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 (50μL·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 Ⅲslowed 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.
国家科技期刊平台
NSTL
万方数据
维普
