| Soil microbial diversity is an important factor affecting the growth of traditional chines medicinal materials,and the phenolic acid exudes during the continuous cropping of medicinal materials have significant impact of microbial diversity in rhizosphere soil.Coptis chinensis is a widely used traditional chinese medicinal material.However,the continuous cropping obstacle seriously limited the yield and quality of Coptis chinensis.The concentration of phenolic acid such as ferulic acid,p-coumaric acid and protocatechuic acid in the planted soil is increasing year by year.The accumulation of phenolic acid is considered to be the main cause of the continuous cropping disorder of Coptis chinensis.However,the effect of phenolic acid on soil microbial diversity in soil of Coptis chinensis is still unclear.With the application of high-throughput sequencing technology in soil microbial diversity research,it is possible to systematically analyze changes in soil microorganism and environmental factors.In addition,the phenolic acid degradation process in the natural environment is very slow.Screening high-efficiency phenolic acid degradation strains from Coptis chinensis rhizosphere soil and elucidating its degradation mechanism will lay foundation for the biodegradation of phenolic acids and the restoration of microbial diversity.Objective:In this study,Illumina high-throughput sequencing technology was used to analyze the changes of microbial community structure about the rhizosphere soil of Coptis chinensis in different planting years and to study the effects of planting Coptis chinensis on microbial diversity and soil physicochemical property.At the same time,combined with the culture method,the microbes in the soil of Coptis chinensis were isolated and the phenolic acid degradation strains were screened.The phenolic acid degradation pathway and related genes were explored by genomic sequence analysis.Methods:1.The soil physicochemical indicators were detected and the gDNA of the rhizosphere soil of Coptis chinensis was extracted,the 16S/ITS amplicons were subjected to high-throughput sequencing.Clustering analysis,alpha diversity analysis,beta diversity analysis,and correlation analysis between microbes and environmental factors were carried out to analyze the dynamic changes of soil microbial community structure under different planting patterns and planting years.2.The microbes in the rhizosphere soil of Coptis chinensis were separated by culture method,and the ferulic acid was used as the sole carbon source for screening.Microbes which can degradating ferulic acid were screened and identified.The degradation ability of these strains of phenolic acid was further tested by high performance liquid chromatography,and the effects of substrate type,carbon source concentration,culture time and soil environment on ferulic acid drop were tested.3.The second-generation sequencing technology was used to determine the genome map of the degradation microbe S.olivochromogenes A035,and BLAST was used to analyze the phenolic acid degradation related genes.Quantitative PCR was used to detect the expression of ferulic acid degradation genes in this strain,and the protein encoded by fcs/ech was analyzed in other species of Streptomyces.sp.4.The fcs/ech knockout strain of S.olivochromogenes A035 was constructed to determine its role in the catabolism of ferulic acid.The Ferulyl-CoA synthetase(FCS)and Enoyl-CoA hydratase/aldolase(ECHA)encoded by fcs/ech were heterologously expressed in E.coli,and the functions in vitor of these two key enzymes for degradation were determined.Result:1.The results of amplicon sequencing showed that the abundance of microorganism in rhizosphere soil increased significantly with the age of planting.The diversity of bacteria in the fifth year of continuous cropping was the highest,and the diversity of fungi decreased first year and then increased.The abundance of Acidobacteria,Mortierella and Ascomycota was enriched with increasing in continuous cropping.The ? diversity analysis showed that the microbial differentiation coefficient in the soil approached zero as the planting years of Coptis chinensis increased.Soil physicochemical indicators showed that the soil pH of Coptis chinensis was low,and the soil microbes were positively correlated with soil organic matter and alkali nitrogen content.The absolute abundance of ferulic acid degradation microbes,screened in this study,were highest in the fifth year in rhizosphere soil.2.A total of 14 phenolic acid degradation microbes were screened from the rhizosphere soil of Coptis chinensis.One strain of fungi with high-efficiency degradation of ferulic acid and 4 strains of actinomycetes were screened from the microorganisms isolated from the rhizosphere soil of Coptis chinensis.They were identified as Penicillium daleae?Streptomyces olivochromo?Streptomyces aurantiacus and Nocardia sp.by ITS and 16 S sequence,respectively.Next,the degradation characteristics of ferulic acid of Penicillium daleae F-0056 and Streptomyces olivochromogenes A035 were tested.When the amount of ferulic acid was 200?g/ml,the degradation rate of ferulic acid was 87.5% after 72 hours culture of P.daleae F-0056.In the soil environment,the degradation rate of P.daleae F-0056 on ferulic acid reached to 71.8% on the 6th day.And it was found that the strain S.olivochromogenes A035 grew less when cultured in cinnamic acid and benzoic acid,and grew most in vanillic acid and p-hydroxybenzoic acid.Then the degradation effect of ferulic acid in the soil which added S.olivochromogenes A035 was significantly better than control,ferulic acid in the soil was completely degraded on the 24 th days.3.Consistent with the broad-spectrum phenolic acid degradation ability of S.olivochromogenes A035,the genome of this strain contain genes encoding key enzyme of phenolic acid degradation pathway such as coenzyme A ligase,coenzyme A acetalase,benzoic acid dioxygenase,protocatechuic acid dioxygenase,etc.When S.olivochromogenes A035 was cultured for 12 h with ferulic acid as the sole carbon source,the relative expression of ferulic acid degradation genes was significantly up-regulated compared with that in glucose,and the fcs and ech were up-regulated 1033 times and 1250 times,respectively.4.The FCS was heterologously expressed in E.coli BL21.The in vitro enzyme activity assay was performed on the purified protein to obtain the transforming product feruloyl-CoA,which indicated that the fcs gene plays an important role in ferulic acid conversion.The FCS and ECHA,two enzymes were heterologously expressed in E.coli BL21,and the E.coli cells were sonicated to obtain a crude enzyme,which was able to decompose the substrate ferulic acid and produce the product vanillic acid.Indicated that the degradation of ferulic acid in S.olivochromogenes A035 is related to the two enzymes FCS/ECHA.Conclusion:The above results indicated that the soil of Coptis chinensis was acidic and the soil microbial composition was positively correlated with soil pH.The longer the planting time,the greater the influence on the microbial community structure in the soil,and the phenolic acid degrading microbes were enriched in the soil with longer planting years.The S.olivochromogenes A035,screened from rhizosphere soil,has a broad spectrum of phenolic acid degradation ability,which can efficiently degrade ferulic acid in soil environment.Ferulic acid significantly induced transcription of phenolic acid degradation genes in this strain,indicating that the strain degraded ferulic acid through the classical FCS/ECHA pathways.This study enriched the phenolic acid degradation microbes resource base and laid the foundation for the repair of Coptis chinensis continuous cropping obstacles. |
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