大麦极限糊精酶抑制物含量和热稳定性的基因型和环境效应研究

Limit dextrinase(LD)activity in malt barley is closely associated with LD inhibitor (LDI) and thermostability. It is an effective approach of improving malt quality to reduce LDI content and enhance LD thermostability. In this project, LD will be extracted and purified, and then the methodology of measuring LDI content will be developed. About 150 barley genotypes with LD activities having been assayed in the previous study will be used for determining genotypic difference in LDI content and its change during malting. Moreover around 70 genotypes with large difference in LDI content will be analyzed for their LD thermostability. Based on a patent we developed some years ago and controlled environments, the effect of temperaure, light and water conditions on LDI and LD thermostability will be investigated. Meanwhile we will determine the effect of notrogen level and timing, harvesting time on LDI and LD thermostability under field conditions. In addition, a DH population will be used to locate QTLs controlling LD thermostability, and to identify the SNP loci associated with LDI content and LD thermostability in malt barley. It may be expected that the results obtained in this project will determine genotypic and environmental variation of barley grain and malt LDI content, and LD thermostability, and illustrate genetics and its molecular mechanisms of LDI synthesis and decomposition as well as the genotypic difference in LD thermostability.

啤用大麦麦芽极限糊精酶（LD）活性与极限糊精酶抑制物（LDI）和热稳定性密切相关，减少LDI和增强LD热稳定性是改善麦芽品质的重要途径。本研究在提取、纯化LD并建立LDI定量分析技术的基础上，拟以150份LD活性已明确的大麦基因型为材料，鉴定LDI含量及在制麦过程中变化的基因型差异，并分析其中约70份LDI含量差异明显的基因型的LD热稳定性；利用建立的离体穗培养技术，在受控环境下研究温度、光照、水分等条件以及在大田条件下研究氮素水平与运筹、收获期等主要农艺因子对LDI和LD热稳定性的影响；以已构建的DH群体为材料，分析该群体各株系的LDI含量和LD热稳定性，定位影响LD热稳定性的QTL位点，鉴定与LDI含量和LD热稳定性相关联的SNP位点。本研究结果可望明确大麦籽粒和麦芽LDI含量、LD热稳定性的基因型与环境效应及其与LD活性的关系，阐明LDI合成和降解、LD热稳定性的遗传控制与分子机理。

制麦是酿酒工业中至关重要的第一步，制麦过程中籽粒内的生化变化主要是淀粉和多糖在一些降解酶的作用下水解为小分子的糖类，降解酶主要有α-淀粉酶、β-淀粉酶 、极限糊精酶（LD）。极限糊精酶是淀粉降解完全程度的限速酶，因此LD与麦芽浸出率关系密切。LD在籽粒中有多种存在形式，较大一部分是以1:1的结合态与极限糊精酶抑制物相结合，这样使其活性受到抑制。同时，LD易受高温的影响而失活。迄今，有关LD的热稳定性和LDI含量的基因型与环境差异知之甚少。本课题研究了麦芽LD抑制物含量和LD热稳定性以及LD在制麦过程中降解的基因型差异、环境因素对大麦LDI合成和LD热稳定性的影响，并分析了麦芽LDI和LD热稳定性相关基因的定位。取得的主要结果有：鉴定到LD活性高且热稳定性强的大麦种质;确定了2个与LDI含量有关的DArT标记（bpb-8347、 bpb-8399）和31个SNP标记;发现LD含量和糖化力、库尔巴哈值、粘度和可溶性氮含量相关显著，而LDI和分析的麦芽品质性状之间的相关性均不显著；明确了制麦过程中添加GA和ABA对麦芽品质的影响，GA促进淀粉降解相关蛋白的表达，而ABA促进淀粉合成相关蛋白的表达。研究结果初步阐明了大麦籽粒和麦芽LDI含量、LD热稳定性的基因型与环境效应及其与LD活性的关系，鉴定到的特异种质和相关分子标记可用于啤用大麦品质育种实践。