Screening And Identification Of Swainsonine-degrading Bacteria In The Endophytic Bacteria From Oxytropis Kansuensis Bunge

Locoweed, the most serious poisonous plants which are harm to the sustainable development of animal husbandry in the world, is the common name of toxic plants of the genera Astragalus sp. and Oxytropis sp. The main toxic ingredient of locoweed is swainsonine (SW). SW, an inhibitor of alpha-mannosidase in cells, can alter metabolism of glycoprotein, lead to cellular vacuolation, and finally result in animal death. One of the locoweed, Oxytropis kansuensis Bunge, widely distributes in the pastoral pasture of northwestern Sichuan and Gansu province. Researches showed that locoweed is one of the Leguminosae plants, contains rich nutrition composition and mineral elements. If locoweed can be utilized reasonably, not only in the locoweed poisoning will be controled, but also the shortage of forage grass will be solved. Bacteria which can degrade SW much effectively were isolated from soil embedding locoweed by Wang Yan et al. and endophytic fungal which can produce SW was also isolated from Oxytropis kansuensis Bunge by Yu Yong tao et al. This research includes screening and isolation of SW degrading bacteria from endophytic bacteria of Oxytropis kansuensis Bunge, identification of the SW degrading bacteria. It establishes the foundation for the research on the relationship between endophytic bacteria and SW in locoweed.1. Determination of SW content in Oxytropis kansuensis Bunge from pasture region of northwestern Sichuan provinceDrying and grinding the sample of Oxytropis kansuensis Bunge and useing 75% ethanol as the extraction agent, SW was extracted by Soxhlet extraction method and detected by gas chromatography. The result showed that the contentration of SW in the sample was 65.405μg/g. It proved that the Oxytropis kansuensis Bunge in the pastoral pasture of northwestern Sichuan province was one of locoweeds, offering the source of bacteria for the isolation of endophytic bacteria from locoweed.2. Isolation and cultivation of endophytic bacteria in Oxytropis kansuensis BungeTaken the fresh and healthy samples of Oxytropis kansuensis Bunge without lesion, surface sterilized with 70% ethanol and Sodium hypochlorite, clipped and bruised them, and imbibed the fluid into the common broths for enrichment culture, the enrichment medium was spreaded into solid medium to isolate endophytic bacteria. The result showed that 83 strains of endophytic bacteria were isolated from Oxytropis kansuensis Bunge by morphological observation. This experiment isolated and cultured endophytic bacteria from Oxytropis kansuensis Bunge. which was as the foundation for screening of SW-degrading bacteria in locoweed.3. Screening of SW-degrading bacteria in endophytic bacteria and determining the degradation abilityFive strains were found which could grow in the mineral solid medium where SW was the only carbon source via enrichment culture. And strain Ab was able to degrade SW stably with 3 replications, whose degradation rate was 11.5771% within 24 hours by gas chromatography when the concentration of SW was 100 mg/L. The SW degradation rate could not be improved by extending cultivation time, while it had high tolerance of SW (1g/L).4. Identification of the SW-degrading bacteriaThe strain was identified by morphological observation, physiologic and biochemical tests,16S rDNA gene sequencing and phylogenetic characteristics analysis. The results showed that strain Ab was gram-negative bacteria. The phylogenetic tree showed that it mostly closed to Brevundimonas diminuta of Caulobacteraceae, and finally named as Brevundimonas diminuta Ab.16S rDNA sequence of the strain has been submitted to GenBank database and accession numbers was HM625914.