黏质沙雷氏菌次生代谢物对TMV的抑制机理

【目的】分离、纯化和鉴定黏质沙雷氏菌2A2次生代谢物中具有抑制TMV侵染力的活性成分，明确其对TMV的抑制机理。【方法】通过病毒生物学测定，确定黏质沙雷氏菌2A2菌株对TMV的抑制效率。为明确黏质沙雷氏菌2A2发酵液次生代谢物中抑制TMV侵染力的活性成分，通过TLC和硅胶柱层析对其发酵液进行了分离纯化，获得主要的次生代谢物，通过局部枯斑法测定各代谢物的生物学活性，筛选可显著抑制TMV侵染活性的物质，经NMR分析，确定其成分结构，进而确定物质组分。为进一步揭示代谢物对TMV的抑制机理，利用透射电子显微镜探索代谢物对TMV粒体形态的影响，TMV粒体与代谢物甲醇溶液混合30 min后，于透射电子显微镜下观察TMV粒体形态。同时，用代谢物甲醇溶液喷施处理寄主下部3片叶，共设5个处理：处理Ⅰ，喷施代谢物24 h后，接种TMV；处理Ⅱ，代谢物与等体积TMV汁液混合30 min后，接种叶片；处理Ⅲ，先接种TMV，24 h后喷施代谢物；处理Ⅳ，宁南霉素（50 μL•mL-1）与等体积TMV汁液混合30 min后，接种下部叶3片做阳性对照；处理Ⅴ，无菌水与等体积TMV汁液混合30 min后，接种下部叶3片做空白对照。分别在次生代谢物诱导和TMV侵染后1、3、5、7、9 d，取未经任何处理的上部叶，TRIZOL法快速提取总RNA并反转录cDNA，利用real-time RT-PCR分析样品PR基因和TMV的RNA相对表达量，进而明确代谢物处理寄主对寄主PR基因表达的影响和对TMV在寄主体内复制增殖的抑制作用。【结果】黏质沙雷氏菌2A2菌株对TMV具有显著的抑制作用。对其发酵液进行分离纯化，获得3种主要代谢物，通过局部枯斑法测定，编号为BJH-2的代谢物可显著抑制TMV的侵染活性，经NMR分析，确定了该代谢物为灵菌红素（prodigiosin）。透射电子显微镜结果显示灵菌红素可显著破坏TMV粒体，使TMV典型的杆状病毒粒体降解断裂成排列紊乱的短杆状。real-time RT-PCR结果表明，灵菌红素处理植株后，处理Ⅰ、处理Ⅱ、处理Ⅲ可诱导寄主体内PR基因表达随着时间逐渐上调，第7 天左右，三者处理的寄主体内PR1、PR2、PR4、PR5表达上调至高峰，显著高于空白对照，亦高于处理Ⅳ的阳性对照，从而提高系统抗性；灵菌红素处理植株后，处理Ⅰ、处理Ⅱ、处理Ⅲ寄主体内TMV RNA的表达量随着时间上调减缓，在接种TMV后的第9天，三者处理的寄主体内的TMV RNA含量分别是空白对照的11.98%、5.23%、15.90%，显著低于空白对照，亦低于处理Ⅳ的阳性对照。【结论】灵菌红素既对TMV在寄主体内的复制增殖具有抑制作用，又可诱导寄主产生系统抗性，整体效果高于宁南霉素，可作为防治植物病毒新的生物制剂。

Secondary Metabolites Produced by Serratia marcescens as an Inhibitor Against TMV

【Objective】The objective of this study is to separate and purify the secondary metabolites produced by Serratia marcescens and identify the active constituent significantly against TMV (Tobacco mosaic virus), and to reveal the inhibition mechanism. 【Method】 The inhibition activity of S. marcescens 2A2 against TMV was identified by using virus biology measurement. In order to indicate the antiviral components of secondary metabolites, the fermentation liquor of S. marcescens 2A2 was separated and purified with TLC (thin layer chromatography) and silica gel column chromatography, and the main secondary metabolites were acquired. The secondary metabolite having significantly inhibitory effect on TMV infection was screened by local lesion method, its constituent structure was analyzed with NMR (nuclear magnetic resonance), and then the material composition was determined. In order to reveal the inhibition mechanism of metabolite inhibiting TMV, the effect of metabolite on TMV morphology was observed under transmission electron microscope (TEM). TMV particles were mixed with the metabolite methanol solution for 30 minutes before the TEM observation. Meanwhile, the metabolite methanol solution was used to spray three lower leaves of inoculated tobacco and five different treatments were set as follows:Ⅰ, inoculating TMV 24 h after metabolite spraying; Ⅱ, mixing metabolite with isopyknic TMV for 30 minutes before inoculation; Ⅲ, inoculating TMV 24 h before metabolites spraying; Ⅳ, mixing ningnanmycin (50 μL•mL-1) with isopyknic TMV for 30 minutes before inoculating three lower leaves and using this as the positive control; Ⅴ, mixing sterile water with isopyknic TMV for 30 minutes before inoculating the three lower leaves and using this as blank control. The upper tobacco leaves without any treatment were taken 1, 3, 5, 7 and 9 days after secondary metabolite induction and TMV inoculation, and total RNA was extracted using the TRIZOL method and reverse transcribed to cDNA. Real-time PCR assay was used to analyze PR gene of sample and TMV RNA expression, furthermore to make clear the effect of metabolite treatment on PR gene family of host and the inhibition effect on TMV multiplication.【Result】S. marcescens 2A2 had a significant inhibitory effect against TMV. Three main secondary metabolites were acquired through separating and purifying the fermentation liquor. The secondary metabolite named as BJH-2 showed a significantly inhibitory effect on TMV infection, and then BJH-2 metabolite was determined as prodigiosin with NMR. Observation results of transmission electron microscope indicated that prodigiosin significantly damaged TMV particles and broken the typical rod-shaped viral particles into disorganized short rod-shaped ones. RT-PCR showed that treatmentsⅠ, Ⅱ and Ⅲ induced the up-regulation of PR gene expression in host along with the time after prodigiosin treatment, and on the 7th day, the expression of PR1, PR2, PR4 and PR5 was up-regulated to the peak which was significantly higher than that of blank control and also higher than that of the positive control. Meanwhile, treatmentsⅠ, Ⅱ and Ⅲ slowed down the up-regulation of TMV RNA expression in host over time after prodigiosin treatment. On the 9th day after TMV inoculation, TMV RNA contents of host in treatmentsⅠ, Ⅱ and Ⅲ were only 11.98%, 5.23% and 15.90% of the blank control, respectively, which were all significantly lower than that of the blank control and also lower than that of the positive control in treatment Ⅳ.【Conclusion】Prodigiosin can not only inhibit TMV replication in host but also induce host increasing system resistance, which is more effective than ningnanmycin. Therefore, prodigiosin can be used as a new biological agent to prevent and control plant virus.