【作者】 郭宁；

【导师】 王晓武；

【作者基本信息】 中国农业科学院 ， 蔬菜学， 2014， 博士

【摘要】 白菜类作物（Brassica rapa L.）属于十字花科（Cruciferae）芸薹属（Brassica），包含许多重要的蔬菜和油用作物，在我国农业生产和居民膳食结构中占有举足轻重的地位。伴随着生活水平的逐步提高，人们对蔬菜的营养品质愈加重视。花青苷和黄酮醇苷作为重要的黄酮类植物次生代谢产物，广泛存在于水果和蔬菜中。研究表明这两类物质不仅对于植物的生长发育至关重要，而且具有抗氧化、抗肿瘤和改善心血管等有益于健康的功效。因此富含这两类活性物质的果蔬越来越受到消费者的青睐。研究植物体内花青苷和黄酮醇苷合成的遗传机制以及培育富含这两类物质的果蔬已成为研究热点。本研究通过液相色谱和质谱（LC-MS）技术对白菜中花青苷和黄酮醇苷类物质进行定性定量分析，揭示了这两类次生代谢产物在白菜中的自然变异特点；利用比较基因组学的方法对白菜中花青苷和黄酮醇苷合成基因进行鉴定，并在此基础上利用表达谱和定量PCR技术对合成基因在白菜中的表达特性进行研究；利用F2遗传分离群体对紫菜薹花青苷积累基因进行QTL定位；利用构建“超级池”高通量重测序分析的方法定位紫色小白菜叶片紫色基因；通过表达谱与代谢谱数据相关性和全基因组关联分析，鉴定与白菜黄酮醇苷各组分和总量相关的基因。主要研究结果如下：1.利用UFLC-UV-Q-Trip-MS和UPLC-Q-TOF-MS技术，分别对紫色白菜叶片中花青苷和95份不同类型白菜材料叶片中黄酮醇苷进行定性定量分析。在三种紫色白菜中共鉴定出23种花青苷，其中紫菜薹和紫色小白菜含有17种不同酰基化取代的矢车菊素-3-双/三葡萄糖苷-5-葡萄糖苷；紫芜菁检测出6种不同酰基化取代的天竺葵素-3-双葡萄糖苷-5-葡萄糖苷。95份不同类型白菜间黄酮醇苷类物质的组成没有差异，共鉴定出27种黄酮醇苷，包括15种山奈酚苷、10种槲皮素苷和2种异鼠李素苷，但不同材料间黄酮醇苷的总量和不同组分的含量均存在显著差异。2.在白菜基因组测序的基础上，利用比较基因组学根据拟南芥花青苷和黄酮醇苷生物合成基因在白菜中鉴定出73个同源基因。研究发现，花青苷和黄酮醇苷合成基因在白菜3倍化复制后发生了过保留；白菜花青苷和黄酮醇苷合成结构基因主要通过全基因组和串联重复复制方式扩增，更多的上游结构基因被保留下来；通过比较花青苷和黄酮醇苷合成正调节与负调节基因的扩增与保留情况，发现负调节基因在白菜中扩增与保留的比例更高。3.对95份材料花青苷和黄酮醇苷合成基因的表达谱以及代表性材料中主要结构基因qPCR分析发现，花青苷合成特异的后期结构基因主要在紫色材料中表达，绿色材料中花青苷合成负调控基因的表达水平更高。BrF3’H基因在紫菜薹和紫芜菁之间的表达差异解释了这两种紫色材料积累不同类型花青苷的原因。BrFLS1和BrDFR在紫色和绿色材料中的表达差异体现了不同材料中花青苷和黄酮醇苷的含量差异和积累特性。4.通过QTL分析在A09连锁群上发现控制紫菜薹花青苷积累的主效QTL位点，定位于1.9Mb的区域内。在定位区域中找到了拟南芥中AtEGL3的共线性基因BrEGL3.1和BrEGL3.2，重测序分析发现BrEGL3.1在亲本间存在较大变异，可能为控制紫菜薹花青苷积累的候选基因。5.利用超级池（SuperBSA）高通量重测序的方法成功将紫色小白菜叶片紫色性状基因定位到A03染色体末端1.5Mb的区域，开发优化了一种通过Pooling-sequence进行基因定位的方法。6.利用95份不同类型白菜材料的黄酮醇苷定性定量分析的代谢谱数据、基因数字化表达谱数据和全基因组重测序鉴定出的高密度SNP变异结果，通过表达谱与代谢谱相关性分析和全基因组关联分析鉴定了与白菜中黄酮醇苷类物质合成相关的基因，为进一步研究白菜黄酮醇苷合成关键基因和遗传机制奠定基础。更多还原

【Abstract】 Brassica rapa, comprising a variety of vegetables and oil crops, is one of the most consumedvegetable which plays an important role in people’s daily diets in China and throughout East Asia. Withthe improvement of living standard, people pay more attention to the nutritional qualities of vegetables.Anthocyanins and flavonol glycosides, which are two major groups of flavonoid compounds, performseveral key biological functions in plants as well as play beneficial health roles as potential protectivefactors against cancer and heart disease. Because of the nutritional value and beneficial health effects,vegetables which are rich in these two compounds are increasingly popular and received considerableresearch attention. So it is necessary to characterize the composition and reveal the genetic mechanismof anthocyanins and flavonol glycosides in B. rapa.In this study, the profiles and the contents of anthocyanins and flavonol glycosides were identifiedin the leaves of different accessions to reveal the natural variation of these two metabolites in B. rapa.Anthocyanin and flavonol glycoside biosynthetic genes have been identified by comparative genomicanalysis between B. rapa and Arabidopsis thaliana. QTL mapping analysis was conducted for the genescontrolling anthocyanin biosynthesis in Zicaitai (Brassica rapa L. ssp. chinensis var. purpurea). Amethod combining super-BSA and high throughput re-sequence was used to map the gene for purplepigmentation in leaves of purple pakchoi (Brassica rapa L. ssp. chinensis). The candidate genesinvolved in flavonol glycosides biosynthesis of B. rapa were identified by correlation analysis betweenexpression and metabolic profiling, as well as genome-wide association study (GWAS).The main results of this dissertation are as follows:1. To investigate the metabolic profiles of anthocyanins and flavonol glycosides in the leaves of B.rapa accessions, an analytical method combining UFLC-UV-Q-Trap-MS and UPLC-Q-TOF-MS wasperformed. In total,23anthocyanins were detected and identified in the leaves of Zicaitai, purple turnipand purple pakchoi.17different acylated cyanidin3-di/triglucoside-5-glucosides were detected inZicaitai and purple pakchoi, while six different acylated pelargonidin3-diglucoside-5-glucosides wereidentified in purple turnip.27flavonol glycosides containing15kaempferol glycosides,10quercetinglycosides and two isorhamnetin glycosides were identified in95accessions. The contents of flavonolglycosides were significant difference among the95accessions which accumulated the same27components.2. Comparative genomic analyses between A. thaliana and B. rapa on a genome-wide level wasconducted to elucidate the anthocyanin and flavonol glycosides biosynthetic pathway in B. rapa. In total,we identified73genes in B. rapa as orthologs of41biosynthetic genes in A. thaliana. In B. rapa, theanthocyanin and flavonol glycosides biosynthetic genes have expanded and most genes exist in morethan one copy. The anthocyanin biosynthetic structural genes have expanded through whole genome andtandem duplication in B. rapa. More structural genes located upstream of the anthocyanin biosyntheticpathway have been retained than downstream. More negative regulatory genes have been retained than positive ones in the biosynthetic pathway, which may help us to understand the morphologicalcharacteristics and genetic basis of these two metabolites in B. rapa.3. The expression profile and qPCR analysis showed that the expression levels of late biosyntheticgenes were higher in purple accessions than in green ones, and the negative regulatory genes ofanthocyanin biosynthesis were expressed higher in green accessions. The different expression patternsof BrF3’H between Zicaitai and purple turnip could explain that different kinds of anthocyanins wereidentified in these two purple cultivars. BrFLS1and BrDFR could reveal the metabolic characteristic ofanthocyanins and flavonol glycosides in puple or green accessions.4. To determine the genetic basis controlling anthocyanin accumulation in Zicaitai, we conductedQTL analysis. A major QTL was identified for total anthocyanin content on chromosome A09whichexplained56.7%of phenotypic variation. Two genes, BrEGL3.1and BrEGL3.2, which are syntenicorthologs of AtEGL3, are candidate genes for a key role in the control of anthocyanin accumulation inZicaitai.5. Two DNA pools which contained900green lines and200purple lines from a F2populationcrossed by purple pakchoi and green Caixin were resequenced to map the gene for the purplepigmentation of pakchoi leaves. The selected signal was found in a1.5Mb region at the end ofchromosome A03, and two anthocyanin biosynthetic genes were identified.6. The candidate genes involved in flavonol glycosides biosynthesis of B. rapa were identified bycorrelation analysis between expression and metabolic profiling, as well as genome-wide associationstudy (GWAS), which will promote the cloning and functional analysis of the genes controlling flavonolglycosides accumulation and the understanding of the biological process of flavonol glycosidesbiosynthesis in B. rapa, as well as offer genetic resources and theoretical foundation for thedevelopment of new varieties with enhanced health-promoting properties by molecular breeding.更多还原