基于转录组测序筛选大麦籽粒黄酮醇及其衍生物代谢酶基因

The flavonols and their derivatives of barley grain have some medicinal and health value, their accumulation in barley grain is related to the flavonol synthesis gene, flavonol methylation gene, flavonol glycosylation gene and the other metabolic genes, these metabolic genes show the genetic diversity in different plant. In order to screen the flavonols and their derivatives metabolic genes of local barley grain in Chinese, the 10 barley varieties grains in four development stages will be used to built three transcriptome library in the project, which contains some special flavonols respectively. The transcriptome sequencing, sequence alignment, Unigene annotation and the others techniques will be used to select the core sequence of flavonols and their derivatives metabolic candidate genes and their gene markers. By these gene markers, the expression of metabolic candidate genes in different barley varieties grain will be detected, and the relation of the expression of these candidate genes to the content of different flavonols or their derivatives will be detected respectively, and then the core sequence of flavonols and their derivatives metabolic candidate genes will be verified again. The 3′ and 5′RACE technique will be employed to amplify the whole sequence of these candidate genes. These whole sequences of candidate genes will be aligned respectively, and verified again according to the sequence alignment results of these candidate genes. Finally, these candidate genes will be cloned, and then expressed in different E.coli strains, the catalytic properties of candidate gene encoded protease will be tested in different catalytic reaction. According to the catalytic properties of candidate gene encoded protease, the flavonols or their derivatives metabolic candidate genes will be confirmed finally. Through a comprehensive analysis of the above information, the genetic diversity of these metabolic genes will be elucidated in some extent, and some theoretical guidance can be provided to further research the molecular mechanism of flavonols and their derivatives accumulation in different barley grain, and improve their metabolic pathways too.

大麦籽粒黄酮醇及其衍生物具有一定的药用和保健价值，其积累与黄酮醇合成、黄酮醇甲基化和糖基化等代谢酶基因相关，其代谢酶基因在不同植物中具有遗传多样性。为了筛选我国地方大麦籽粒黄酮醇及其衍生物代谢酶基因，本项目以10个地方大麦品种4个发育阶段的籽粒为材料，构建3种与不同黄酮醇化合物对应的转录组文库。采用转录组测序、序列比对和Unigene注释等技术筛选黄酮醇及其衍生物代谢酶基因核心序列和基因内标记。通过基因内标记检测不同大麦籽粒中代谢酶候选基因的表达量、分析其表达量和黄酮醇及其衍生物含量的相关性，验证候选基因核心序列。再利用3′和5′端扩增被验证候选基因全序列、比对并筛选候选基因全序列。最后采用大肠杆菌克隆并表达候选基因，检测候选基因编码蛋白酶的催化特性，确认黄酮醇及其衍生物代谢酶基因。解析代谢酶基因遗传多样性，为揭示不同大麦籽粒中黄酮醇及其衍生物的积累机制、改良其代谢途径提供一定的理论支持。

大麦籽粒黄酮类化合物具有多种生理功能和一定的药用与保健价值，其积累与黄酮醇合成酶等代谢酶基因相关，但在不同材料中其代谢类基因存在遗传多样性。项目拟检测大麦成熟籽粒中黄酮类化合物成分及其含量差异、并构建其指纹图谱构建和检测技术体，检测大麦成熟籽粒中差异表达的黄酮类化合物相关代谢酶基因、筛选黄酮类化合物代谢酶基因，分离克隆大麦成熟籽粒黄酮类化合物代谢酶基因的编码区序列并开发其特异标记，分析特异性基因标记与黄酮类化合物含量的相关性，验证所克隆基因的功能。通过本项目实施，构建了大麦成熟籽粒黄酮类化合物的高效液相色谱、高效液相色谱-串联质谱检测技术体系。在8个大麦成熟籽粒中检测245种包括黄酮、黄烷酮、黄酮醇、异黄酮和花色苷等含量不同的黄酮化合物，其中木犀草素、芹菜素和圣草酚等20多种化合物是首次发现的大麦黄酮类化合物新成分。筛选到成熟籽粒中黄酮类化合物成分及其含量差异明显的云南高世代材料、北青7号和淳安六棱胭脂大麦等8个大麦材料。筛选到与大麦籽粒黄酮类化合物代谢相关的840个涉及次生代谢物合成、RNA剪接与修饰以及碳代谢等不同功能的差异表达基因，其中黄酮类化合物代谢酶基因8个。开发了黄酮类化合物8个代谢酶基因的特异性标记、基于多态性基因特异性标记和SSR标记初步定位了黄酮类化合物含量的175个QTLs，有效证明了QTLs的连锁遗传是导致大麦成熟籽粒中黄酮醇化合物含量相关的主要原因。克隆了大麦籽粒中黄酮类化合物3个代谢酶基因编码区全长序列，分析3个代谢酶基因的遗传多样性，获得3个基因的原核表达蛋白、初步验证了3个基因的功能。初步建立了大麦黄酮类化合物代谢酶基因的原核表达、表达蛋白的提取和纯化技术体系。本项目研究成果为完善大麦黄酮类化合物代谢途径、解析大麦成熟籽粒中黄酮类化合物差异积累的遗传机制，创制基于大麦籽粒的黄酮类化合物专用反应器、选育黄酮类化合物专用保健品种并延长大麦产业链等研究提供了理论依据、技术与材料支撑。