不同硬度小麦储藏期抗霉变特性差异与分子机理研究

The control of grain spoilage by mould is the focus of grain storage management， which is concerned to the grain quality and food safety. During grain storage, Aspergillus glaucus group are the most predominant fungal species in wheat at the beginning of the storage and considered as an index of advanced deterioration, which could thrive at lower moisture content. Then, the growth of Aspergillus glaucus would cuase the alternation of other fungal ecological groups to further deteriorate the grain quality or even produce harmful metabolites, such as mycotoxins. In our previous study, it was found that soft wheat could significantly resist the growth of Aspergillus glaucus group. This property is of great importance for enlonging the storage period of wheat and guranteeing the food safety. However, the intrinsic mechanism of the mould resistance of wheat is still unclear yet. Recently, It has been found that the physiological contents between hard wheat and soft wheat are significantlt different, which might be the intrinsic properties causing the difference of microbial spoilage of hard wheat and soft wheat. To verify this hypothesis, the stuctures of bran, embryo and endosperm of hard wheat and soft wheat and the growth of Aspergillus glaucus on these tissues will be studied by scanning electron microscopy. The proteomics analysis was carried out for the unique proteins in hard wheat and soft wheat using two-dimensional electrophoresis and MALDI-TOF MS techniques, some mould resistant proteins are respected to be identified in this process. In addition, HPLC, protein purification, mould resistance as well as PCR techniques are used to identify the mould resistant substances and determine their amount, the genes encoding mould resistant proteins are also expected to be identified. In this study, the differences of the tissue stucture and physiological contents in soft and hard wheat cultivars and their effect on the growth of Aspergillus glaucus will be investigated to explore the mechanism of mould resistance of wheat. This study will contribute experimental data for fully explaining the mould resistant mechanism of wheat and would also lay the ground work to wheat safe storage and molecular breeding.

粮食储藏过程中霉菌的生长可严重影响粮食品质，也与食品安全性密切相关。因此，防治霉菌是粮食安全储藏的主要任务。在常规储藏过程中，粮食霉变主要由灰绿曲霉等起始性霉菌引发，该菌的生长可改变粮堆生态环境并引起霉菌类群演替，最终导致粮食劣变及产生危及食品安全的真菌毒素。我们在前期研究中发现储藏期软质小麦对灰绿曲霉有较强抗性，这一特性对阻断储粮真菌发生具有重要的意义，但目前尚不能解释不同硬度小麦品种抗霉变差异的机理。本项目拟采用扫描电镜技术研究霉菌在不同硬度小麦的种皮、胚和胚乳中生长特性；采用双相电泳和MALDI-TOF MS技术研究不同硬度小麦各组织中蛋白质组学差异；采用高效液相色谱、蛋白质纯化技术、抑菌实验和PCR等技术分析不同硬度小麦中的抗菌成分及其含量并确定抗菌蛋白的分子标记。通过对不同硬度小麦抗霉菌相关特性的系统研究从分子水平上揭示小麦抗霉变作用的内在机制，为小麦安全储藏和育种提供理论基础。

粮食储藏过程中霉菌的生长可严重影响粮食品质，也与食品安全性密切相关。因此，防治霉菌是粮食安全储藏的主要任务。在常规储藏过程中，粮食霉变主要由灰绿曲霉等起始性霉菌引发，该菌的生长可改变粮堆生态环境并引起霉菌类群演替，最终导致粮食劣变及产生危及食品安全的真菌毒素。我们在前期研究中发现储藏期软质小麦对灰绿曲霉有较强抗性，这一特性对阻断储粮真菌发生具有重要的意义。本项目对不同硬度小麦储藏期抗霉变特性差异与分子机理进行了研究。本项目研究了霉菌在不同硬度小麦表面生长的主要部位及其差异；采用双相电泳和MALDI-TOF MS技术研究了不同硬度小麦各组织中蛋白质组学差异，初步确定与软质小麦抗霉变特性相关的病程相关蛋白；对不同硬度小麦中采用高效液相色谱、蛋白质纯化技术、抑菌实验和PCR等技术分析不同硬度小麦中的抗菌成分及其含量并确定抗菌蛋白的分子标记。研究结果明确了不同硬度小麦在偏高水分储藏条件下对灰绿曲霉的抑制作用显著差异，霉变主要发生在小麦胚部。对软质小麦扬麦15和硬质小麦扬麦16的胚部的蛋白质组学分析结果表明两种小麦胚部中含有的特异性蛋白质存在显著差异。硬质小麦扬麦16中存在15种特异性蛋白，而软质小麦扬麦15中存在56个特异性蛋白。由于小麦基因组测序工作尚未全部完成，多数特异性蛋白的功能尚无法确定。但可以确定软质小麦杨麦15中存在多种特异性蛋白具有抗真菌活性，如puroindoline B、木聚糖酶抑制蛋白、淀粉酶抑制蛋白、wheatwin 1、serpin、过氧化物酶等。软质小麦胚部中的几丁质酶、过氧化物酶、木聚糖酶抑制蛋白、beta-1,3-葡聚糖酶等抗真菌蛋白的活性均高于硬质小麦胚部，抗真菌蛋白的活性与小麦储藏期间抗霉变能力具有较高的相关性。从小麦RNA中扩增出了以上几种抗真菌蛋白基因并实现了大肠杆菌表达，初步证明了小麦wheatwin1 蛋白对黄曲霉生长和产毒具有抑制。通过本项目对不同硬度小麦抗霉菌相关特性的系统研究，从分子水平上明确了小麦霉变小麦抗霉变作用的内在机制，为小麦安全储藏和育种提供理论基础。