Study On Genetic Diversity Of Oenococcus Oeni Isolated From Chinese Wine

There are few researches on the classification and identification of Oenococcus oeni in China for the present. In this study, a rapid and effective response system had been established to identify and distinguish Oenococcus oeni isolated from the main wine production areas of China via the research of Species-specific PCR, Sequence analysis of 16S rRNA and single enzyme AFLP markers. The main results are listed as follows:1. Optimization and establishment of the identification system of Oenococcus oeni by species-specific PCRSpecies-specific PCR is a rapid method used frequently was applied to identify 22 strains of Oenococcus oeni isolated from main wine production areas. In the study, The DNA could be extracted directly by the optimized DNA extraction method, then used for a stable amplification without any purification. The amplification results were stable. Optimization of the reaction system and reaction conditions were done to make the bacterial suspension and colonies used for PCR amplification directly as a DNA template in the further study. The clear and stable amplification results indicated that it was an effective and reliable method. Further way by optimizing the reaction system and reaction conditions was done to make the bacterial suspension and colonies can be used as a DNA template for directly PCR amplify. The results showed that the amplification is stable. The method is fast and reliable, it has improved the efficiency of identification. The reaction system (20μL) included: 10×PCR buffer 2.0μL, dNTPs 1.6μL(2.5 mmol/L), Mg2+ 1.6μL(25 mmol/L), each primer 0.8μL(10 umol/L), bacterium suspension 2.0μL or a bit of colonies, Taq DNA polymerase 0.1μL(5 U/μL). Reaction conditions: initial denaturation at 94℃for 5 min, 35 cycles at 94℃for 45 s, 64℃for 2 min, 72℃for 2 min. and the final extension at 72℃for 7 min.2. Further characterization of Oenococcus oeni by 16S rRNA gene sequences analysis16S rRNA gene sequences between 41-705 bits of 22 strains of bacteria were determined. The results showed that the 16S rRNA gene sequence similarity of 22 strains of bacteria and model strain were all above 99.8 %, comparing with the model strain of Oenococcus oeni from GeneBank database. It further proved that the 22 strains of bacteria were Oenococcus oeni. Multiple sequence alignment analysis showed that the similarity of every two strains were all above 99.8 %, it indicated that the 16S rRNA gene of Oenococcus oeni was conservative.The phylogenetic tree constructed by using the 16S rRNA gene sequence of 22 Oenococcus oeni strains and model strains of 7 genera of streptococcaceae showed that: Oenococcus oeni,Leuconostoc mesenteroides,Weissella viridescens belonged to the same cluster, the relationship of Leuconostoc mesenteroides and Oenococcus oeni is closest.3. Establishment of SE-AFLP reaction system to study the genetic diversity of Oenococcus oeniIn order to study the genetic diversity of the 22 Oenococcus oeni strains, digestion and ligation conditions were screened, and amplification system was also optimized. The most suitable SE-AFLP reaction system established by us was: 10μL (200ng) DNA was digested with 3U HindⅢat 37℃for 12 h, ligation mixture was kept at 22℃for 12 h, selective amplification was set up with 5μL restriction-ligation sample DNA which has been diluted 5 times, HindⅢprimer 0.75μL (10μmol/L), Taq DNA polymerase 0.2μL(5 U/μL), 10×PCR buffer 2.5μL, dNTP 1μL (2.5 mM), MgC12 2μL(25 mM), ddH2O was added up to 25μL. Under this optimization system, clear, steady and higher polymorphic Oenococcus oeni SE-AFLP bands were obtained.4. Study the genetic diversity of the 22 Oenococcus oeni strains by SE-AFLPIn the experiment, each primer can amplify bands between 16 and 23. the average bands of each primer were 19.5 bands. Among the 4 primers's amplified bands, 49 bands were polymorphic bands, which accounted for 62.8%. Similarity analysis showed that genetic similarity coefficient between different strains has large difference. Results of the cluster analysis indicated that 22 Oenococcus oeni strains can be divided into two major groups when the genetic similarity coefficient was 0.690. All the results showed that abundant genetic diversities exist in 22 strains of Oenococcus oeni isolated from main wine production areas of China. The SE-AFLP bands generated by four selected primers can discriminate 22 strains of Oenococcus oeni. SE-AFLP molecular marker can be used to differentiate Oenococcus oeni strains.