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NRPS-A在侧孢短芽孢杆菌S62-9抗菌肽生物合成中的功能分析

标题: NRPS-A在侧孢短芽孢杆菌S62-9抗菌肽生物合成中的功能分析
英文标题: Functional Analysis of NRPS-A in the Biosynthesis of Antimicrobial Peptides from Brevibacillus Laterosporus S62-9
作者: 刘洋
出版时间: 2018-06-02
所在大学: 河北农业大学
关键词: 侧孢短芽孢杆菌,抗菌肽,非核糖体肽合成酶,NRPS-A,TAIL-PCR
英文关键词: Brevibacillus laterosporus,antibacterial peptide,non-ribosomal peptide synthetase,NRPS-A,TAIL-PCR
论文级别: 硕士
学位: 硕士
导师: 郭润芳
专业: 微生物学
主题: NRPS-A在侧孢短芽孢杆菌S62-9抗菌肽生物合成中的功能分析
页码: 67
摘要: 抗生素在被发现至今已被应用到生活的各个领域中,但抗生素的滥用也带来了一系列的问题,对于食品安全造成了严重的危害,因此对于新型的抗菌物质的寻找,以代替抗生素和解决抗生素残留问题是目前亟待解决的问题。由侧孢短芽孢杆菌(Brevibacillus laterosporus)S62-9菌株产生的抗菌肽BBL,具有抑菌谱广、耐酸碱性、耐高温性等优点,并且不易残留、抗药性产生几率小的优点,在食品防腐剂和饲料添加剂的方面具有广阔的应用前景。目前有关侧孢短芽孢杆菌抗菌肽的遗传信息、合成机制、免疫机制以及编码基因等方面相关的报道极少。侧孢短芽孢杆菌S62-9遗传信息定位在质粒上,已经完成了质粒的测序工作,得到了contigs。在contig4中发现了一个不完整的非核糖体肽合成酶NRPS-A基因。为了挖掘NRPS-A在抗菌肽BBL合成中的功能,本文通过TAIL-PCR技术获得NRPS-A的上游序列,通过基因簇序列分析和比对分析挖掘NRPS-A的功能以及其它功能基因,通过同源重组敲除的方式对NRPS-A基因进行敲除,验证该基因的功能。研究结果为探究NRPS-A基因在抗菌肽BBL合成中的功能提供基础,为编码基因的的解读与抗菌肽的合成途径的研究提供研究基础,也为利用基因工程的手段构建或改造抗菌肽BBL的高产菌株提供一个前提。现论文的主要研究结果如下:TAIL-PCR扩增非核糖体肽合成酶通过TAIL-PCR对侧孢短芽孢杆菌S62-9的质粒基因组中的非核糖体肽合成酶NRPS-A基因的上游序列进行扩增,得到了4条扩增条带。经过DNAMAN比对后得到1条2.74kb大小的序列。序列分析后得到了NRPS-A基因、pp-binding基因以及一个ABC基因;基因比对后,contig5和contig4拼接成了一个完整的基因决定簇。完整的A结构域基因,编码524个氨基酸;1个pp-binding基因编码83个氨基酸。生物信息学分析对拼接好的决定基因簇通过序列比对、同源性分析、底物特异性分析,基因簇分为合成、修饰、转运3个主要功能结构。同时将相关的基因进归类,分为合成结构BblG、转运结构BblF、以及其他的修饰结构。经过分析,合成相关结构由NRPS-A和pp-binding组成,属于一种非典型的非核糖体肽合成酶模块。NRPSA是非核糖体肽合成酶中的腺苷化结构域,其特异性底物为缬氨酸,构建了NRPS-A蛋白的三维构象,推测它在抗菌肽BBL合成中负责识别并活化特异性的氨基酸;ppbinding基因是聚酮合酶中的酰基载体蛋白,负责接受活化的酰化氨基酸;BblF功能域由1个ATP结合区域,2个跨膜区域组成的ABC转运蛋白系统,负责抗菌肽BBL的转运。NRPS-A敲除菌株的筛选与验证通过同源重组敲除的方法敲除NRPS-A基因的部分编码序列,随后的多步筛选得到了缺失突变体pAH-21和pAH-25。在PCR验证显示缺失突变体p AH-21和pAH-25已经完成敲除,敲除NRPS-A后菌株无抑菌圈的产生,说明NRPS-A基因与抗菌肽BBL的合成有直接关系。本文通过TAIL-PCR扩增得到一个非典型的非核糖体肽合成酶,分析了NRPS-A基因在抗菌肽BBL合成中的功能,基因敲除试验表明NRPS-A与抗菌肽BBL合成具有直接的关系,为抗菌肽BBL的合成途径与合成机理的研究提供基础。
英文摘要: Antibiotics have been widely applied to various fields of life so far,meanwhile,the abuse of antibiotics also has brought a range of problems,causing serious harm to food safety,so searching for new types of antibacterial substances to replace antibiotics and solving the problem of antibiotic residues needs to be solved urgently.The antibacterial peptide BBL produced by Brevibacillus laterosporus S62-9 has the advantages of wide spectrum of inhibition,acid and alkali resistance,and high temperature resistance.In addition,due to the less occurrence of residue and drug resistance,it shows a broad application prospects in terms of food preservatives and feed additives.At present,there are few reports on genetic information,synthetic mechanism,immune mechanism,and coding genes of Brevibacillus laterosporus antibacterial peptides.The fore research finding showed the genetic information of Brevibacillus laterosporus S62-9 was located on the plasmid,and some contigs were obtained from the sequencing of the plasmid,and an incomplete non-ribosomal peptide synthetase NRPS-A gene was found in contig4.In order to understand the function of NRPS-A in the synthesis of antimicrobial peptide BBL,the upstream sequence of NRPS-A was amplified by TAIL-PCR.The functions of NRPS-A and other genes were predicted and analyzed by gene clustering and alignment.Knocking out the NRPS-A gene by homologous recombination verified the function of this gene.The research results provide the proof for exploring the function of NRPS-A gene in the synthesis of antibacterial peptide BBL,and provide basis for the interpretation of the encoded gene and the synthesis pathway of antibacterial peptide,and,and also provide a prerequisite for the high yield strains by genetically reconstructing antibacterial peptide BBL.The main research findings of this paper are as follows.TAIL-PCR amplification of non-ribosomal peptide synthetase The upstream sequence of the non-ribosomal peptide synthetase NRPS-A gene in the plasmid genome of Brevibacillus laterosporus S62-9 was amplified by TAIL-PCR to obtain four amplified bands.After DNAMAN alignment,a 2.74 kb size sequence was obtained.After sequence analysis,the NRPS-A gene,pp-binding gene and an ABC gene were found.After gene alignment,contig5 and contig4 were spliced into a complete gene determinant.An entire NRPS-A domain encodes 524 amino acids,and a pp-binding gene encodes 83 amino acids.Bioinformatics Analysis The spliced determinant gene clusters were divided into three major functional domains: synthesis,modification,and transport through sequence alignment,homology analysis,and substrate specificity analysis.At the same time,related genes were classified into synthetic gene BblG,transporter gene BblF,and other modified genes.After analysis,the synthesis-related genes consisted of NRPS-A and pp-binding,which belonged to an atypical non-ribosomal peptide synthetase module,NRPS-A is an adenylation domain in non-ribosomal peptide synthetase,its specific substrate is Valine,and its three-dimensional conformation of NRPS-A protein was constructed and speculated that it is responsible for recognition and activation specific amino acids in the synthesis of antimicrobial peptide BBL.The pp-binding gene is an acyl carrier protein in polyketide synthase and responsible for the activated acylated amino acid.BblF gene consists of an ATP binding region,two transmembrane regions of ABC transport system,responsible for the transport of antimicrobial peptide BBL.Screening and validation of NRPS-A knockout strains Partial coding sequence of NRPS-A gene was knocked out by homologous recombination knockout,followed by multiple-step screening to obtain deletion mutants pAH-21 and pAH-25.The PCR verification showed that the deletion mutant pAH-21 and pAH-25 had already been knocked out the target gene.No inhibition zone surrounding the mutant colony indicated that the synthesis of NRPS-A gene had a direct relationship with the synthesis of antimicrobial peptide BBL.In this paper,a non-typical non-ribosomal peptide synthetase was amplified by TAILPCR,and the function of NRPS-A gene in the synthesis of antibacterial peptide BBL was analyzed.Knockout test showed that NRPS-A has direct synthesis with antibacterial peptide BBL.The relationship provides a basis for the study of the synthesis pathway and synthesis mechanism of antimicrobial peptide BBL.