The Genetic Analysis Of Plantaricin Locus Of Lactobacillus Plantarum And Heterologous Expression Of Genes PlnEF

Lactobacillus plantarum can survive the harsh winemaking conditions to conduct malolactic fermentation in winemaking process.It is capable of producing plantaricin to inhibit or kill the undesirable Lactic acid bacteria in wine.Meanwhile,the plantaricin could inhibit the spoilage in wine thus reducing the use of sulfur dioxide.Therefore,it can be significant to screen the plantaricin-producing Lactobacillus plantarum strains in future.In this paper,firstly,through the induction of co-culture in MRS broth culture,Lactobacillus plantarum XJ25 and PC520 strains produced bacteriocin successfully,of fourteen experimental strains.Secondly,based on the sequencing draft genome,the plantaricin(pln)loci of the strains XJ25 and PC520 were studied and compared genetically to get the genetic maps of the plantaricin.Finally,to obtain the recombinant proteins in vitro experiment,the expression vectors for genes pln E and pln F were constructed successfully.Moreover,some purified methods were served for the purification of the related proteins.The result showed that:1)Of fourteen Lactobacillus plantarum strains,the range of inhibitive zone of strains XJ25 and PC520 was wide,and the great inhibition effect of these strains was showed.The feature of co-culture in L.plantarum has been common.It has been displayed that these two strains were capable of producing bacteriocin via the inductive effect of Enterococcus faecalis XJ26 M which was important to induce the production of plantaricin as the environmental factor.It was showed that these seven strains,through 16 S r RNA and rec A genes PCR amplification,were identified as L.plantarum,moreover the analysis of RAPD of these different twelve random PCR fragments has showed the polymorphism for the genome of these strains.2)It has been found that the structures of the plantaricin locus of these two strains were similar,consisting of six operons-pln EFI,pln JKLR,pln GHSTUVW,pln ABCD,pln MNOP,pln WXY.The regulatory operon had three components– pln ABCD.Meanwhile,the novel orf1 with unknown function has been found in these two strains.However,pln Y and pln S genes were missing in the PC520 pln locus,and mutations of two amino acids(A-S,V-I)were detected at sites 14 and 42,respectively,on the protein encoded by XJ25 pln F gene.3)Via the one step cloning technique,the primers were designed to insert the DNA fragments successfully with high transformation efficiency.The optimal condition has been found for the induction of recombinant protein,namely,18 ?,0.6 m M IPTG,12 h,for the extensive culturing of these strains.4)After the AKTA protein purification analysis and ultra-filtration treatment to eliminate extra proteins,these protein concentrations ranged from 600-1000 ng/ul,which need further purification treatments.After purified,they displayed the inhibitive activity,and the proteins Pln E and Pln F may interact with different antibacterial effect.