黏质沙雷氏菌次生代谢物对TMV的抑制机理  

Secondary Metabolites Produced by Serratia marcescens as an Inhibitor Against TMV

作　　者：毕建华[1] 杨金广[1] 欧阳明安[2] 李锡宏 张长华 申莉莉[1] 钱玉梅[1] 王盼[1] 王升平[1] 王凤龙[1] 

机构地区：[1]烟草行业烟草病虫害监测与综合治理重点实验室/中国农业科学院烟草研究所,山东青岛266101 [2]福建农林大学植物病毒研究所,福州350000 [3]湖北省烟草科学研究所,武汉430030 [4]贵州省烟草公司遵义市公司,贵州遵义563000

出　　处：《中国农业科学》2014年第5期912-922,共11页Scientia Agricultura Sinica

基　　金：国家烟草专卖局重点项目(TS-06-20110039);湖北省烟草公司科技重点项目(027Y2013-006)

摘　　要：【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(50μL·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,【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-t

关 键 词：灵菌红素 黏质沙雷氏菌 烟草花叶病毒 拮抗作用 抑制机理 

分 类 号：S476[农业科学—农业昆虫与害虫防治]