辣椒溶杆菌全基因组序列特征及其pks-nrps基因簇的初步功能分析
发布时间：2018-12-18 00:07
【摘要】：辣椒溶杆菌(Lysobacter capsici)X2-3是本实验室从小麦根际土壤中分离到的一株溶杆菌菌株。研究发现该菌株对多种病原真菌及卵菌具有很强的拮抗活性,说明其在农业的生物防治方面具有广阔的应用前景。为了解该菌基因组的序列特征,及与抗真菌作用相关基因(簇)的结构和功能特点,本研究利用高通量测序的方法,获取了该菌的全基因组序列,利用生物信息学方法对基因组整体特征及成分进行了初步分析,并且对其中存在的抗真菌物质的基因簇进行了序列分析和功能验证,获得了pks-nrps基因簇内部片段插入突变体,并对突变体进行了拮抗活性的检测。本文的研究结果如下:1、本研究采用Illumina高通量测序技术,对L.capsici X2-3的进行了全基因组测序。通过双端测序共获得5,931,492个读长,经拼接后,产生13个contigs,组装成3个scaffords,基因组大小为6,126,365 bp,测序平均深度为285倍,GC含量为66.79%。使用GeneMarkS软件共预测到5,117个基因,利用GO、KEGG、COG、NR等数据库分别对预测基因进行了注释和代谢通路分析。GO数据库注释结果显示,参与催化活性、代谢进程、细胞进程及结合功能的基因在数量上占有绝对优势;KEGG数据库注释结果表明,代谢涉及的基因数量最多,其次是参与环境信息处理和遗传信息处理涉及的基因,生物体系统基因数量所占的比例最少;COG数据库的功能分类结果表明,参与代谢的蛋白数目最多,其次是参与细胞进程与信号转导及信息存储与处理的蛋白。2、分析了L.capsici X2-3基因组中非核糖体多肽合成酶基因簇的序列。通过序列比对发现,基因组中共存在6个PKS(polyketide synthases)/NRPS(nonribosomal peptide synthetases)基因簇,使用软件PKS-NRPS Analysis对基因组中的PKS/N RPS基因簇进行了模块划分及结构域分析,使用软件NRPS predicor对所有NRPS模块的A结构域进行了底物特异性预测。3、利用重组载体pEX18GM通过同源重组的方法,获得了pks-nrps基因簇的内部片段插入突变体,并对突变体进行了拮抗活性检测。检测结果显示,插入位点位于结构域KS及A内部的突变体完全丧失了对真菌及卵菌的拮抗活性,而插入点位于结构域之间的突变体保持与野生型相同的拮抗活性,这表明,该基因簇负责催化抗真菌物质的生物合成。
[Abstract]:Lysobacillus capsici (Lysobacter capsici) X 2-3 was isolated from wheat rhizosphere soil in our laboratory. It was found that the strain had strong antagonistic activity against a variety of pathogenic fungi and oocytes, which indicated that the strain had a broad application prospect in the biological control of agriculture. In order to understand the sequence characteristics of the bacteria genome and the structural and functional characteristics of the genes (clusters) associated with antifungal action, the whole genome sequence of the fungus was obtained by high-throughput sequencing. Bioinformatics method was used to analyze the overall characteristics and components of the genome. The gene clusters of antifungal substances were sequenced and functional verified. The internal fragments of pks-nrps gene cluster were inserted into mutants. The antagonistic activity of the mutant was tested. The results are as follows: 1. The whole genome of L.capsici X2-3 was sequenced by Illumina high throughput sequencing technique. A total of 5931492 reading lengths were obtained by two-terminal sequencing. After splicing, 13 contigs, were assembled into 3 scaffords, genomes with an average depth of 285 times and GC content of 66.79%. A total of 5117 genes were predicted by GeneMarkS software. The predicted genes were annotated and metabolic pathways were analyzed by using GO,KEGG,COG,NR and other databases. The results of GO database annotation showed that 5117 genes were involved in the process of catalytic activity and metabolism. The genes of cell progression and binding function have the absolute advantage in quantity. The results of KEGG database annotation showed that the number of genes involved in metabolism was the most, followed by the genes involved in environmental information processing and genetic information processing, and the proportion of genes involved in biological systems was the least. The results of functional classification of COG database showed that the number of proteins involved in metabolism was the most, followed by protein. 2, which was involved in cell process and signal transduction and information storage and processing. The sequence of non ribosomal polypeptide synthase gene cluster in L.capsici X 2-3 genome was analyzed. Six PKS (polyketide synthases) / NRPS (nonribosomal peptide synthetases) gene clusters were found in the genome by sequence alignment. The PKS/N RPS gene clusters in the genome were divided into modules and analyzed by using software PKS-NRPS Analysis. The substrate specificity of A domain of all NRPS modules was predicted by software NRPS predicor. By means of homologous recombination of recombinant vector pEX18GM, the internal fragments of pks-nrps gene cluster were inserted into mutants. The antagonistic activity of the mutant was tested. The results showed that the mutants with insertion sites located within the domain KS and A completely lost the antagonistic activity against fungi and oocytes, while the intergenic mutants with insertion sites between the domains maintained the same antagonistic activity as the wild type. The gene cluster is responsible for the biosynthesis of antifungal substances.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S476
本文编号：2384979
[Abstract]:Lysobacillus capsici (Lysobacter capsici) X 2-3 was isolated from wheat rhizosphere soil in our laboratory. It was found that the strain had strong antagonistic activity against a variety of pathogenic fungi and oocytes, which indicated that the strain had a broad application prospect in the biological control of agriculture. In order to understand the sequence characteristics of the bacteria genome and the structural and functional characteristics of the genes (clusters) associated with antifungal action, the whole genome sequence of the fungus was obtained by high-throughput sequencing. Bioinformatics method was used to analyze the overall characteristics and components of the genome. The gene clusters of antifungal substances were sequenced and functional verified. The internal fragments of pks-nrps gene cluster were inserted into mutants. The antagonistic activity of the mutant was tested. The results are as follows: 1. The whole genome of L.capsici X2-3 was sequenced by Illumina high throughput sequencing technique. A total of 5931492 reading lengths were obtained by two-terminal sequencing. After splicing, 13 contigs, were assembled into 3 scaffords, genomes with an average depth of 285 times and GC content of 66.79%. A total of 5117 genes were predicted by GeneMarkS software. The predicted genes were annotated and metabolic pathways were analyzed by using GO,KEGG,COG,NR and other databases. The results of GO database annotation showed that 5117 genes were involved in the process of catalytic activity and metabolism. The genes of cell progression and binding function have the absolute advantage in quantity. The results of KEGG database annotation showed that the number of genes involved in metabolism was the most, followed by the genes involved in environmental information processing and genetic information processing, and the proportion of genes involved in biological systems was the least. The results of functional classification of COG database showed that the number of proteins involved in metabolism was the most, followed by protein. 2, which was involved in cell process and signal transduction and information storage and processing. The sequence of non ribosomal polypeptide synthase gene cluster in L.capsici X 2-3 genome was analyzed. Six PKS (polyketide synthases) / NRPS (nonribosomal peptide synthetases) gene clusters were found in the genome by sequence alignment. The PKS/N RPS gene clusters in the genome were divided into modules and analyzed by using software PKS-NRPS Analysis. The substrate specificity of A domain of all NRPS modules was predicted by software NRPS predicor. By means of homologous recombination of recombinant vector pEX18GM, the internal fragments of pks-nrps gene cluster were inserted into mutants. The antagonistic activity of the mutant was tested. The results showed that the mutants with insertion sites located within the domain KS and A completely lost the antagonistic activity against fungi and oocytes, while the intergenic mutants with insertion sites between the domains maintained the same antagonistic activity as the wild type. The gene cluster is responsible for the biosynthesis of antifungal substances.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S476
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