采后苹果褐腐病生防菌Z0-J高效定殖果面的物质基础与分子机制

Decay in fruits and vegetables, caused by post-harvest diseases, results in a serious enormous loss and has become a global problem. More and more beneficial microorganisms are considered as potential biological agent, but only a few have been applied because of the unclear colonization-limiting factors and mechanism. In previous research work, an endophytic Pseudomonas sp. Z0-J stably colonized on apple peels, which has a significant effect on postharvest brown rot control and contains the chemotaxis key gene cheA, was obtained. The project will focus on limiting factors and molecular mechanisms of biocontrol agent Z0-J colonization on apple surface to conduct the following researches：1) to find out the relationship between the colonization dynamic characteristics of Z0-J and its antiseptic effect; 2) to determine the effect of apple epidermis components, especially chemoattractants, on the effective colonization of Z0-J using HPLC combined with electron microscopy; 3) to reveal the association between colonization-related gene function and Z0-J effective colonization based on RNA-Seq technology, bioinformatics and gene knockout methods. The results will provide basis for the improvement of engineering bacteria, development of biological control agent and scientific application of biocontrol agent Z0-J, as well as provide examples and foundations for in-depth understanding of the colonization mechanism of biocontrol bacteria.

采后病害引致果蔬腐烂变质造成的巨大损耗己成为全球性问题，越来越多有益微生物成为潜在生防因子，但仅极少数得以应用，究其原因是生防菌的定殖影响因子及分子机制不清。本项目组前期获得一株稳定定殖于苹果果皮，对采后褐腐病防效显著且含有趋化性关键基因 cheA 的内生假单胞菌 Z0-J。锁定上述科学问题，本项目采用宏观与微观相结合、多学科交叉的方式，拟从微生态学、生理学和分子生物学三个方面开展以下研究：1）弄清生防假单胞 Z0-J 的定殖动态特征与其防腐效果的关系；2)利用液相色谱结合电镜技术探明苹果表皮物质成分，尤其是趋化诱导物对Z0-J 有效定殖的影响；3）利用 RNA-Seq 技术和生物信息学方法，结合基因敲除手段揭示定殖相关基因功能与Z0-J 有效定殖采后苹果的关联性。研究结果将为生防菌Z0-J的工程菌改良、制剂开发与科学合理施用提供依据，也对深入认识采后果蔬生防菌的定殖机制提供范例和基础。

采后病害引致果蔬腐烂变质造成的巨大损耗己成为全球性问题，越来越多有益微生物成为潜在生防因子，但仅极少数得以应用，究其原因是生防菌的定殖影响因子及分子机制不清。本项目组前期获得一株稳定定殖于苹果果皮，对采后褐腐病防效显著且含有趋化性关键基因 cheA 的内生假单胞菌 Z0-J。锁定上述科学问题，本项目采用宏观与微观相结合、多学科交叉的方式，从微生态学、生理学和分子生物学三个方面开展研究，探明生防菌 Z0-J 成功定殖的生理生化基础及分子机制，为果蔬采后病害的生物防治提供科学依据和理论指导。取得的研究成果如下：.（1）明确了采后富士苹果（Malus domestica Borkh cv. Red Fuji）进行针刺接种，在菌体发酵液原液浓度条件下防腐保鲜效果达到85.2%（9 d），处理10 d 后，生防Z0-J菌株仍能稳定定殖于果实表面，表明生防假单胞Z0-J 在苹果表皮的有效定殖与生防效果直接相关；.（2）探明了不同苹果组分都对 Z0-J 定殖量均产生显著促进作用，尤其是葡萄糖对 Z0-J 定殖量的增加最为显著。苹果表皮检测出17种氨基酸，9种有机酸，趋化性试验结果表明上述氨基酸和有机酸对菌株Z0-J菌体定殖量有不同程度影响；.（3）在上述结果基础上，利用原位试验进一步证明葡萄糖、天冬氨酸和苹果酸等物质对生防菌具有显著的趋化诱导作用；.（3）转录组分析结果表明具有COG分类的差异表达基因总数为676个，其中受到趋化物质诱导后菌株Z0-J定殖过程中与运动能力，DNA复制、重组与修复等相关生理过程的差异基因明显上调表达。基因敲除结果显示，趋化性相关基因cheA直接参与生防菌株的高效定殖，并影响生防保鲜效果。