矮牵牛FLC亚家族MADS-box基因功能演化机制解析

The FLOWERING LOCUS C (FLC) subfamily of MADS-box genes play key roles in flowering regulation of higher plants, and may have conservative functions in vernalization-mediated flowering pathway. However, we almost have no any knowledge on the functions of.FLC homologs in tropical and subtropical plants that do not need vernalization to promote flowering. In this research programme, a genome-wide functional and evolutionary analysis of the FLC-like genes in petunia, a subtropical species with high ornamental and landscaping values, will be conducted. Firstly, the FLC-like genes are identified by BLAST searching the petunia genome, transcriptome and EST database with the sequences of FLC-clade genes in Arabidopsis and Solanaceae species such as tomato and tobacco. These genes are then cloned and sequenced, followed by the construction of overexpression vectors, gene silencing (i.e. VIGS or amiRNA) vectors and/or gene knockout (i.e. CRISPR/Cas9) vectors, which are used for Agrobacterium-mediated transformation of Arabidopsis and/or petunia. At the same time, the W138 dTph1 transposon insertion library are screened for possible mutants of FLC-like genes. The functions of petunia FLC homologs are.characterized based on the phenotype changes of the transgenic plants and/or mutants. On the other hand, the spatio-temporal expression patterns of the FLC-like genes and the effects of cold temperature and day length of illumination on expression of the genes will be investigated by real-time PCR and/or in-situ hybridization. The transcription regulators that interact with the FLC-like proteins with significant functions and that can bind to the cis-element of the FLC-like genes will also be studied by yeast two-hybrid and one-hybrid screening, respectively. Moreover, digital gene expression profiling (DGE) analysis between the wild-type petunia and the transgenic plants or mutants is.used to search for downstream genes regulated by the FLC-like genes. The functions and molecular regulatory mechanism of petunia FLC-like genes will be revealed by these analyses, which could provide important experimental evidences for the existing meaning of the FLC-like genes in the subtropical species that do not require vernalizaiton to promote flowering. The results of this study will offer further understanding of the evolution of FLC-like genes in higher plants, and also could promote petunia breeding for flowering in cold temperature.

FLC亚家族MADS-box基因对植物开花时间起着关键调控作用，而且在春化需求型植物中具有功能保守性，然而在无春化需求的热带、亚热带植物中，其功能基本一无所知。本项目以极具观赏和园林应用价值的亚热带植物矮牵牛为材料，对其FLC-like基因的功能演化进行研究：一方面通过搜索基因组与转录组数据库确定矮牵牛FLC-like基因成员及编码序列，构建相关表达载体，转化拟南芥和矮牵牛，创造目标基因过量表达和功能缺失植株，明确基因功能；另一方面，对FLC-like基因的时空表达模式进行分析，研究不同低温和光周期条件对其表达的调控作用，同时对功能重要的FLC-like基因的互作因子及上下游调控基因进行筛选和分析，阐释其作用机制。研究结果可望揭示无春化需求植物FLC-like基因存在的意义，增加对FLC类MADS-box基因功能演化的认识，同时为矮牵牛性状改良提供依据，具有十分重要的科学意义和实用价值。

本项目以矮牵牛基因组和转录组数据为基础，全面鉴定了MADS-box基因家族成员，其中包括6个FLC-like基因（PMADS6、PMADS15、PMADS15-like、PMADS17、PMADS17-like和PhAGL27）；以矮牵牛W115株系为材料，克隆了6个FLC-like基因的全长gDNA和CDS序列，明确了其基因结构和序列特征，分析了它们在矮牵牛根、茎、叶、芽、花、果实、种子等不同组织器官以及顶芽和叶片不同发育阶段的时空表达模式，并且研究了不同低温条件对其表达的调控作用；构建了6个FLC-like基因的相关表达载体，包括超表载体、RNAi载体、amiRNAi载体等，转化拟南芥和矮牵牛，创造了目标基因过量表达以及部分基因功能缺失的转基因植株，明确了基因功能；对FLC-like基因的启动子序列进行了分析，发现了响应低温等顺式作用元件，同时对FLC-like基因的互作因子及PMADS17-like基因的下游调控通路进行了分析，初步阐释了其作用机制。此外，还从基因组水平全面鉴定了矮牵牛ALOG和TCP转录因子基因家族成员，并从W115 株系中克隆了11个ALOG家族基因（PhLSHs）的编码序列，分析了ALOG和TCP家族的基因结构、保守结构域及motifs；通过qPCR技术，研究了PhLSHs和PhTCPs基因在矮牵牛不同组织器官中的表达模式，以及PhTCPs基因在大、小花矮牵牛中的表达差异；通过构建超表和amiRNAi载体，转化拟南芥和矮牵牛，鉴定了PhLSH3a/3b/4、PhLSH7a/7b、PhLSH10a/10b/10c以及部分PhTCPs基因的功能。.项目研究结果在一定程度上揭示了矮牵牛FLC-like基因存在的生物学意义，增加了我们对高等植物FLC类MADS-box基因功能演化的认识，同时鉴定了矮牵牛ALOG和TCP基因家族成员，明确了其表达模式及部分基因功能，为矮牵牛的相关性状改良提供了科学依据和基因资源。