灯盏花酚类物质合成遗传网络及其与营养物质的互作机制研究

Based the nutrition intervention model which has been constructed in our present NSFC project, the transcriptome sequncing will carry out via next generation sequncing platform using HisSeq2000 in Erigeron breviscapus (Vant.) Hand-Mazz. To acquire more information about E.breviscapus transcriptome,total RNA from differential treatments,differential vevelopemnt stages and various tissues will be used to build the transcriptome cDNA sequencing bank(basical bank).After sequnencing the basical bank and filtered the no-usage sequences, sequence assembly, sequence alignment, gene annotation and metabolic pathway annotationget will be carried out in the project, the globle information of E.breviscapus transcriptome will be analysis,especially the genetic networks associated with secondary metabolic pathways which involving effective components (phenols and flavonoids) will be more detail.And the phenolic compounds metabolic pathways network will be construted. Secondly, in terms of nutrition intervention mode, the combination of DGE from differential treatemts, differential developmental stages and various tissues with phenols metabolic phenotype identified by HPLC and HPLC-PDA－MS, and expression profiling of key genes and concerning controlling elements by real time quantitative PCR testing, the project will study the interacting mechanism between phenolics metabolism associated genes,determine the key functional gene and concerning controlling elements in phenolic metabolism, and environment,and the phenolic accumulation profiling and gene expression profiling during phenolic compounds biosythesis will be get after finished the project. After finish the project, not only get a number of functional genes associated with the effective component synthesis, the parsing phenolic metabolic network in the project, to lay the foundation for the next multi-gene metabolic genetic engineering and molecular assisted breeding in E.breviscapus;but also provide basic data for efficient cultivation in E.breviscapus.

利用申请人所执行的在研基金项目中已构建的营养干预模式，利用本单位新购进的HisSeq2000对灯盏花表达组进行序列测定，通过序列拼接、序列比对、基因注释及代谢途径注释，宏观上解析灯盏花的表达组信息，确定与灯盏花功能组分合成代谢的相关遗传信息，构建其功能组分合成相关的代谢网络。其次，就营养干预模式下，灯盏花不同发育阶段的数字表达谱及酚类物质的代谢表型变化特征的测定，同时结合不同发育阶段相关基因的相关基因掌握灯盏花有效组分的积累机制及其遗传控制机制，探求灯盏花酚类代谢与环境的遗传互作机制。本课题的开展，不仅能获得一批与灯盏花有效组分合成相关的功能基因序列，解析灯盏花酚类代谢网络，同时通过营养干预与代谢关联基因表达数据的整合，探索灯盏花有效组分的积累模式，并用于高效栽培模式建立的指导。此外，实验还能获得灯盏花的SSR标记，为灯盏花的分子鉴定、分子育种及分子辅助育种，下一步的遗传作图奠定基础。

项目执行过程中，根据申请书的计划内容，开展两批次、20个样本的转录组测定及其基本分析，完成了54个样品的灯盏花有效组分测定，通过转录组与灯盏花代谢产物的测定，已初步锁定灯盏花中参与酚类物质合成代谢的相关基因43个，参与调控的基因23个，确定了灯盏花有效组分合成的基本途径，氮磷钾对灯盏花有效组分积累的调控并非与直接调控相关基因的表达，而是通过磷酸肌醇介导调控。除氮、磷、钾组合处理的转录组测定及基本分析，项目克隆了5个可能参与有效组分的基因克隆（myb、chi、fns、、abc）、进行了两个基因的转基因实验及其评价（chi、myb）、构建了一个基因的原核表达载体构建（ubgat），获得了分属在5361个unigene中的SSR标记8360，分属30000多个unigene如中的SNP位点65355个，获得可能参与到551个基因的表达的Micro-RNA396个，培养了近80个个体的灯盏花单倍体群体。此外，还获得396个Micro-RNA，这些Micro-RNA可能参与到551个基因的表达。这些基础数据的获取将为灯盏花的分子育种、代谢基因工程奠定良好的基础。