一种新型抗菌肽基因决定簇的克隆与序列结构特征
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摘要
侧孢短芽孢杆菌(Brevibacillus laterosporus)S62-9菌株产生的抗菌肽BBL属于非核糖体合成肽,其生物合成涉及到一系列相关基因。为了获得完整的抗菌肽BBL基因决定簇,本研究基于部分已知的NRPS-A结构域基因序列,以S62-9菌株质粒DNA为模板,采用TAIL-PCR技术对NRPS-A结构域的上游进行扩增,得到一个大小为2.74kb的序列,该序列包含NRPS-A的5′末端序列、pp-binding序列以及1个ABC转运蛋白基因。该序列进一步与已知序列进行序列拼接及软件分析比对,最终获得一个大小为22.2kb的基因簇,该基因簇包含13个Bbl功能结构域,根据功能该基因簇分为3类功能相关基因:一类是编码转运相关蛋白,BblE包含3个ABC转运蛋白,负责抗菌肽向胞外渗透运输;二是催化短肽合成基因,Bbl F包含了pp-binding结构域和NRPS-A结构域,与氨基酸的装载和缩合有关,负责短肽的合成;三是肽链修饰相关基因,Bbl D、Bbl E、Bbl M、Bbl K等基因负责肽的修饰。
Antimicrobial peptide BBL produced by Brevibacillus laterosporus S62-9 strain belongs to non-ribosomal synthesis peptide,the synthesis of which involves a range of related genes.In order to get the complete antimicrobial peptide gene cluster,the primers were designed based on the partially known NRPS-A domain gene sequence,and TAIL-PCR was carried out using plasmid DNA of S62-9 as a template.The results showed that a length of2.74 kb sequence was amplified,containing 5′-ter minal sequence of NRPS-A,the pp-binding sequence and one ABC transporter.This sequence was further aligned with the known sequence and analyzed by software,and a size of 22.2 kb gene cluster containing 13 Bbl domains was obtained,which was classified into 3 functional groups:groupⅠ(Bbl E)encoded the related transport proteins,containing 3 ABC transporters,which were responsible for transmembrane transport of BBL;groupⅡ(Bbl F)included a PP-binding domain and a NRPS-A domain,and were responsible for the synthesis of BBL;groupⅢ(Bbl D、Bbl E、Bbl M、Bbl K Etc.)played an important role in the modification for BBL.
Antimicrobial peptide BBL produced by Brevibacillus laterosporus S62-9 strain belongs to non-ribosomal synthesis peptide,the synthesis of which involves a range of related genes.In order to get the complete antimicrobial peptide gene cluster,the primers were designed based on the partially known NRPS-A domain gene sequence,and TAIL-PCR was carried out using plasmid DNA of S62-9 as a template.The results showed that a length of2.74 kb sequence was amplified,containing 5′-ter minal sequence of NRPS-A,the pp-binding sequence and one ABC transporter.This sequence was further aligned with the known sequence and analyzed by software,and a size of 22.2 kb gene cluster containing 13 Bbl domains was obtained,which was classified into 3 functional groups:groupⅠ(Bbl E)encoded the related transport proteins,containing 3 ABC transporters,which were responsible for transmembrane transport of BBL;groupⅡ(Bbl F)included a PP-binding domain and a NRPS-A domain,and were responsible for the synthesis of BBL;groupⅢ(Bbl D、Bbl E、Bbl M、Bbl K Etc.)played an important role in the modification for BBL.
引文
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[21]Pavlidou M,Pross E K,Musiol E M,et al.The phosphopantetheinyl transferase KirP activates the ACP and PCP domains of the kirromycin NRPS/PKSof Streptomyces collinus Tü365[J].Fems Microbiology Letters,2011,319(1):26-33.
[22]肖长清,周洁.乳链菌肽Nisin的生物合成及表达调控机制[J].生物学杂志,2010,27(5):88-90.
[2]郑宗明,顾晓波,俞海青,等.非核糖体肽合成酶主要结构域的研究进展[J].中国抗生素杂志,2005,30(2):120-124.
[3]肖吉,张光涛,朱义广,等.海洋微生物次级代谢产物生物合成的研究进展[J].中国抗生素杂志,2012,37(4):241-253.
[4]Marahiel M A.A structural model for multimodular NRPS assembly lines[J].Natural Product Reports,2016,33(2):136-140.
[5]Tokuoka M,Seshime Y,Fujii I,et al.Identification of a novel polyketide synthase-nonribosomal peptide synthetase(PKS-NRPS)gene required for the biosynthesis of cyclopiazonic acid in Aspergillus oryzae[J].Fungal Genetics&Biology,2008,45(12):1608-1615.
[6]Seshime Y,Juvvadi P R,Tokuoka M,et al.Functional expression of the Aspergillus flavus,PKS-NRPS hybrid CpaA involved in the biosynthesis of cyclopiazonic acid[J].Bioorganic&Medicinal Chemistry Letters,2009,19(12):3288-3292.
[7]杨倩,于宏伟,郭润芳,等.侧孢短芽孢杆菌S62-9产抗菌物质的分离纯化及部分特性的研究[J].河北农业大学学报,2010,33(2):74-78.
[8]张楠,刘洋,于宏伟,等.含抗菌肽基因的天然质粒在枯草杆菌中的表达[J].食品科学,2017,38(8):24-29.
[9]张楠,刘洋,柯晓静,等.侧孢短芽孢杆菌S62-9抗菌肽基因的初步定位[J].河北农业大学学报,2016,39(5):82-86.
[10]Jia X,Lin X,Chen J.Linear and exponential TAIL-PCR:a method for efficient and quick amplification of flanking sequences adjacent to Tn5transposon insertion sites[J].Amb Express,V2017,7(1):195.
[11]戴宝新,冯惠勇,李天明,等.利用TAIL-PCR克隆Gluconobacter suboxydans葡萄糖脱氢酶基因及其生物信息学分析[J].食品科学,2014,35(1):194-198.
[12]Zhong X,Tian Y,Niu G,et al.Assembly and features of secondary metabolite biosynthetic gene clusters in Streptomyces ansochromogenes[J].Scientia Sinica Vitae,2013,56(7):609-618.
[13]朴玉华,徐彦胜,胡国全.改进TAIL-PCR方法克隆产甲烷古菌的mcr基因簇[J].应用与环境生物学报,2013,19(2):356-359.
[14]杨立勇,刘灶长,周立国,等.高效、新T-DNA侧翼序列分离技术-Actail-PCR[J].中国农业科学,2009,42(4):1447-1451.
[15]Choi J,Jeon J,Lee Y H.Identification of T-DNA integration sites:TAIL-PCR and sequence analysis[J].Fungal Biology,2015:217-222.
[16]Amsaveni R,Sureshkumar M,Aravinth A,et al.Production of Non-Ribosomal Peptide Synthetase(NRPS)-Dependent Siderophore by Aeromonas Isolates[J].Iranian Biomedical Journal,2016,20(4):235-240.
[17]王刚.生防菌淡紫拟青霉的基因组及其抗生素leucinostatins生物合成途径的研究[D].北京:中国农业科学院,2016.
[18]潘海学,唐功利.非核糖体肽合成酶催化的非常规装配模式[J].微生物学通报,2013,40(10):1783-1795.
[19]房耀维,刘姝,王淑军,等.海洋稀有放线菌Salinispora arenicola CNP193基因组新颖PKS和NRPS基因簇的发掘[J].海洋科学,2014,38(12):48-57.
[20]黎志凤,Etienne,Nguimbi,等.埃博霉素(Epothilones)的PKS/NRPS杂合基因簇[J].生物工程学报,2003,19(5):511-515.
[21]Pavlidou M,Pross E K,Musiol E M,et al.The phosphopantetheinyl transferase KirP activates the ACP and PCP domains of the kirromycin NRPS/PKSof Streptomyces collinus Tü365[J].Fems Microbiology Letters,2011,319(1):26-33.
[22]肖长清,周洁.乳链菌肽Nisin的生物合成及表达调控机制[J].生物学杂志,2010,27(5):88-90.