面粉颜色相关性状分子基础及关键基因Psy1功能研究

发布时间：2018-04-21 23:06

本文选题：QTL定位 + RNAi　；参考：《中国农业大学》2016年博士论文

【摘要】：小麦面粉颜色相关性状包括面粉亮度(L*)、红度(a*)、黄度(b*)及黄色素含量(YPC),是影响面制品色泽的重要因素,已成为小麦品质改良的重要内容。对面粉颜色性状研究多集中于QTL定位、基因克隆及功能标记开发,而关键基因Psyl功能验证及遗传调控机理研究还鲜有报道。由于作图群体及分子标记局限性,大量面粉颜色性状基因未被发现。本研究利用小麦90K SNP芯片,对藁城8901/周麦16RIL体和247份小麦品种进行QTL定位和全基因组关联分析,揭示面粉颜色相关性状的分子基础；采用RNAi、RNA-Seq和TILLING技术相结合的实验策略,研究影响面粉颜色关键基因Psyl功能及遗传调控机理。主要结果如下：1.应用小麦90K SNP芯片,分析藁城8901/周麦16 RIL群体,构建高密度遗传图谱,对面粉颜色相关性状进行QTL定位及候选基因发掘。整个遗传图谱包含2205个骨架标记,覆盖普通小麦21条染色体,总长度为2859.2 cM,每条染色体平均长度为136.2 cM,标记间平均遗传距离为1.3cM。共检测到52个影响面粉颜色性状的QTL,位于第1、2、5、7同源群染色体和3B、4A、4B、6B染色体上,单个QTL解释5.3-32.2%的表型变异。与前人研究相比,发现5个影响a。,2个影响b*,1个影响L*和1个影响YPC的新QTL。发现7个面粉颜色性状的候选基因,一个为Pinb-D1基因,其余6个基因均与萜类化合物骨架合成途径相关。由此可见,基于90K SNP芯片构建高密度遗传图谱是进行复杂数量性状QTL定位及候选基因发掘的有效工具。2.利用小麦90K SNP芯片,对247份普通小麦品种进行面粉颜色相关性状全基因组关联分析,共检测到19个显著关联位点,包含1个影响a*和1个影响L*的新位点。发现6个影响面粉颜色性状的候选基因,即Pinb-D1、Psy-A1、Psy-B1和NADP-依赖性苹果酸酶、细胞质型苹果酸脱氢酶及抗坏血酸过氧化物酶编码基因,证实应用90K SNP芯片对复杂数量性状进行全基因组关联分析的高效性和可靠性。3.RNAi转基因植株籽Psy1表达显著降低(54-76%),YPC显著下降(26-35%),验证Psyl对小麦籽粒黄色素合成具有重要影响。利用RNA-Seq技术对3个RNAi效果较好的转基因株系(273-2A、275-3A和279-1A)进行转录组测序,发现次级代谢途径和核心代谢途径上的多个候选基因协同响应Psy1基因表达下调。应用TILLING技术对Psyl功能分析显示,天冬氨酸富集区(DXXXD)是PSYl重要功能结构域,其附近保守核苷酸通过调控基因表达、酶活性及选择性剪接等方式影响黄色素合成。RNAi转基因植株及EMS突变体Psy1基因表达分析结果均显示,花后14天是小麦籽粒Psyl基因表达遗传调控的关键时期。优异EMS突变体为小麦面粉颜色及营养品质的遗传改良提供重要种质资源。
[Abstract]:The relative characters of wheat flour color including the brightness of wheat flour, the degree of red, the content of yellow pigment and the content of yellow pigment YPC are the important factors affecting the color and color of flour products, which have become the important content of wheat quality improvement. The studies on color traits of flour were mainly focused on QTL mapping, gene cloning and functional marker development. However, the functional verification of key gene Psyl and the mechanism of genetic regulation were rarely reported. Due to the limitations of mapping population and molecular markers, a large number of flour color trait genes were not found. In this study, wheat 90K SNP microarray was used to localize and analyze the whole genome of Gaocheng 8901 / Zhoumai 16RIL body and 247wheat cultivars to reveal the molecular basis of flour color related traits, and the experimental strategy of combining RNA-Seq with TILLING technique was used. To study the function and genetic regulation mechanism of flour color key gene Psyl. The main results are as follows: 1. Wheat (Triticum aestivum) 90K SNP microarray was used to analyze the population of 8901 / Zhoumai 16 RIL in Gaocheng, and to construct a high-density genetic map, and to map the color related traits of wheat flour by QTL and explore the candidate genes. The whole genetic map contains 2205 skeleton markers covering 21 chromosomes of common wheat, the total length is 2859.2 cm, the average length of each chromosome is 136.2 cm, and the average genetic distance between markers is 1.3 cm. A total of 52 QTLs affecting the color traits of flour were detected, which were located on the chromosomes of homologous group 1 and chromosome 3BX 4A4 B, and the phenotypic variation of 5.3-32 2% was explained by a single QTL. Compared with previous studies, it was found that five effects, two effects, one effect on L * and one new QTL affecting YPC were found. Seven candidate genes for flour color traits were found, one of which was Pinb-D1 gene, and the other 6 genes were all related to the skeleton synthesis pathway of terpenes. Therefore, constructing high density genetic map based on 90K SNP chip is an effective tool for QTL mapping and candidate gene discovery of complex quantitative traits. Using wheat 90K SNP microarray, a total of 19 significant loci were detected for the whole genome association analysis of wheat flour color related traits, including one new locus affecting a * and one new affecting L *. Six candidate genes affecting flour color traits, namely Pinb-D1 Psy-A1 Psy-B1 and NADP-dependent malonase, cytoplasmic malate dehydrogenase and ascorbic acid peroxidase encoding genes were found. It was confirmed that the high efficiency and reliability of the whole genome association analysis of complex quantitative traits with 90K SNP microarray. 3. The expression of Psy1 in seeds of RNAi transgenic plants was significantly decreased by 54-760.YPC significantly decreased from 26 to 355.It was proved that Psyl had an important effect on the synthesis of yellow pigment in wheat grains. The transcriptome sequencing of three transgenic lines with good RNAi effect was carried out by RNA-Seq. It was found that several candidate genes in the secondary metabolic pathway and the core metabolic pathway responded to the down-regulation of Psy1 gene expression. The functional analysis of Psyl by TILLING showed that Aspartic acid rich region (DXXXD) is an important functional domain of PSYl, and its nearby conserved nucleotides regulate gene expression. The effects of enzyme activity and selective splicing on the expression of Psy1 gene in xanthine synthesis. RNAi transgenic plants and EMS mutants showed that 14 days after anthesis was the key period for genetic regulation of Psyl gene expression in wheat grains. Excellent EMS mutants provide important germplasm resources for genetic improvement of wheat flour color and nutritional quality.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S512.1

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