【作者】 李林；

【导师】 刘同坤；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2020， 硕士

【摘要】 抽薹开花是白菜一个重要的农艺性状。先期抽薹不仅降低了白菜的产量,而且影响其商品性和食用价值。目前对拟南芥的开花途径已经有很深入的研究,并通过遗传和分子研究鉴定出许多关键的开花基因,这些基因被称为“开花响应元件”,分配到不同的调控途径。其中CO、FT、FLC基因就是主要的“开花响应元件”。光周期途径是影响开花的重要途径,CO是光周期途径关键基因,CDF1过表达拟南芥中CO的表达量下降,开花延迟。FKF1、GI分别与CDF1相互作用形成FKF1-GI-CDF1复合体调节CO的表达。在不结球白菜中,BrCDF1蛋白能够与BrFKF1蛋白相互作用,而且不结球白菜与拟南芥都是十字花科植物,因此推测FKF1-CDF1-CO-FT这条途径在不结球白菜中也是保守的。本研究对不结球白菜BrCDF1基因功能进行探索,并验证其与CO启动子之间的调控关系,以及其在光周期途径中所起的作用。春化途径是影响开花的另一个重要途径,是春化途径重要的基因家族,但是有关白菜在MAF/FLC基因家族的进化机制和功能的研究是有限的。为了进一步探索白菜在开花过程中MAF/FLC家族的进化历史和机制,本研究对MAF/FLC家族的进化模式,足迹和保守性进行分析。主要研究结果如下:1.以不结球白菜‘苏州青’为材料,采用酵母单杂交和β-半乳糖苷酶试验(ONPG)分析BrCDF1转录因子与CO启动子是否能够结合。结果显示,BrCDF1转录因子和CO启动子之间具有较高的结合活性;实时荧光定量RT-PCR结果表明,BrCDF1基因在过表达转基因植株中的相对表达量比野生型植株高;CO、FT基因在过表达转基因植株中的相对表达量比野生型低,同时BrCDF1过表达植株较野生型晚开花。这表明BrCDF1转录因子能与CO启动子结合,从而抑制CO、FT基因的表达,进而影响不结球白菜光周期开花途径。2.以白菜‘Chiifu-401-42’为材料,通过对拟南芥和白菜MAF/FLC基因的系统进化树和氨基酸基序进行分析,结果表明,BrMAF/FLCs与MAF/FLCs具有高度的序列同源性。利用BiFC和Y2H实验进一步研究了 BrMAF/FLCs与MAF/FLCs在进化过程中的功能保守性。BiFC实验表明,BrMAF2是一种核蛋白。根据相似的序列和细胞内表达模式,BrMAF2是拟南芥MAF2的直系同源基因,表明MAF/FLC家族的功能可能在白菜和拟南芥之间是保守的。Y2H实验表明BrSVP和BrMAF1,BrMAF2,BrMAF4存在相互作用,但是BrMAF3在白菜中不能与BrMAF1,BrMAF4和BrFLC相互作用,这与拟南芥中的研究结果不一致。以上结果表明,BrMAF和AtMAF之间具有序列高度相似性,但BrMAF基因家族仍然存在功能分化。为了进一步研究白菜MAF/FLC表达模式保守性,检测了BrMAF/FLC基因在白菜的时空表达。结果显示,BrMAF1,BrMAF2,BrMAF3和BrFLC3在茎和叶中高表达,而BrMAF4,BrFLC1和BrFLC2在根中优先表达。利用实时定量RT-PCR来研究春化期间BrMAF/FLC基因的表达,结果表明,在春化期间,BrMAF2和BrFLC2表达量减少。但BrMAF1,BrMAF3和BrMAF4的表达水平较春化前升高,随后下降。上述研究结果进一步证实了白菜BrMAF/FLC基因在基因复制过程中发生了功能分化。基因序列高同源性并不意味着功能高保守性。本研究的结果有助于更好地理解MAF/FLC在植物进化过程中的作用机制及功能。更多还原

【Abstract】 Bolting and flowering is an important agronomic trait in rapa.Early bolting not only reduces the yield of Brassica rapa,but also affects its commerciality and edible value.At present,the flowering pathway of Arabidopsis has been deeply studied,and many key flowering genes have been identified through genetic and molecular studies.These genes are called "flowering response elements" and are assigned to different regulatory pathways.Among them,CO,FT and FLC genes are the main "flowering response elements".The photoperiod pathway is an important pathway that affects flowering.CO is a key gene of the photoperiod pathway.Overexpression of CDF1 in Arabidopsis thaliana decreases the expression level of CO and delays flowering.FKF1 and GI interact with CDF1 to form FKF1-GI-CDF1 complex to regulate CO expression.In non-heading Chinese cabbage,BrCDF1 protein can interact with BrFKF1 protein,and non-heading Chinese cabbage and Arabidopsis are both cruciferous plants,so it is speculated that FKF1-CDF1-CO-FT is a pathway in non-heading Chinese cabbage is also conservative.This study explored the function of BrCDF1 gene in non-heading Chinese cabbage and verified its regulatory relationship with the CO promoter,as well as its role in the photoperiod pathway.Vernalization pathway is another important pathway that affects flowering.MAF/FLC is an important gene family for vernalization pathway,but information about the evolution and function of Brassica rapa in the MAF/FLC gene family is limited.In order to further explore the evolutionary history and mechanism of the MAF/FLC family during flowering of Brassica rapa,this study analyzes the evolutionary pattern,footprint and conservatism of the MAF/FLC family.The main findings are as follows:1.Using non-heading Chinese cabbage ’Suzhouqing’ as the material,using yeast single hybridization and β-galactosidase test(ONPG)to analyze whether BrCDF1 transcription factor can bind to the CO promoter.The results show that BrCDF1 transcription factor and CO start It has high binding activity;real-time fluorescence quantitative RT-PCR results show that the relative expression of BrCDF1 gene in overexpressing transgenic plants is higher than that of wild type plants;the relative expression of CO and FT genes in overexpressing transgenic plants is higher than that of wild type At the same time,BrCDF1 overexpressing plants bloomed later than wild type.This indicates that BrCDF1 transcription factor can bind to the CO promoter,thereby inhibiting the expression of CO and FT genes,which in turn affects the photoperiod flowering pathway of non-heading Chinese cabbage.2.Using Brassica rapa ’Chiifu-401-42’ as material,the phylogenetic tree and amino acid motifs of MAF/FLC genes in Arabidopsis and Brassica rapa were analyzed.The results showed that BrMAF/FLCs and MAF/FLCs have a high sequence Homology.BiFC and Y2H experiments were used to further study the functional conservation of BrMAF/FLCs and MAF/FLCs during evolution.BiFC experiments show that BrMAF2 is a nuclear protein.Based on similar sequences and intracellular expression patterns,BrMAF2 is an ortholog of Arabidopsis MAF2,suggesting that the function of the MAF/FLC family may be conserved between Brassica rapa and Arabidopsis.The Y2H experiment showed that BrSVP interacts with BrMAF1,BrMAF2,and BrMAF4,but BrMAF3 cannot interact with BrMAF1,BrMAF4,and BrFLC in Brassica rapa,which is inconsistent with the results in Arabidopsis.The above results indicate that and have a high degree of sequence similarity,but the BrMAF gene family still has functional differentiation.To further study the conservativeness of the expression pattern of Brassica rapa MAF/FLC,the temporal and spatial expression of BrMAF/FLC gene in Brassica rapa was detected.The results showed that BrMAF1,BrMAF2,BrMAF3 and BrFLC3 were highly expressed in stems and leaves,while BrMAF4,BrFLC1 and BrFLC2 were preferentially expressed in roots.Real-time quantitative RT-PCR was used to study the expression of BrMAF/FLC gene during vernalization.The results showed that during vernalization,BrMAF2 and BrFLC2 decreased.However,the levels of BrMAF1,BrMAF3 and BrMAF4 increased from before vernalization and then decreased.The above research results further confirmed that the Brassica rapa BrMAF/FLC gene has undergone functional differentiation during gene replication.High homology of gene sequences does not mean high conservation of functions.The results of this study help to better understand the mechanism and function of MAF/FLC in plant evolution.更多还原