【作者】 王薇；

【导师】 王玉刚；

【作者基本信息】 沈阳农业大学 ， 蔬菜学， 2023， 硕士

【摘要】 白菜类作物（Brassica rapa L）广泛分布于中东亚地区和欧洲,主要包括大白菜、小白菜等多种蔬菜作物和部分油料作物。抽薹是白菜类作物重要农艺性状,抽薹早晚直接影响此类作物最终收获产品器官（叶球或种子）的产量和质量,以及种植季节和区域。开花是白菜类作物从营养生长过渡到生殖生长的关键阶段,研究此类作物的开花分子机制以及开花调控网络具有重要的理论价值和实践意义。AP1（APETATAL1）对开花时间呈现正向调控,同时促进花的形态建成,是开花调控网络的关键整合因子。但在白菜中该基因的功能和分子机制尚未明确。所以本试验对白菜BrAP1基因功能进行验证,并对其调控开花时间的分子机制进行初步研究。主要研究结果如下:1.白菜BrAP1基因的挖掘前期对染色体片段代换系CSSL16与轮回亲本Rc Br进行表型鉴定,发现在开花时间上有显著差异,利用RNA-seq技术进行转录组分析并筛选开花相关基因。结果表明白菜AP1基因（包含三个同源基因:Bra A02g018970.3C、Bra A07g030470.3C和Bra A07g034100.3C）为差异表达基因,实时荧光定量PCR验证了此结果,在接续的试验中对差异最显著的AP1基因（Bra A07g034100.3C）进行特异敲除,从而对白菜BrAP1基因进行功能验证。2.白菜BrAP1基因的敲除验证及相关基因表达分析对T₁代基因编辑植株与野生型Rc Br,进行抽薹开花表型以及靶点序列鉴定。结果表明,T₁代基因编辑植株表现为明显的晚抽薹表型,并且在靶点处发生了不同程度的碱基替换。对BrAP1相关基因在T₁代基因编辑植株与野生型中的表达量进行分析,结果表明白菜Br FD、Br FT和BrAP1基因,在T₁代编辑植株中表达水平与野生型相比显著下调,而开花抑制基因Br CEN则显著上调。3.BrAP1亚细胞定位与互作蛋白验证为探究BrAP1基因在细胞中的表达位置,利用绿色荧光蛋白对BrAP1进行亚细胞定位,结果可知,白菜BrAP1基因主要在烟草细胞核中表达且表达效率极强,说明BrAP1基因有转录因子的一般特性。利用酵母双杂交,对BrAP1进行互作蛋白一对一验证,发现有两个蛋白与BrAP1互作,分别是Br CEN和Br FT蛋白。Br FT和Br FD也相互作用。研究结果有助于丰富白菜类作物抽薹开花的分子调控网络,同时为培育不同适应性品种提供理论依据。更多还原

【Abstract】 Brassica rapa has long been cultivated in Central of East Asia and Europe,mainly as a vegetable foodstuff,such as Chinese cabbage and Pak-choi,and to a less extent to produce fodder and oilseed.Among the agronomic traits in B.rapa,flowering time（FT）is important because it affects the yield of seeds and the harvested crop’s commercial quality.FT is the critical period for the transition from vegetative growth to reproductive growth,therefore,it is important to explore the molecular mechanism and the regulatory network of flowering in B.rapa.AP1（APETATAL1）as a key integration factor in the flowering regulatory network,which positively regulated flowering time and promoted flower morphogenesis.Our study verified the function of BrAP1 and preliminary study on mechanism to regulate flowering in B.rapa.The main research results are as follows:1.Identification of the BrAP1 in B.rapaThe two parents,chromosome segment substitution line CSSL16 and the recurrent parent Rc Br,showed significant differences in flowering time under multiple environments.Transcriptome analysis was utilized to identified the flowering time related genes.The results showed that BrAP1（three homologous genes,Bra A02g018970.3C,Bra A07g030470.3C and Bra A07g034100.3C）was differentially expressed in B.rapa.Among the three homologous genes,Bra A07g034100.3C showed the most significant expression level.Therefore,Bra A07g034100.3C as the research object in consecutive experiments and verified the function of BrAP1 in B.rapa.2.Knockout verification of BrAP1 and related gene expression analysisPhenotypic evaluation and target sequence of T₁ transgenic plants,compared with wild type Rc Br.The T₁ transgenic plants showed significantly late flowering and had bases substitutions at the target locus.Quantitative real-time PCR was used to detected the BrAP1related genes expression level,the expression levels of Br FD,Br FT and BrAP1 were significantly decreased in ap1 mutant.But the expression level of Br CEN was significantly increased.3.Subcellular localization and interaction proteins verification of BrAP1In order to explore the expression position of BrAP1 in cells,constructed a vector containing GFP was used for subcellular localization of BrAP1.The results demonstrated that BrAP1 had a high expression efficiency and mainly expressed in the nucleus of tobacco,this result indicate that BrAP1 has the general characteristics of transcription factor.Y2H was used to identify the potential interact proteins of BrAP1.Br CEN and Br FT were found to interacted with BrAP1,meanwhile Br FT and Br FD also interaction.The results contribute to enrich the molecular regulatory network of flowering in B.rapa,and provide a theoretical basis for cultivating different varieties and enhancing their adaptability.更多还原