小麦储藏生理性变质的特性与机理研究

Many physiological deterioration phenomena, such as enzyme activity decrease, weak viability and depressive respiration, were regularly observed in the period of wheat storage, and this deterioration directly led to the decline seed and processing quality. At present, a great deal of research work on the stored wheat physiology nearly focused on the cell metabolism, whereas the deterioration mechanism of wheat deterioration is not very clear yet. Fortunately, our previous study found that wheat physiological deterioration may occur in a particular organization, and possibly caused by the wheat internal physiological activity. To verify this hypothesis, we first in this study determined the wheat tissue location by the living tissue staining techniques, in which the physiological deterioration occurred initially. Then the proteomics analysis was carried out for the deteriorated location using 2-DE and MALDI-TOF MS techniques, some functional proteins responsible for physiological deterioration were expected to be identified in this process. The gene, Tri-EXPB1, which expressed coleoptile cell wall expansin protein, was also used as target gene for survey the effect of wheat deterioration on gene transcript activity by fluorescence quantitative PCR technique. In order to understand RNA metabolic activity and the DNA degrade frequency, protein electrophoresis and zymogram staining technique were used. In addition, agarose gel electrophoresis, nuclear magnetic resonance as well as scanning electron microscopy are used to study the structure damage of macromolecular including nucleic acid, protein and starch grains. In this study, proteomics analysis, gene expression, and nucleic acid metabolism are investigated to explore the physiological deterioration characteristics and mechanism of stored wheat. This study will contribute experimental data for fully explaining the physiological mechanism of stored wheat, and also possibly laid the groundwork to delay wheat deterioration and improve the wheat storage methods.

小麦在储藏过程中会发生酶活性下降、生命力减弱、呼吸衰退等生理变质现象，导致其种用品质和加工品质持续变劣。目前，国内外对小麦储藏生理的研究多停留在细胞代谢水平，对其变质的分子机理还不十分明确。研究发现，小麦生理劣变可能发生在特定组织，生理活动是引起小麦发生变质的内因。本项目首先通过活性染色技术确定小麦生理变质的组织区位；运用2-DE和MALDI-TOF MS技术进行生理劣变区蛋白组学分析，鉴定生理劣变的相关蛋白并分析其功能；采用荧光定量PCR技术研究小麦Tri-EXPB1基因表达水平，探究生理变质对基因转录活性的影响；采用蛋白电泳及酶谱染色技术研究RNase与Nuclease的活性，了解RNA代谢活性与DNA降解情况。此外，采用电泳、核磁共振、扫描电镜等技术研究小麦劣变区核酸、蛋白及淀粉粒等大分子结构变化。本项目分别从蛋白组学、基因表达、大分子结构等分子层次研究了小麦生理变质的特性及机理。

小麦籽粒是具有生物活性的有机体，在储藏过程中会发生酶活性下降、生命力减弱、呼吸衰退等生理变质现象，导致其种用品质和加工品质持续变劣。本项目通过活性染色技术、蛋白组学技术、基因检测技术、生化分析手段等，探究了小麦储藏变质过程中的基本特性和其生理劣变发生的机理。. 研究发现：（1）小麦生理性劣变最先发生在根冠部位，继而沿胚轴向胚芽方向发展。（2）在小麦劣变发生过程中，鉴定得到36个上调蛋白和126个下调蛋白。上调蛋白主要与核糖体及组蛋白相关的蛋白，可能参与了贮存蛋白的降解；下调蛋白主要参与了能量代谢以及对抗外界环境压力。（3）新收获小麦核酸酶活性较高；完成后熟作用的小麦核酸酶活性较低，当储藏温度升高时，酶活性短期内有升高趋势，但随储藏时间延长，核酸酶活性又逐渐降低。（4）安全水分条件下的小麦在常温储藏时Tri-EXPB1基因表达水平较低，变化不明显；高温储藏时，该基因随小麦储藏时间延长表达量不断降低；Tri-EXPB1基因表达量变化与小麦发芽率变化高度相关；（5）新收获小麦呼吸作用较强；小麦随储藏时间延长，呼吸逐渐减弱；高温劣变时小麦呼吸作用先升高后降低。常温储藏条件下小麦的ATP含量变化不大；新收获小麦ATP含量较储藏12个月小麦的高，但随储藏时间延长，两者都逐渐降低。（6）小麦储藏劣变过程中，胚部大分子蛋白逐渐减少，蛋白呈降解趋势；球形大淀粉粒不断减少，小淀粉粒储藏开始阶段多呈球形，随后不规则形态逐渐增多。（7）新收获小麦籽粒中脱氢酶、过氧化氢酶和过氧化物酶活性均略高于已储藏12个月的小麦。在储藏过程中，三种酶的活性随储藏时间延长持续下降。. 上述研究结果表明，小麦在储藏劣变过程中，其各项生理因素都在发生作用，生理变质是多种生理活动综合作用的结果。本研究为理解小麦储藏变质机理，调控储藏品质提供了基础。