SA信号分子在拮抗酵母诱导樱桃番茄采后抗病性中的作用研究

Application of antagonistic yeast to induce disease resistance and then strengthening defense capability to disease is an effective way to keep postharvest fruits and vegetables healthy and fresh. In this research, Pichia guilliermondii will be selected as the antagonistic yeast, with cherry tomato fruit as the study material. Firstly, salicylic acid (SA) content in the fruit will be determined during storage period to clear whether the increasing endogenous SA is induced by antagonistic yeast. On this basis, to reveal the fact that antagonistic yeast depending on SA-mediated signal transduction pathway to induce disease resistance of cherry tomato fruit, effect of antagonistic yeast on the key enzymes (PAL and BA2H) of endogenous SA biosynthesis, resistant substances (chitinase, β-1, 3-glucanase, total phenolic and lignin) related to SA-signaling pathway and expression level of defense gene (PR-1) responding to SA will be investigated. Finally, influence of key enzymes′ (in the biosynthesis of SA) gene silencing to the biological control effect of antagonistic yeast will be researched, which may verify that SA is an essential signaling molecule in the antagonistic yeast-induced disease resistance of postharvest cherry tomato fruit. Conclusion of this research will further enrich the mechanism that antagonistic yeast inducing disease resistance of postharvest fruits and vegetables, and provide a theoretical basis on the future study for signal transduction in disease resistance. We hope to accelerate the application of biological control agent in the field of postharvest preservation for fruits and vegetables.

拮抗酵母诱导果蔬抗病性以增强其对病害的防御能力是果蔬采后防病保鲜的有效途径。本项目选择Pichia guilliermondii为实验用拮抗酵母、樱桃番茄果实为实验对象，首先检测果实贮藏期间內源水杨酸(SA)的含量，以明确拮抗酵母是否具有诱导内源SA上升的作用；其次，考察拮抗酵母对果实內源SA生物合成关键酶（苯丙氨酸解氨酶和苯甲酸2-羟化酶）、SA信号途径相关抗病物质（几丁质酶、β-1, 3葡聚糖酶、总酚、木质素）和响应SA的防卫基因（PR-1）表达水平的影响，揭示该酵母可以通过SA所介导的信号转导途径来诱导抗病性；最后，研究果实內源SA生物合成关键酶基因沉默对拮抗酵母病害防治效果的影响，以证实SA是拮抗酵母诱导樱桃番茄采后抗病性中不可或缺的信号分子。项目研究将进一步丰富拮抗酵母诱导果蔬采后抗病性的作用机理，并为深入研究抗病信号转导途径奠定理论基础，以期加速生防制剂的商业化应用。

樱桃番茄在我国种植广泛，于我国蔬果产业中占有重要地位。樱桃番茄采后由于病原微生物的侵染而造成的病害是其贮存运输中损失严重的重要原因。拮抗酵母能够有效地防治樱桃番茄采后病害，并且以其安全无毒、持久性强、抗菌谱广、不会诱发病原菌耐药性等优点而成为生物防治领域的重点研究对象。本项目选择Pichia guilliermondii为实验用拮抗酵母，首先检测贮藏期间樱桃番茄果实内源水杨酸(SA)的含量，实验结果表明108 CFU/mL的拮抗酵母菌悬液能够在接种后的24 h即诱导果实内源SA含量的升高，揭示了P. guilliermondii具有诱导樱桃番茄果实内源SA上升的作用。其次，考察拮抗酵母对果实内源SA生物合成关键酶—苯丙氨酸解氨酶（PAL）、苯甲酸2-羟化酶（BA2H）、异分支酸合酶（ICS）；SA信号途径相关抗病物质（几丁质酶、β-1, 3葡聚糖酶、总酚、木质素）和响应SA的防卫基因（PR-1）的影响，实验结果表明经108 CFU/mL拮抗酵母菌悬液处理的果实在贮藏期的6-72 h内，其PAL和BA2H活性均显著提高，且该组果实PAL和ICS基因的表达水平也显著增强；此外，108 CFU/mL的拮抗酵母菌悬液能够使樱桃番茄果实在贮藏期间几丁质酶和β-1, 3葡聚糖酶活性升高、总酚及木质素含量上升，并显著地增强其病程相关蛋白基因（PR-1）在贮藏期间的表达量。本部分从果实内源SA生物合成、SA信号途径抗病物质积累和防卫基因表达三方面，揭示了P. guilliermondii可通过SA介导的信号转导途径诱导樱桃番茄的采后抗病性。最后，研究果实内源SA生物合成关键酶基因沉默对P. guilliermondii病害防治效果的影响。本部分实验采用农杆菌侵染诱导的方法来稳定性沉默樱桃番茄的PAL基因，但采用qRT-PCR方法并未在首批实验获得的再生苗中检测到PAL基因的干扰片段出现，目前在积极进行第二批转化实验，以期获得具有遗传性的转基因植物以实现稳定性的基因沉默，并验证SA是P. guilliermondii诱导樱桃番茄采后抗病性中不可或缺的信号分子。本项目研究成果进一步丰富了拮抗酵母诱导果蔬采后抗病性的作用机理，并为深入研究抗病信号转导途径奠定了理论基础，有助于加速生防制剂的商业化应用。