Cloning And Bioinformatic Analysis Of The Marine Actinomycetes NRPS Gene

Nature microorganisms especially actinomycetes can synthesize polypeptide secondary metabolites with medicinal value by non-ribosomal pathways.Nonribosomal peptide synthetases(NRPS)catalyte the biosynthesis of ribosomal peptide and NRPS gene often exists in the secondary metabolites gene clusters.NRPS,a kind of multifunctional protein complex,is responsible for the specific recognition,activation and translocation of amino acid substrate,then condensate them in specific order to form non-ribosomal peptide.Domain A is responsible for the identification of amino acid activation and domain T is just like a carrier,and domain C is responsible for the synthesis of peptide bonds.So studies on the structure of different domain of NRPS gene will lay a foundation for the researches on secondary metabolites biosyntic gene cluster.In this experiment,five marine actinomycetes were isolated and one of them exhibited high bacteriostatic effect and presumpted L1 as Streptomyces globisporu though physi-biochemical and molecular biology identification.Two pairs of primers were designed and used to amplify the domain C and domain A sequences of non-ribosomal peptide synthetase genes.The parameters of the PCR amplification were optimized and the optimal annealing temperature for domain C gene was 60 ?,the amplified fragments N1 was 414 bp.The optimal annealing temperature for domain A gene was 63 ?,the amplified fragment A1 was 715 bp.Bioinformational analysis of N1 showed that its similarity with the non-ribosomal peptide synthetase(sequence ID:WP 46386756.1)was 58%.Blastx alignment found that the deduced amino acid sequence contained a condensation superfamily core binding region which was a unique region for NRPS catalytic peptide bond formation.Sequence analysis of A1 fragment showed that its similarity with the non-ribosomal peptide synthetase(sequence ID:WP 46386756.1)was 99%.Blastx alignment found that the deduced amino acid sequence contained an AFD_class_I superfamily core region,which was responsible for the catalytic formation of ATP.It showed that the amplified target fragments were the C domain and the A domain gene sequence of the non-ribosomal peptide synthetase gene.