小麦后熟过程中麦谷蛋白大聚体变化介导的品质改善机理

The wheat postharvest maturing begins immediately after harvest, continues during storage, and depends on time, ambient conditions of storage and the grain moisture content. During the wheat maturation a number of biochemical and colloidal changes occur until the final technological maturity is reached. Newly harvested wheat has poor milling and baking quality, therefore, the wheat postharvest maturation is a necessary part of improvement of their technological quality. However, to our knowledge, in the available literature of recent date there are little data on mechanism of quality improvement during wheat postharvest. Glutenin macropolymer (GMP) is the SDS-insoluble subfraction of wheat gluten and one of the largest protein-based biopolymers in nature. The amount and structure of glutenin macropolymer in flours are strongly correlated with dough characteristics and bread quality. Freshly harvested wheats with different gluten strength are stored in 4 kinds of main grain econological region in the present study. Optimal quality index representing the degree of wheat maturation is summed up after analyzing physicochemical properties of wheat kernel and its processing and eating qualities. Content, composition and structure of glutenin macropolymer are analyzed by means of electrophoresis and chromatographic separation technology. The characteristics of particle size distribution and variation of particle formation of glutenin macropolymer are studied using laser particle size analyzer and confocal scanning laser microscopy. A solution of Fluorescein isothiocyanate (FITC) was used for non-covalent labeling. Relation of content, particle size distribution and accumulation variation of glutenin macropolymer to wheat quality improvement is investigated. Expression level and its difference of GMP in wheats with different gluten strength are compared during the wheat maturation by proteomics analysis. According to variation and degree of Post-translation modification including phosphorylation, glycosylation and methylation of GMP, the key protein and pathway improving wheat quality will be illustrated. In the end of program, we will establish molecule-controlling mechanism of GMP - different protein - quality improvement during wheat postharvest maturation. It can lay a theoretical foundation for making an intensive study in quality improvement during wheat postharvest maturation.

小麦后熟是收获后籽粒内部一系列生化及胶体反应、品质逐步改善并达到最佳状态的过程，但对后熟中品质改善的机理缺乏深入研究。本研究以不同筋力小麦为材料，模拟我国4个主要储粮生态区域储藏，从与品质关系最为密切的麦谷蛋白大聚体(GMP)入手，在分析籽粒理化特性及加工品质的基础上，总结归纳表征小麦后熟程度的最佳品质代表指标；利用电泳及色谱分离技术分析GMP含量及组成变化，利用激光粒度分析、FITG标记结合CLSM技术研究GMP的粒度分布特征及其形成的动态变化规律，探讨GMP含量、粒度分布及其积累特征与小麦品质改善之间的内在关系；利用蛋白质组学，比对不同筋力小麦后熟过程中GMP表达水平及差异，结合GMP糖基化、甲基化和磷酸化等翻译后修饰种类和程度，阐述小麦品质改善的关键蛋白和作用途径，探讨“GMP -差异蛋白-小麦后熟过程品质改善”的分子控制机理。研究可为深入探讨后熟过程中小麦品质改善机理奠定理论基础。

本研究以新收获的漯麦18为原料，模拟四种条件进行储藏：15℃/ 50％RH（WT1）；20℃/ 65％RH（WT2）；28℃/ 75％RH（WT3）；35℃/ 85％RH（WT4）。研究后熟过程中麦谷蛋白大聚体（GMP）与后熟之间的内在关系，研究表明：利用湿面筋含量、面筋指数、面团流变学特性等传统的品质指标变化分析，发现后熟过程中，随着储藏时间延长，小麦品质改善，同时利用GlutoPeak分析，并和品质指标进行回归分析，发现在储藏过程中，峰值时间、EnMT值、面团流变学特性与后熟时间呈极显著相关关系，GlutoPeak峰值时间、EnMT值可作为小麦后熟品质改善的最佳代表指标。后熟过程中麦谷蛋白尤其是GMP的变化明显，而且游离SH含量随着储藏时间的延长而降低， S-S含量在整个后熟过程中逐渐升高。SDS-PAGE图谱中GMP分子分为HMW-GS和LMW-GS。储藏4周后，WT4样品的HMW-GS区域产生了新谱带（约145 kDa），LMW-GS区域部分<20 kDa的谱带消失。HMW-GS区域谱带颜色随后熟期的延长而逐步加深，说明部分LMW-/HMW-GS亚基间通过S-S连接形成复杂的GMP分子。随着后熟的进行，2-D 图谱中HMW-GS区域点数增加，颜色逐渐加深，B-、C-和D-LMW-GS亚基的点数则逐渐减少，颜色变浅，其中C-LMW-GS亚基的点变化尤为明显，说明GMP聚合过程中C-LMW-GS的贡献最大。四种样品SE-HPLC图谱中清、球蛋白含量均随储藏时间延长而逐渐减少、醇溶蛋白含量降低，麦谷蛋白特别是GMP含量显著增加，其中WT4变化最为明显，第12周时，WT4中GMP增加了17.69%，而WT1只增加了9.40%。RP-HPLC分析发现：储藏第12周时WT4中HMW-GS增加了1.86%，HMW-GS/LMW-GS比值增加0.41，同期WT1增加了0.9%，HMW-GS/LMW-GS比值只增加0.02。小麦后熟过程中蛋白质特别是麦谷蛋白通过S-S交联形成GMP，其中C-LMW-GS是聚合形成GMP的主要参与者，而且受储藏条件的影响，高温、高湿有利于GMP分子的形成，从而形成适宜的面筋网络结构，这是控制小麦后熟过程中品质改善的主要原因。