小麦胚部病程相关蛋白抑制储藏真菌生长及产毒的机理研究

The growth of fungi could deteriorate the grain quality and even produce mycotoxins during grain storage, which could severely affect the edible quality and safety of cereal grains. Owing to the characteristics of grain structure, the growth of fungi and mycotoxin production usually occur firstly in the grain embryo. In our previous study, it was found that the soft wheat showed higher storage tolerance to fungal growth and mycotoxin accumulation, which is associated with more pathogenesis-related proteins (PRs) in its embryo. However, the inhibitory mechanisms of pathogenesis-related proteins on the growth of storage fungi and mycotoxin production are still unclear. In current study, proteomic analysis of wheat embryo and wheat storage test would be performed to reveal the regular existence of PRs among different wheat cultivars. The relationship between PRs in wheat embryo and fungal growth as well as mycotoxin production would be analyzed to reveal the PR(s) in wheat embryo, which is/are responsible for the inhibition of fungal growth and mycotoxin production in stored wheat. The wheat PRs would be prepared through protein overexpression in E.coli and the effect of PRs on spore germination, cell morphology, energy metabolism of storage fungi would be analyzed. The effect of PRs on expression level of critical mycotoxin-related genes in toxigenic fungi would be analyzed and the proteome of toxigenic fungi would be analyzed to reveal the changes of mycotoxin-related proteins before/after treated with PRs. The factors affect the action of PRs, such as moisture content, temperature, aging degree, would be analyzed and their mechanisms would be demonstrated. Through the systematic study above, the molecular mechanisms of PRs in wheat embryo inhibiting fungal growth and mycotoxin production could be elucidated, which would provide valid theoretical foundation for the safe storage and differential storage of different wheat cultivars as well as molecular breeding of storage tolerant wheat.

储粮中真菌生长及真菌毒素积累可严重影响粮食品质和食用安全。由于粮食籽粒结构特性，储粮中真菌生长和真菌毒素积累一般最先发生在籽粒胚部。前期研究发现软质小麦籽粒胚部含有的病程相关蛋白对其储藏霉变及毒素积累具有抑制作用，但其作用机理尚不清楚。本研究拟通过对小麦储藏和蛋白质组学分析，研究不同品种小麦胚部病程相关蛋白的存在规律并确定抑制储粮真菌生长和产毒的主要病程相关蛋白；制备病程相关蛋白并分析其对储藏真菌的孢子萌发、细胞形态、能量代谢以及产毒真菌中与真菌毒素生成相关的转录组、蛋白质组和关键基因表达水平等方面的影响，阐明其对储藏真菌生长及产毒的作用机理；分析不同储藏条件下影响小麦胚部病程相关蛋白抑制储藏真菌生长及毒素积累作用效果的主要因素并阐明其影响机理。本项目通过以上系统研究从分子水平上揭示病程相关蛋白在储藏期小麦抗霉变和抗毒素生成作用中的分子机制，为小麦安全储藏、分类储藏和分子育种提供理论基础。

储粮真菌生长及真菌毒素积累可严重影响粮食品质和食用安全。储粮中真菌生长和真菌毒素积累一般最先发生在籽粒胚部，前期研究表明软质小麦储藏抗霉变特性可能与其胚部含有的病程相关蛋白有关。为了阐明病程相关蛋白对储粮真菌生长和产毒的抑制作用，本研究对软质和硬质小麦胚部蛋白质组以及病程相关蛋白的种类和活性进行了比较分析。结果表明软质小麦胚部含有较高活性的wheatwin 1、beta-1,3-葡聚糖酶、几丁质酶、beta-1,4-甘露聚糖酶、过氧化物酶、淀粉酶抑制剂和木聚糖酶抑制剂等多种已报道具有抗真菌作用的病程相关蛋白，在储藏过程中软质小麦胚部病程相关蛋白的活性下降较慢。这表明软质小麦胚部的病程相关蛋白是其籽粒内部抗真菌机制的重要组成部分。为了研究病程相关蛋白对储藏真菌生长和产毒的的抑制作用，我们利用分子生物学技术从软质小麦基因组DNA中扩增出了的wheatwin 1、beta-1,3-葡聚糖酶、几丁质酶和内切-1,4-β-甘露聚糖酶等四种病程相关蛋白的编码基因并将其在大肠杆菌或毕赤酵母中进行异源表达以大量制备病程相关蛋白。随后，我们分析了这四种病程相关蛋白对储粮常见真菌真菌的生长及产毒的抑制作用。研究表明Wheatwin1在低浓度时可促进黄曲霉产毒量的增加，在浓度达到90μg/mL时则可以抑制黄曲霉生长并减少黄曲霉毒素的积累。wheatwin1处理黄曲霉可调控黄曲霉产毒的关键基因的表达水平。Beta-1,3-葡聚糖酶、几丁质酶和内切-1,4-β-甘露聚糖酶对小麦霉变常见真菌禾谷镰刀菌、链格孢霉、灰绿曲霉、黄曲霉、黑曲霉和扩展青霉等真菌的菌丝生长具有不同程度的抑制作用。这些病程相关蛋白可破坏储粮真菌的细胞壁以抑制真菌生长。病程相关蛋白对储藏真菌的抑制效果与其酶学活性和真菌细胞壁组分相关，其对不同储粮真菌的抑制效果差异主要是由于不同真菌细胞壁中葡聚糖、几丁质和甘露聚糖等组分的含量不同。在小麦粉中添加beta-1,3-葡聚糖酶、几丁质酶和内切-1,4-β-甘露聚糖酶可有效抑制黄曲霉生长和黄曲霉毒素B1的产生。本项目研究从分子水平上揭示病程相关蛋白在储藏期小麦抗霉变和抗毒素生成作用中的分子机制，为小麦分类储藏和耐储藏小麦的分子育种提供理论基础。