小麦后熟期Puroindoline蛋白的生化特性与抗霉菌作用机制研究

The Puroindoline (PIN) is a critical protein involved in wheat hardness and frequently found abundant in endosperm and aleurone layer of mature seed. Previous results showed that PIN not only regulate the formation of wheat hardness during seed development but also effectively inhibit the growth of many plant pathogens. The after-ripening period is a key stage for wheat hardness formation, however, the role of PIN based on adhesion with the lipid membrane and its impact on membrane structure are not yet clear, the biochemical properties therefore need more investigation. In addition, the fungal resistance of PIN also remain to be elucidate.. In our work, newly harvested wheat with different hardness properties will be selected to mimic after-ripening in a simulated grain storehouse，the PIN protein was subsequently extracted and purified during storage. The quantity of PIN binding with starch granules, effects on structure of artificial liposome membrane and its fungus resistance were studied. By using overlap PCR technology, the important active center of PIN protein TRD domain was carried out site-directed mutation, and therefore the role of PIN in lipid binding effect was evaluated. The influence of PIN on morphology of hyphae and spores of Aspergillus glaucus and Aspergillus flavus, the two main pathogenic fungi frequently involved in cereal spoilage during grain storage, are evaluated with electron microscope. Additionally, the fungal cell wall synthesis, structure and permeability of cell membrane, respiration of mitochondria, synthesis and degradation of nucleic acid of these fungi affected by PIN protein will be investigated. RNA-seq technology is also employed to elucidate the regulation of PIN protein on vegetative growth and metabolism of aflatoxin of Aspergillus flavus. In our work, the resistance mechanism of PIN protein will be studied from morphology, physiology and genetics of the test fungi.. In view of this, our work will not only enrich the evidence for the hypothesis of wheat hardness formation, but also support molecular breeding of endurable storage wheat and potentially develop new preservative used in grain storage.

Puroindoline蛋白（PIN蛋白）不仅影响小麦籽粒硬度形成，还能有效抑制许多植物病原菌的生长。目前，国内外对PIN蛋白在小麦后熟期的生化特性及其抗霉菌的作用机制还不十分明确。本项目选取新收获小麦在模拟实验仓中储藏，后熟过程中分离纯化PIN蛋白，研究其与淀粉粒结合作用、对脂质体膜结构的影响及抗菌活性；采用PCR技术定点突变PIN蛋白TRD结构域，分析其与脂膜的结合作用；采用电镜观察后熟期小麦PIN蛋白对储粮主要危害性霉菌灰绿曲霉和产毒黄曲霉菌丝与孢子形态的影响；测定PIN蛋白对菌丝生长、细胞壁合成、细胞膜结构稳定性与通透性、线粒体呼吸作用、DNA合成等方面的影响；采用转录组学技术研究PIN蛋白对霉菌营养生长及产毒代谢的调控作用。本项目从PIN蛋白生化特性入手，从形态学、生理学和遗传学方面解析PIN蛋白抗霉菌的作用机制，为耐储藏小麦品种的选育及新型储粮防霉剂开发提供科学依据。

Puroindoline蛋白（PIN蛋白）是小麦籽粒中的天然抗菌蛋白，对部分细菌和植物病原真菌有较好的抑制作用。本项目从软质小麦品种扬麦15中提取PIN蛋白，测定了PIN蛋白对黄曲霉及储粮中常见危害霉菌的抑制作用。扩增pin基因序列并在大肠杆菌中表达PIN融合蛋白。分析蛋白特性，并综合运用扫描电镜、透射电镜、激光共聚焦显微镜等，结合转录组分析方法，从形态学、生理学及遗传学等方面研究了PIN蛋白对黄曲霉和灰绿曲霉的抑制作用机理。.研究发现，小麦籽粒PIN蛋白分子量约13 kDa，可有效抑制黄曲霉对大米、小麦和玉米的侵染，对常见储粮危害性霉菌如灰绿曲霉、链格孢霉、禾谷镰刀菌、青霉、白曲霉、棕曲霉及黑曲霉等均具有较明显的生长抑制作用。经大肠杆菌异源表达，获得具有抗菌活性的PIN融合蛋白，其分子量约43 kDa，对上述常见储粮霉菌的抗菌效果较好。PINA融合蛋白可耐受70℃高温处理30min，在pH6.0~10.0缓冲液中仍保持活性稳定；在紫外照射条件下，PINA蛋白抗菌作用不变，能耐受K+、Mg2+等多种金属离子。.研究了PIN蛋白对储粮霉菌的作用机制。结果表明，该蛋白作用黄曲霉和灰绿曲霉孢子时，能使其细胞壁凹陷，引起细胞膜透性增加、线粒体膜电位和细胞核损伤。能不同程度抑制孢子萌发，使菌丝生长和形态发生异常。经PIN蛋白处理，黄曲霉原生质体表面出现塌陷和孔洞。PINB蛋白可干扰黄曲霉的细胞壁、细胞膜、氧化应激、孢子形成及毒素合成等相关基因表达，aflmfs1和aflmfs2是PINB蛋白影响黄曲霉生长和产毒的关键基因；黄曲霉aflmfs1、aflmfs2及aflmfs1aflmfs2基因缺失突变株的孢子和菌丝形态、细胞膜和蛋白质合成异常，AFB1毒素产生量减少，同时，PINB蛋白对基因缺失突变株的抑制作用减弱。本研究为耐储藏小麦品种分子选育及新型储粮防霉剂的开发提供了科学依据。