Isolation&Identification Of Nicotine Degrading Endophyte In Tobacoo And Its Application Research

Profit in tobacco industry is always high,but the nicotine in bobacco do harm to people’s health. The nicotine content can be reduced by farming and chemical methods but effect doesn’t obvious. It is an attempt of new technology that use Tobacco endophytes reduce nicotine content.The experiment choose the tobacco plant in Hunan Liuyang Guandu tobacco base. Five endophytes that isolated from it were got. One of them was determined as bacillus that can reduce the nicotine content. The experiment adopted ultraviolet rays to improve its ability to reduce nicotine content, and apply it to reduce the nicotine content existed in tobacco plants. As respect to such application, some related studies were done and following conclusion was got.1. Endophyte were isolated from healthy tobacco plants, and the experiment chose nicotine as selective medium. By primary and secondary screening, a tobacco endophyte with ability of reducing nicotine was gained, which can reduce the nicotine content by75.2%in1.01mg/mL liquid culture in4days. What’s more, through morphological observation, the colony had followed characteristics:irregular shape, gear edge, milky white, rough surface with folds and concave, opaque, bacillus, paired growth, both ends rounded. Combined with sequence analysis of16SrDNA the result implied that this bacterial strain’s16SrDNA gene sequence shared99%homology with Bacillus spss’. Therefore, the strain was identified as Bacillus sp.2. By CaCl2method, tobacco endophyte separated was used to prepare competent cells. Amp gene of PUC18plasmid as antibiotic marker was used to mark tobacco endophyte. So, it had the same antibiotic marker with plasmid. Then, the experiment made use of marked endophyte to infect tobacco plant and it could be detected in22th day in tobacco plant, but cannot in32th day. For that, foliar spray and root watering were confirmed as optimum infection way.3. Tobacco endophyte Tll was acted by80s UV radiation. And bacterial strain U32was gained which could reduce nicotine content by89%, the rate of reducing was14%higher than original strain Tll. The experiment used bacterial strain W-2-4(from soil) and Tll (from high efficiency nornicotine bacterial U32which come from Tll mutagenesis) to act on tobacco plant. Compared with the control group, the fifth day’s sample showed that the nicotine content in tobacco was reduced by25.6%. The result of comparison of the tenth days’with control group, the content was dropped by16.8%. With W-2-4effect, there was no significant difference between the fifth days’and control group. Additionally, The nicotine content was declined by17.6%in the comparison between the tenth days’and control group, this result identified with the maximum ratio of reducing nicotine of W-2-4in seventh day.