抗坏血酸诱导卡利比克毕赤酵母对氧化胁迫的应答调控机制研究

Antagonistic yeasts have shown great potential as an alternative to synthetic fungicides for the control of postharvest decay of fruits. However bio-control efficacy of antagonistic yeasts were reduced as the growth and activity were restrained by oxidative stress on the surface and wounds of fruits. Ascorbic acid can effectively improve the tolerance of antagonistic yeasts to oxidative stress, so as to enhance their bio-control efficacy to postharvest decay of fruits. However, the mechanisms of improving the tolerance of antagonistic yeasts to oxidative stress by ascorbic acid, especially the mechanisms of response and regulation of antagonistic yeasts to oxidative stress induced by ascorbic acid are unclear yet. In this study, a Pichia carribica strain which was isolated by our research team is selected to be the model strain. 2D-electrophoresis being combined with mass spectrometry was applied in analyzing the differentially expressed proteins of yeast induced by ascorbic acid, transcriptome sequencing technology was applied in determining the differentially expressed genes of yeast. The construction of yeast mutant strain which is knocked out related gene was applied in proving the definite function of related genes. The key genes involve in improving the tolerance to oxidative stress of yeast by ascorbic acid was discovered by the means of bioinformatics and molecular genetics, so as to reveal the mechanisms of improving the tolerance of antagonistic yeasts to oxidative stress by ascorbic acid in molecular level. The expected research results will provide theoretic evidence for the application of antagonistic yeasts to control postharvest decay of fruits.

利用拮抗酵母代替化学杀菌剂防治水果采后病害已显示出良好的应用前景。然而由于氧化胁迫导致拮抗酵母在水果表面及伤口的生长和活性受到抑制，导致拮抗酵母生物防治效力降低。抗坏血酸能有效的增强拮抗酵母对氧化胁迫的耐受性，提高其对水果采后病害的生防效力。然而，抗坏血酸提高拮抗酵母对氧化胁迫耐受性的机制，特别是抗坏血酸诱导拮抗酵母对氧化胁迫的应答调控机制尚不明确。本研究拟以课题组分离的卡利比克毕赤酵母为模式菌株，利用二维电泳结合质谱技术分析筛选抗坏血酸诱导卡利比克毕赤酵母的差异表达蛋白，利用转录组测序技术筛选酵母差异表达基因，构建相关基因的敲除突变体菌株从而验证相关基因的功能，通过生物信息学和分子遗传学的手段探明参与抗坏血酸提高酵母对氧化胁迫耐受性的关键基因，从分子水平上阐明抗坏血酸提高拮抗酵母对氧化胁迫耐受性的机制。研究成果可为拮抗酵母防治水果采后病害的实际应用提供理论支撑。

利用拮抗酵母代替化学杀菌剂防治水果采后病害已显示出良好的应用前景。然而由于氧化胁迫导致拮抗酵母在水果表面及伤口的生长及活性受到抑制，引起拮抗酵母生物防治效力降低。抗坏血酸能有效增强拮抗酵母对氧化胁迫的耐受性，提高其对水果采后病害的生防效力。然而，抗坏血酸提高拮抗酵母对氧化胁迫耐受性的机制，特别是抗坏血酸诱导拮抗酵母对氧化胁迫的应答调控机制尚不明确。因此，本课题以卡利比克毕赤酵母为模式菌株，研究了氧化胁迫条件下抗坏血酸诱导和未诱导的卡利比克毕赤酵母对苹果采后青霉病控制效果，氧化胁迫条件下抗坏血酸诱导提高卡利比克毕赤酵母生防效力的生理机制和分子机制。课题的研究结果表明，250µg/mL抗坏血酸诱导培养卡利比克毕赤酵母对苹果青霉病的控制效果最好，并能显著提高氧化胁迫条件下卡利比克毕赤酵母在苹果表面的定殖能力，降低活性氧积累量和丙二醛含量，提高过氧化氢酶、多酚氧化酶和过氧化物酶等氧化还原酶活性。蛋白质组学分析结果表明：抗坏血酸诱导培养卡利比克毕赤酵母，在氧化胁迫条件下，其参与能量代谢、糖代谢和与抗氧化酶直接作用等途径的蛋白上调表达。转录组学分析结果表明：抗坏血酸诱导培养卡利比克毕赤酵母，在氧化胁迫条件下，其涉及细胞壁和能量合成、多糖水解及抗氧化等途径的基因上调表达。上述结果表明，抗坏血酸诱导培养可以调节拮抗酵母能量代谢、糖代谢及抗氧化代谢，从而提高其对氧化胁迫的耐受性，提高其对水果采后病害的生物防治效力。研究结果为抗坏血酸诱导拮抗酵母对氧化胁迫的应答调控提供了理论支持。