基于CRISPR/Cas9基因编辑技术的栾树叶色变异分子机制研究

This project aimed at the scientific problems of the molecular mechanism of leaf color variation in Koelreuteria paniculata Laxm. In this study, to find out the main accumulated pigment component in leaves of Jinye Luan, we choose Beijing Luan (green leaf) and Jinye Luan (golden leaf) which is a mutant of K. paniculata, as research materials, will employ high performance liquid chromatography-mass spectrometry (HPLC-MS) method to analyze flavones/anthocyanins components in the leaves of both materials. On this basis, RNA-seq technology will also employed to provide a comprehensive overview of transcriptional changes between Beijing Luan and Jinye Luan, to explore the participation of key regulatory genes of colored pigment formation. Candidate genes will be screened by conjoint analysis of.transcriptome data and flavonoids/ anthocyanins data. Furthermore, functional analysis of candidate genes will be performed by using gene editing technology--CRISPR/Cas9. sgRNAs of target candidate genes will be designed and constructed into CRISPR/Cas9-sgRNA expression vector, and will be transferred into K. paniculata by means of Embryogenic Callus Regeneration System which had been established in our laboratory. Subsequently, a series of molecular identification and physiological and chemical analysis, together phonotypical variations will be.performed. This study not only has very important significance to elucidate the molecular and physiological mechanism for leaf color variation in K. paniculata, but also has a broad application prospect for new varieties with high ornamental value and high medicinal value by gene editing breeding approach, while for other similar mutants of tree species, such as Sophora japonica(Jinye Huai) and Ulmus pumila (Jinye Yu) are of high reference value.

本项目针对栾树叶色变异机制这一科学问题，以北京栾和锦叶栾为研究材料，采用高效液相色谱-质谱联用(HPLC-MS)的方法，分析北京栾、锦叶栾叶片代谢物成分差异，发现锦叶栾叶片色素主要成分；在此基础上，借助RNA-seq技术分析北京栾、锦叶栾的基因差异表达规律，发掘参与彩叶色素形成的关键调控基因；采用基因编辑技术--CRISPR/Cas9，参照RNA-seq数据分析结果，以黄酮类/花青素合成途径关键调控基因为靶基因设计sgRNAs，构建成CRISPR/Cas9-sgRNAs表达载体，利用已建立的栾树胚性愈伤组织转化体系进行遗传转化，对转化植株进行系列分子检测、生理生化分析，开展栾树叶色变异形成的分子生理机制研究。该研究不仅对于阐明栾树叶色变异的分子机制具有十分重要的意义，而且对于通过基因编辑技术选育观赏价值高、药用价值高的栾树新品种具有广阔的应用前景，对于金叶槐、金叶榆的相关研究具有借鉴价值。

本研究以栾树（北京栾）和锦叶栾为试材，采用基因编辑技术，探究栾树叶色变异的分子机制。锦叶栾是一种用于园林绿化的突变品种，具有金色的叶片表型。我们对栾树野生型和突变体叶片进行了生理、解剖、microRNA测序、转录组学和代谢组学分析，并使用基因编辑技术验证了候选基因的功能。与野生型相比，突变体的叶绿素a (Chl a)和叶绿素b (Chl b)含量分别降低76.05%和44.32%，类胡萝卜素含量显著增加。叶片超微结构显示突变体叶绿体形态异常，基粒片层数减少。在联合组学测定分析中，共鉴定出参与色素代谢、叶绿体发育和光合作用的59个差异表达基因(DEGs) 和40个转录因子(TF)。我们发现参与叶绿体发育和叶绿素合成的叶色变异候选基因GLK和petC表达量显著下调，其中，在毛白杨中使用基因编辑技术敲除候选基因GLK，获得了黄绿色的转基因株系。此外，PSY和PDS基因上调，NCED基因下调促进类胡萝卜素的积累。多种查尔酮和黄酮醇基因在突变体中表达上调。结果表明，突变体中类胡萝卜素与叶绿素的比值提高了75%以上，查尔酮和黄酮醇的积累共同导致了突变体金黄色叶片的表型。为深入探究栾树相关分子机制，我们基于体胚发生方式为栾树建立快速有效的再生途径，且初步建立了其遗传转化体系，不仅为栾树良种繁育和保存提供了条件，且为栾树后续的遗传研宄和分子育种提供了基础。