甘蓝黄色花瓣基因cpc-1的图位克隆及功能解析

In view of the important role of flower color in attracting insect pollination and so on, focusing on the scientific problem of typical flower color variation between the two subspecies of cabbage and Chinese kale, the materials of this project included inbred lines of yellow-petalled cabbage YL-1 and white-petalled Chinese kale 11-192. The components and contents of carotenoids in petals were determined by ultra performance liquid chromatography and mass spectrometry (UPLC-PDA-Q-TOF/MS). Based on the previous inheritance analysis and fine-mapping result, The carotenoids content, re-sequencing and RNA expression analysis were combined to predict the candidate genes, which were then verificated by function complementation and CRISPR techniques. The nucleotide variation and evolution of the yellow-flowered genes cpc-1 between cabbage and Chinese kale were analyzed. The promotor sequence was cloned and its spatiotemporal expression was identified. Yeast two-hybrid technique was used to explore the proteins interactive with CPC-1. This project belongs to an integrated cross-disciplinary research to connect physiology, heredity and evolution. Cloning of the yellow-petalled genes in cabbage would provide an important reference for elucidating the genetic basis and mechanism of yellow flower formation, and guide the breeding of different flower color inbred lines and infer their evolutionary relationship.

针对花色在吸引昆虫授粉等方面的重要作用，围绕结球甘蓝与芥蓝两个变种间存在的典型花色变异这一科学问题，本项目以黄花结球甘蓝自交系YL-1、白花芥蓝自交系11-192为试材，利用超高效液相色谱和质谱法（UPLC-PDA-Q-TOF/MS）对花瓣的类胡萝卜素成分和含量进行测定；在已有遗传规律分析及精细定位的基础上，结合类胡萝卜素含量测定、重测序及基因表达结果预测候选基因；采用功能互补、CRISPR技术验证候选基因的功能，揭示黄花基因cpc-1在变种间的变异并进行变种进化分析；克隆启动子序列并进行时空表达特性分析，利用酵母双杂交技术挖掘与cpc-1基因互作的蛋白。本研究属于连接生理、遗传、进化方面的一个综合交叉学科，甘蓝黄花基因的图位克隆为解析甘蓝黄花形成的遗传基础及作用机理提供重要依据，对指导不同花色材料育种、推测它们的进化关系有重要意义。

针对花色在吸引昆虫授粉等方面的重要作用，围绕结球甘蓝与芥蓝两个变种间存在的典型花色变异这一科学问题。本项目利用黄花结球甘蓝自交系YL-1和白花芥蓝自交系11-192，对其花瓣的类胡萝卜素含量进行测定，发现在YL-1中显著升高；通过连锁分析，将花色基因cpc-1精细定位于207 Kb区间，并将Bol029878定为候选基因，命名为BoCCD4；通过功能互补，发现11-192的BoCCD4基因能够挽救YL-1黄花表型；通过序列分析，发现YL-1在BoCCD4编码区存在3个InDel和34个SNP；通过表达分析，发现BoCCD4仅在白花组织中表达；通过转录组分析和酵母双杂试验，挖掘到BoCCD4的3个潜在互作蛋白和2个潜在调控因子。本研究为解析甘蓝黄花形成的遗传基础及作用机理提供重要依据，对指导不同花色材料育种、推测它们的进化关系有重要意义。