Isolation,Identification And Metabolites Of Symbiotic Bacteria Of Mongolian Medicine Parmelia Saxatilis (L.) Ach

Objective: This topic selected two medium,three temperature culture separation Symbiotic bacteria of Mongolian medicine Pamela saxatilis(L.)Ach and establish the best experimental conditions for culture Mongolian medicine Pamela saxatilis(L.)Ach symbiotic bacteria.The metabolites of cultured Mongolian medicine Pamela saxatilis(L.)Ach symbiotic bacteria were identified by HPLC and compared with those of Mongolian medicine Pamela saxatilis(L.)Ach.This study is of great significance for later research,utilization and development of metabolites of Mongolian medicine Pamela saxatilis(L.)Ach and Symbiotic bacteria of Mongolian medicine Pamela saxatilis(L.)Ach resources protection.Method: 1.Wash the lichen body of the Mongolian medicine Pamela saxatilis(L.)Ach and place it in the surrounding environment to achieve water balance.Remove the ascus disc,soak in distilled water for 4 hours,and extrude to remove excess water.Use Vaseline to secure the bottom of the petri dish,MY medium over the top of the dish,sealed to prevent contamination.33 copies were prepared by the same method and sealed,and divided into three groups and placed in incubators at 15℃,27℃ and 37℃ respectively.Culture in light condition.The same method was used to prepare 33 WA4 culture-medium,and 11 of them were cultured in 15℃,27℃,37℃ incubators in the dark condition.Culture and isolation of symbiotic bacteria of and establish the best experimental conditions for culture of symbiotic bacteria of Mongolian medicine Pamela saxatilis(L.)Ach.2.Cultured at 27℃ without light for one month.After subculture for two consecutive months,10 bacilli were selected as samples(labeled as sample 1,sample 2,sample 3,sample 4,sample 5,sample 6,sample 7,sample 8,sample 9,sample 10).Ten samples were identified by genomic DNA extraction,amplification,PCR product detection,purification and sequencing.3.Using 2 grams of malt extract,0.2 grams of yeast extract and100 ml of distilled water as liquid medium,the fermentation mycelium was obtained by shock culture for 7 days,and the metabolites were extracted.The metabolites were detected by HPLC and compared with Pamela saxatilis(L.)Ach.Chromatographic conditions: Venusil MP C18column(250 mm×4.6 mm,5 μm).Mobile phase: 0.2% phosphoric acid aqueous solution(A)-acetonitrile(B);Detection wavelength: 274 nm;Flow rate: 1.0m L/min;Column temperature:35℃;Injection volume: 10μl.).Results: The best experimental condition for the culture of symbiotic bacteria of Mongolian medicine Pamela saxatilis(L.)Ach is the dark culture in an incubator at 27℃ on MY medium.Through genomic DNA extraction,amplification,PCR product detection,purification and sequencing,the results were compared and identified three symbiotic bacteria of Mongolian medicine Pamela saxatilis(L.)Ach namely,Alternaria alternata(Symbiotic bacteria 1),Purpureocillium lilacinum(symbiotic bacteria 2),Deconica sp(Symbiotic bacteria 3).Under the same chromatographic conditions,the Pamela saxatilis(L.)Ach of Symbiotic bacteria(Symbiotic bacteria 1)was retained for 3min,and the substances detected at 15 min all existed in Symbiotic bacteria.The retention time of the fermentation Pamela saxatilis(L.)Ach of Symbiotic bacteria(Symbiotic bacteria 1)and the extract of Symbiotic bacteria were 5min,and the substances detected at 76 min were all present in the Mongolian medicine Pamela saxatilis(L.)Ach.The Pamela saxatilis(L.)Ach of Symbiotic bacteria(Symbiotic bacteria 2)was detected at the retention time of 3min,5min and 76 min.The retention time of the fermentation liquid extract of Symbiotic bacteria(Symbiotic bacteria 2)and the extract of Symbiotic bacteria was 3min,and the substances detected at 5min were all present in the Pamela saxatilis(L.)Ach.The Mongolian medicine Pamela saxatilis(L.)Ach of symbiotic bacteria(Symbiotic bacteria 3)were detected at the retention time of 3min,15 min,48min and 76 min.The retention time of the fermentation liquid extract of Symbiotic bacteria(Symbiotic bacteria 3)and the extract of Symbiotic bacteria was 3min,and the substances detected at 5min were all present in the Pamela saxatilis(L.)Ach.Conclusion: In this paper,by studying the culture and isolation of the Pamela saxatilis(L.)Ach symbiotic bacteria,we know that the best experimental condition for the culture of symbiotic bacteria is MY medium in the dark culture box at 27℃.Through genomic DNA extraction,amplification,PCR product detection and purification,sequencing,the results of comparison and identification of three symbiotic bacteria,which laid the foundation for the later research and development of Pamela saxatilis(L.)Ach symbiotic bacteria.The metabolites were extracted from the mycelium by fermentation.The metabolites were detected by HPLC and compared with those of Mongolian medicine Pamela saxatilis(L.)Ach.The results laid a foundation for the development of the metabolites of symbiotic bacteria of in the later stage and had great significance for the protection of stone flower resources.Mongolian medicine Pamela saxatilis(L.)Ach.