水Thr→疏水Ala），并初步证明该基因点突变与抽薹时间的变异密切相关，但确切的分子机理尚不明确。本项目拟利用qRT-PCR与ChIP-PCR技术，分析该基因反义lncRNA COOLAIR的剪接与功能；采用Y2H与BiFC技术，研究点突变影响FLC二聚体的聚合，结合EMSA与LUC双荧光技术，分析其与成花整合因子启动子的互作；利用CRISPR/Cas9系统定点编辑乌菜BcFLC1，与前期获得的拟南芥flc转基因植株，采用qRT-PCR、Western杂交及ChIP-PCR技术，验证点突变BcFLC1基因的功能。研究结果将明确BcFLC1点突变改变乌菜抽薹时间的分子机理，并为乌菜晚抽薹育种提供基础。

先期抽薹不仅影响乌菜商品价值，严重时还会造成种植失败，带来巨大的经济损失。选育晚抽薹新品种，对提高乌菜产量、商品性和食用价值，具有重要的意义。基于前期获得的晚抽薹W13-2及早抽薹W13-1材料，研究发现BcFLC1基因点突变（AA174：T→A）可能影响乌菜抽薹开花时间。本研究利用全转录组测序及qRT-PCR技术，分析两材料之间的差异LncRNA（DELs），共获得60个DEL，其中映射到靶基因BcFLC1的DEL有19个；经过序列比对鉴定，COOLAIR（MSTRG.9105.3）在俩材料中差异4.44倍，表明FLC1基因K-box点突变可降低COOLAIR的转录。利用RNA-Seq技术，分析了两材料的基因转录水平，共获得差异表达基因（DEG）8006个；其中，成花整合因子FT、SOC1等基因转录水平均下调表达，表明BcFLC1基因点突变可影响FT与SOC1的表达延迟其抽薹开花。利用酵母双杂交等技术，分别进行BcFLC1点突变基因与SVP的杂交分析，发现点突变未影响FLC-SVP二聚体的形成，但提高了其聚合强度；表明BcFLC1基因点突变可增强FLC-SVP二聚体的聚合强度，继而可能抑制FT、SOC1等基因的表达，从而延迟开花时间。分别构建BcFLC1-174A点突变基因植物表达载体，遗传转化拟南芥进行功能验证；结果表明，BcFLC1点突变可显著延迟转基因拟南芥抽薹开花时间，并且成花整合因子相关基因的表达水平也显著下调。此外，分析了农杆菌侵染乌菜花药影响因素，优化了共培养条件；结合人工辅助授粉技术，建立了农杆菌介导乌菜花药非组织培养遗传转化技术。研究结果为进一步揭示乌菜抽薹开花分子机制及晚抽薹种质创新提供了理论基础。. 在项目执行期间，发表SCI论文2篇、学术会议论文2篇，授权国家发明专利2项，获得中华农业科技进步奖一等奖1项（10/10）；培养硕士研究生4名。