【摘要】：辣椒溶桿菌(Lysobacter capsici)X2-3是本實驗室從小麥根際土壤中分離到的一株溶桿菌菌株。研究發(fā)現(xiàn)該菌株對多種病原真菌及卵菌具有很強的拮抗活性,說明其在農業(yè)的生物防治方面具有廣闊的應用前景。為了解該菌基因組的序列特征,及與抗真菌作用相關基因(簇)的結構和功能特點,本研究利用高通量測序的方法,獲取了該菌的全基因組序列,利用生物信息學方法對基因組整體特征及成分進行了初步分析,并且對其中存在的抗真菌物質的基因簇進行了序列分析和功能驗證,獲得了pks-nrps基因簇內部片段插入突變體,并對突變體進行了拮抗活性的檢測。本文的研究結果如下:1、本研究采用Illumina高通量測序技術,對L.capsici X2-3的進行了全基因組測序。通過雙端測序共獲得5,931,492個讀長,經(jīng)拼接后,產(chǎn)生13個contigs,組裝成3個scaffords,基因組大小為6,126,365 bp,測序平均深度為285倍,GC含量為66.79%。使用GeneMarkS軟件共預測到5,117個基因,利用GO、KEGG、COG、NR等數(shù)據(jù)庫分別對預測基因進行了注釋和代謝通路分析。GO數(shù)據(jù)庫注釋結果顯示,參與催化活性、代謝進程、細胞進程及結合功能的基因在數(shù)量上占有絕對優(yōu)勢;KEGG數(shù)據(jù)庫注釋結果表明,代謝涉及的基因數(shù)量最多,其次是參與環(huán)境信息處理和遺傳信息處理涉及的基因,生物體系統(tǒng)基因數(shù)量所占的比例最少;COG數(shù)據(jù)庫的功能分類結果表明,參與代謝的蛋白數(shù)目最多,其次是參與細胞進程與信號轉導及信息存儲與處理的蛋白。2、分析了L.capsici X2-3基因組中非核糖體多肽合成酶基因簇的序列。通過序列比對發(fā)現(xiàn),基因組中共存在6個PKS(polyketide synthases)/NRPS(nonribosomal peptide synthetases)基因簇,使用軟件PKS-NRPS Analysis對基因組中的PKS/N RPS基因簇進行了模塊劃分及結構域分析,使用軟件NRPS predicor對所有NRPS模塊的A結構域進行了底物特異性預測。3、利用重組載體pEX18GM通過同源重組的方法,獲得了pks-nrps基因簇的內部片段插入突變體,并對突變體進行了拮抗活性檢測。檢測結果顯示,插入位點位于結構域KS及A內部的突變體完全喪失了對真菌及卵菌的拮抗活性,而插入點位于結構域之間的突變體保持與野生型相同的拮抗活性,這表明,該基因簇負責催化抗真菌物質的生物合成。
[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.
【學位授予單位】：山東農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S476

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