| Nicotine is the main alkaloids in tobacco plants,is also a kind of toxic substance can make the person produces dependency irreversible effects on the environment Through microbial degradation of nicotine is a kind of method of low cost high efficiency,no secondary pollution,so the biological degradation of nicotine technology to reduce the pollution of the nicotine reduce nicotine content Raise the utilization ratio of tobacco waste protection of water resources and ecological environment has important significance.The purpose of this study was to screen and isolate tobacco-degrading bacteria from the long-term tobacco-planting soil in Luoling Township,Luoning County,Henan Province,and to explore their degradation characteristics and degradation products.At the same time,whole-genome sequencing technology and bioinformatics analysis methods were used to understand related degradation genes and infer their degradation pathways,in order to provide new microbial resources for nicotine degradation and environmental pollution control.The main research results are as follows:1.In this study,a strain of nicotine-degrading bacteria was screened and isolated from the soil in Luolin Township,Luoning County,Henan Province.According to the results of morphological,physiological and biochemical identification,16S rRNA gene sequencing and comparison,it was identified as Agrobacterium tumefaciens and named Agrobacterium tumefaciens LN-1.2.High performance liquid chromatography(HPLC)was used to study the fermentation broth of Agrobacterium tumefaciens LN-1 with different degradation time.It was found that the new peak of Agrobacterium tumefaciens LN-1 occurred at 4.51 min and 5.72min at 16h,indicating that nicotine degraded and produced intermediate products.The nicotine absorption peak decreased at 20h,and the area of the new peak increased slightly compared with 16h.The absorption peak of nicotine decreased significantly at 28h,and the area of new peak increased significantly,indicating that the intermediate products were accumulating in the process of nicotine degradation.The absorption peak of nicotine basically disappeared at 30h,the area of new peak was basically unchanged compared with 28h,the content of metabolites was basically stable,and the degradation process of nicotine ended.HPLC results showed that Agrobacterium tumefaciens LN-1 was highly capable of degrading nicotine,which could completely degrade 1 g/L nicotine within 3 0h,and produced several different intermediate metabolites during the degradation process.The growth and degradation curves of Agrobacterium tumefaciens LN-1 showed that the growth of Agrobacterium tumefaciens LN-1 was synchronized with the degradation of nicotine,that is,the carbon and nitrogen sources and energy needed for its growth were obtained through the catabolism of nicotine.3.Four intermediate metabolites were detected by high performance liquid chromatography-mass spectrometry(LC-MS)in the fermentation broth of Agrobacterium tumefaciens LN-1 degrading nicotine,which were 6-hydroxy-3-succinylpyridine(HSP),6-hydroxy-nicotine(6HN)and 3-succinylpyridine(SP).4.In this study,the whole genome of Agrobacterium tumefaciens LN-1 was sequenced-by PacBio RSII platform and BGISEQ-500RS platform,and the genome of Agrobacterium tumefaciens LN-1 was bioinformatics analyzed.The results showed that the genome size of Agrobacterium tumefaciens LN-1 was 5,586,294bp,and GC content was 59.40%,including one ring chromosome(2,908,638 bp),one linear chromosome(2,260,658 bp)and two ring plasmids(233,332 bp,183,666 bp),and this arrangement was similar to that of other Agrobacterium tumefaciens.The genome contained 5404 Open Reading frames(ORFs)with a total length of 4,872.984 bp.The genome contained 130 repeats,including 54 tRNA,4 5SrRNA,4 16SrRNA,4 23SrRNA and 33 SRNA;Functional annotation results showed that the genes involved in biological regulation,cellular processes,metabolic processes,biological process regulation,cellular structural entities,DNA binding,catalytic activity,transporter activity,amino acid transport and metabolism,carbohydrate transport and metabolism,transcription and environmental information processing were more;In addition,a gene cluster was found between 662,156-693,086 bp on the linear chromosome of Agrobacterium tumefaciens LN-1.The gene annotation results and gene sequencing on the chromosome showed that the gene cluster contained hsh,pno,hpo NdhAB and other genes related to nicotine degradation,by analyzing the role of the gene cluster and related genesand combined with the degradation of LC-MS intermediate results,Agrobacterium tumefaciens LN-1 is known to degrade nicotine through a "cross pathway" of pyridine and pyrrolidine pathways(VPP).5.There is also a Para-hydroxybenzoic acid degradation gene cluster in the linear chromosome of Agrobacterium tumefaciens LN-1,which contains pca,pob,fad and other genes related to the degradation of p-hydroxybenzoic acid.It is speculated that Agrobacterium tumefaciens LN-1 can degrade para-hydroxybenzoic acid. |
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