Study Of Endophytic Bacteria Isolated From Peanut Seeds Inhibiting Aflatoxin By Molecular Biology Method

As a traditional method to study the identification of strains,16S rDNAsequencing is used widely, but because of its inherent limitation of low evolutionaryrate, it is difficult to distinguish strains when it’s affinity is close, thus it can notidentify strains at the species level. To solve this problem, our study combined the16S rDNA sequencing technology and the housekeeping gene gyrB analysis to givea comprehensive identification and diversity discuss of the experimental strains.Besides, the rep-PCR amplification of different strains belong to the same genuswas conducted, then its fingerprints structure was evaluated.In this research, seventy endophytic bacteria of peanut seeds from differentprovince were studied by using the polyphasic analysis of conbined16S rDNA andgyrB sequence. The result showed that there were forty-four strains belong toBacillus sp., one belong to Paenibacillus sp., sixteen belong to Serratia sp., eightbelong to Enterobacter sp., accounting for62.9%,1.4%,22.9%,11.4%,1.4%respectively. The study of twelve strains which belong Serratia sp. showed that the16S rDNA sequences similarity diversity range was91.7%to99.9%, the gyrB genewas88.0%to99.4%, the range of latter was wider than the former, having betterdistinctive ability. The evolution distance range of gyrB genes was0.000to0.119,the16S rDNA sequences was0.000-0.079, the former evolutionary distance waslarger than the latter. Despite16S rDNA sequencing is the most traditional andcommonly used method for phylogenetic studies, but because of its low rate ofevolution, the difference between strains is ignored, so it can not identify at thespecies level well. While the housekeeping gene sequencing method overcome thisdisadvantage, drawing attention of more and more researchers.Besides, the experiment of rep-PCR fingerprinting analysis on seventy teststrains was conducted, three different primers were tested for this research: BOX1R,ERIC1R/ERIC2and REP1R-I/REP2-I. The bands range of the BOX1R primer was179-2336bp, polymorphic rate was79.2%, the bands range of ERIC1R/ERIC2primer was38-2476bp, polymorphic rate was90.9%, the bands range ofREP1R-I/REP2-I was34-2622bp, and polymorphic rate was87.5%. The bands ofERIC-PCR amplification was the most clear one, and its polymorphic was also high,although the polymorphic of ERIC-PCR was higher than the BOX-PCR, its bandswere not that clear and the amplification was difficult. Thus when studing therep-PCR fingerprinting technology, primer BOX1R and ERIC1R/ERIC2should beconsidered preferentially. In this study, different method was used to identify bacterias inhibitingAflatoxin, then its diversity was analyzed and its typing structure was alsoevaluated, which laid a theoretical foundation for peanut aflatoxin contamination infields.