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

项目名称： 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

项目编号： No.31460385

项目类型： 地区科学基金项目

立项/批准年度： 2015

项目学科： 食品科学、农学基础与作物学

项目作者： 陈升位

作者单位： 云南农业大学

项目金额： 49万元

中文摘要： 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。

中文关键词： 大麦籽粒；黄酮醇及其衍生物；转录组测序；代谢酶基因；地方大麦品种

英文摘要： 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.

英文关键词： barley grain;flovonol and theirderivitives;transcriptome sequencing;metablic gene;local barley verities