【作者】 杨峰；

【导师】 张大伟；

【作者基本信息】 四川大学 ， 生物学-植物学， 2022， 博士

【摘要】 抽薹和开花是植物为繁衍后代,在长期的进化过程中形成的特性,标志着植物从营养生长向生殖生长转变,是植物发育过程的重要阶段。抽薹和开花性状是典型的数量性状,常受多基因控制,环境对其影响较大,需要将传统遗传学的表型数据同分子生物学的遗传图谱相结合,通过QTL（Quantitative trait loci）定位直接对基因型进行选择和研究。萝卜（Raphanus sativus L.）是我国重要的十字花科蔬菜作物之一,在生产上容易发生“先期抽薹”现象,造成经济损失。萝卜种质资源的遗传变异丰富,抽薹和开花性状变化明显,且同时受到温度和光周期的双重调控影响。因此,萝卜成为了开展植物抽薹和开花基础研究的优异材料。本文以两个抽薹时间相差近120天的萝卜材料为亲本构建的近等基因系作为遗传群体材料,基于高通量测序、生物信息学和分子生物学分析手段,构建了萝卜高质量基因组,结合全基因组和转录组分析,开展了QTL精细定位,深入挖掘萝卜抽薹和开花相关基因功能,进一步解析调控机制。主要研究结果如下:1、利用PacBio CCS测序,构建了晚抽薹萝卜亲本材料C60213的高质量基因组。通过初步组装和去冗余序列,获得序列总长为506.90 Mb,contig N50为5.46 Mb,scaffold N50为5.50 Mb,完成了流式细胞预估基因组98.61%的组装;基于对217个近等基因系F₂子代和2个亲本材料的GBS技术,构建了包含8,070个SNP标记、总长度779.99 cM、平均遗传距离0.10cM的高密度遗传连锁图谱,利用图谱将99.68%的组装结果挂载到了染色体水平。通过基因组注释,发现基因组重复序列和非编码RNA分别占基因组的60.93%、2.24%,注释到52842个非冗余蛋白编码基因,其中共有50797个基因可以预测其功能,占基因总数的96.13%。基因组内部以及与其他二代和三代测序萝卜基因组间比较,发现组装结果有良好的基因组共线性,验证了萝卜在进化过程中发生的全基因组三倍化事件。2、结合高密度萝卜遗传连锁图谱,以及近等基因系F_2:3家系及其亲本在多环境条件下的5组抽薹和开花表型数据,完成QTL定位,获得与抽薹和开花性状密切相关的QTL区间14个。以高质量萝卜基因组为参考,在定位区间鉴定到基因152个,分布在第2、6和7号染色体上。通过比较基因组、转录组数据和QTL定位联合分析,筛选获得3个在不同亲本材料各发育时期显著上调或下调的候选基因,均位于第2号染色体上,命名为RsHAT1、Rs SOK2和Rs MIPS3,并通过荧光定量PCR验证相对基因表达量与预测一致。通过拟南芥进行异源初步验证,发现RsHAT1的显著早花表型并确定其为目的基因用于后续研究。3、拟南芥中存在与萝卜RsHAT1高度同源的基因AT4G17460,该基因编码一个HD-ZIPII转录因子家族成员HAT1。目前,已有报道发现HAT1广泛参与调控拟南芥生长、干旱响应以及花青素合成过程,但关于HAT1调控开花的功能还未知。因此,本论文基于萝卜中挖掘的RsHAT1,进一步在模式植物拟南芥中系统的研究了HAT1调控开花的分子机制。我们发现拟南芥中HAT1可与FLC的激活因子FRI相互作用,并抑制FRI复合体（FRI-FRL-FES1-FLX）的形成,导致FLC不能激活表达,从而促进开花。HAT1蛋白在低温春化条件下处于稳定状态,并在春化过程中逐渐积累,通过与FRI的C末端结合从而抑制FLX和FES1与FRI的互作,使得FRI复合物不能正常形成,此时FLC不能被激活而处于沉默状态,从而介导春化途径促进的开花过程;当植物生长在正常的温度（22℃）,HAT1蛋白处于不稳定状态,处于较低的水平,此时FRI复合物可以正常行使功能,FLC被持续激活和表达,植物不能开花,主要进行营养生长。因此,该模型提出了HAT1介导春化途径诱导开花的新机制,为我们更深入理解植物开花机制提供重要的证据,同时也为更好利用春化途径调节萝卜抽薹和开花时间提供理论指导。综上所述,本文解析获得了晚抽薹萝卜亲本材料C60213高质量基因组,进一步结合QTL精细定位、关键基因筛选、基因功能验证和分子机制解析等方式,获得了一个新的正向调控开花表型的转录因子HAT1,并初步解析了HAT1调控开花的分子机制。这将增进我们对萝卜生长发育重要过程的认识,揭示了萝卜精细调控抽薹和开花的一个新的调节机制,为耐抽薹萝卜育种提供了作用靶点,为下一步开展分子设计育种奠定理论基础。更多还原

【Abstract】 Bolting and flowering were the characteristics of plants formed in the long-term evolution process for the reproduction of offspring,marking the transition of plants from vegetative growth to reproductive growth,and were important stages of plant development.Bolting and flowering traits were typical quantitative traits,which were often controlled by polygenes and greatly influenced by the environment.It was necessary to combine the phenotypic data of traditional genetics with the genetic map of molecular biology,and select and study the genotypes directly by QTL(Quantitative trait loci)mapping.Radish(Raphanus sativus L.)was one of the important cruciferous vegetable crops in China,which was prone to"early bolting"in production,resulting in economic losses.The genetic variation of radish germplasm resources was rich,and the bolting and flowering traits change significantly,which were simultaneously affected by the dual regulation of temperature and photoperiod.Therefore,radish has become an excellent material for basic research on plant bolting and flowering.In this paper,the near-isogenic lines constructed from two radish materials with bolting time difference of nearly 120 days were used as the genetic population materials.Based on high-throughput sequencing,bioinformatics and molecular biology analysis methods,a high-quality radish genome was constructed.Combined with whole genome and transcriptome analysis,QTL fine mapping was carried out to dig deeply the functions of radish bolting and flowering related genes,and further analyze the regulatory mechanism.The main findings were as follows:1.Using PacBio CCS sequencing,a high-quality genome of the late bolting radish parent material C60213 was constructed.Through preliminary assembly and de-redundant sequences,the total length of the obtained sequence was 506.90 Mb,the contig N50 was 5.46 Mb,and the scaffold N50 was 5.50 Mb,completing the assembly of 98.61%of the estimated genome by flow cytometry;based on 217 near-isogenic lines F2 progeny and 2 parent materials of GBS technology,a high-density genetic linkage map containing 8,070 SNP markers with a total length of 779.99 cM and an average genetic distance of 0.10 cM was constructed,and99.68%of the assembly results were mounted to the chromosome level using the map.Through genome annotation,it was found that genomic repetitive sequences and non-coding RNAs accounted for 60.93%and 2.24%of the genome respectively,and 52,842 non-redundant protein-coding genes were annotated,of which 50,797 genes could predict their functions,accounting for 96.13%of the total genes.Good genome collinearity was found in the assembly results within the genome and compared with other 2nd and 3rd-generation sequencing radish genomes.And the genome-wide triploidization event of radish during evolution was verified by genome collinearity comparison.2.Combined with the high-density radish genetic linkage map,and the 5 groups of bolting and flowering phenotype data of the near-isogenic F2:3 family and its parents under multi-environmental conditions,we completed QTL mapping and obtained 14 QTL intervals closely related to bolting and flowering traits.Taking the high-quality radish genome as a reference,152 genes were identified in the mapping interval,which were distributed on 2nd,6th and 7th chromosome.By comparing genome,transcriptome data and QTL mapping combined analysis,3 candidate genes which were significantly up-regulated or down-regulated at each developmental stage of different parental materials were screened and obtained,all of which were located on 2nd chromosome,named RsHAT1,Rs SOK2 and Rs MIPS3.The relative gene expression was verified by real-time fluorescence quantitative PCR to be consistent with the prediction.Through the preliminary heterologous verification of Arabidopsis,a significant early flowering phenotype of RsHAT1 was found and identified as the target gene for subsequent research.3.The gene AT4G17460,which was highly homologous to radish RsHAT1 in Arabidopsis,encodes a member of the HD-ZIPII transcription factor family HAT1.At present,it has been reported that HAT1 was widely involved in the regulation of Arabidopsis growth,drought response and anthocyanin synthesis,but the function of HAT1 in regulating flowering was still unknown.Therefore,based on the RsHAT1 excavated in radish,the molecular mechanism of HAT1 regulation of flowering in the model plant Arabidopsis was further studied systematically in this paper.We found that HAT1 in Arabidopsis could interact with the FLC activator FRI,and inhibit the formation of the FRI complex(FRI-FRL-FES1-FLX),resulting in the inactivation of FLC expression,thereby promoting flowering.HAT1 protein was stable under the condition of low temperature vernalization,and accumulated gradually during vernalization.By binding to the C-terminus of FRI,it inhibited the interaction between FLX and FES1 and FRI,so that the FRI complex could not be formed normally.At this time,FLC could not be activated and was in a silent state,which mediated the flowering process promoted by the vernalization pathway;when plants were grown at normal temperature(22°C),the HAT1 protein was in an unstable state and at a lower level,and the FRI complex could function normally,FLC was continuously activated and expressed,and plants could not flower and mainly undergo vegetative growth.Therefore,this model proposes a new mechanism by HAT1 mediates the vernalization pathway to induce flowering,which provides important evidence for a better understanding of the plant flowering mechanism,and also provides theoretical guidance for better use of vernalization pathway to regulate bolting and flowering time of radish.In summary,this paper analyzed and obtained the high-quality genome of the late bolting radish parent material C60213,and further combined QTL fine mapping,key gene screening,gene function verification and molecular mechanism analysis to obtain a new transcription factor HAT1,which positively regulates flowering phenotype,and preliminarily analyzed the molecular mechanism of HAT1 in regulation of flowering.This will enhance our understanding of the important process of radish growth and development,reveal a new regulatory mechanism of radish’s fine regulation of bolting and flowering,provide a target for bolting-resistant radish breeding,and lay a theoretical foundation for the next step in molecular design breeding.更多还原