Study On The Correlation Of Endogenous Fungus And Its Secondary Metabolites In Rehmannia Glutinosa Libosch

Rehmannia glutinosa Libosch. is perennial herbaceous plants ofscrophulariaceae fanily, is the traditional bulk authentic Chinese herbalmedicine in our country has been regarded as the upper grade traditionalChinese medicinal materials, and known as one of four famous Huai Chinesetraditional medicine. Rehmannia glutinosa contains many of the beneficialactive ingredients, which is the largest research and utilization of iridoidglycosides substances-catalpol. Always, the microbes are the people to get richresources of the active substance, a good therapeutic effect of many antibioticsare derived from microorganisms. Recent studies have found that theendophytic fungus are widely distributed in many plants, but also a wide range,and some endophytic fungus and host plants have the same capacity ofproducing the same or similar active ingredient, therefore, the endophyticfungus on medicinal plants has great potential on the agriculture and thepharmaceutical.Objective: Discussion the correlation of endophytic fungus and its secondarymetabolites in Rehmannia glutinosa and preliminary study the diversity of itsendophytic fungus. Try to screen the active strains which can produce catalpol,and to optimize its fermentation process and the high yield of mutagenesis, inorder to obtain a stable high-yielding mutant strain.Methods: The topic uses the different vegetative organs(roots, leaves) and theroots of different parts(epidermis, phloem, xylem) of Rehmannia glutinosa forthe test material, using the tissue separation to separating, purifying andidentifying the endophytic fungus in it, and studying the species compositionand distribution in different organs or parts; the isolated fugus were beingfermented cultured,through TLC, HPLC and other methods to screen the fuguswhich can produce catalpol, and research their biological charateristics;Optimization of fermentation about the selected high-yield strains by single factor and four factors of three levels orthogonal test; selecting the high-yieldmutations strains through UV mutagenesis, HNO2mutagenesis and compoundmutation methods and combining with HPLC to determinate the content ofcatalpol; Identify CB37strains which produce catalpol by molecular biology.Results:1. The population structure of Rehmannia glutinosa endophytic fungi.There are362endophytic fungus were isolated from the differentvegetative organs or parts of Rehmannia glutinosa.,282strains can producespores which were identified distributed in5orders,6families and11generals,there are97strains belong to Fusarium which accounted for34.4%of allstrains that have been identified, and the Fusatium is the dominant species inRehmannia glutinosa endophytic fungus.2. The study about the secondary metabolites of Rehmannia glutinosaendophytic fungi.Use the methods of TLC and HPLC to detecte the endophytic fungusseparated and purified from the different vegetative organs and parts ofRehmannia glutinosa, found that the endophytic fungus in Rehmanniaglutinosa of different vegetative organs or parts all could produce catalpol, butthe contents is different. There are7strains were screened with a productioncapacity of catalpol, which produced the highest content catalpol strain isNO.CB37, which is isolated from the skin of the Rehmannia glutinosa, and itscatalpol production reched2.31g/L(the catalpol content of the root-skin inRehmannia glutinosa is2.92%)3. The optimal fermentation test of the endophytic fungi producing catalpolCB37.In combination with the single factor and orthogonal experiment method,filtering out the best fermentation conditions for the strain CB37is based onPDB medium, glucose was added10.0g/L, yeast extract15.0g/L, medium startPh7.0,2%inoculun in50mL medium,28℃constant temperature shaking(120r min-1) cultured6d. Compared with the initial culture conditions, mycelialbiomass reached2.73g/L, an increase of7.77%, catalpol production reached2.38g/L, an increase of2.89%. 4. The high yield mutagenic test of the endophytic fungi producingcatalpol CB37.By using the univariate mutagenic of UV mutagenesis, nitrous acidmutagenesis and composite mutagenesis, we get a high-yielding mutant strainB-UH-5, its output of catalpol can reach2.71g/L, compared with the originalstrain CB37yield17.32%. After ten generations subculture, indicating that ithas a certain degree of genetic stability.5. The molecular biological identification of the endophytic fungiproducing catalpol CB37.According to contrast ITS sequence of the test strains CB37and relevantsequence in NCBI database by the BLAST, combined with the method ofmorphology characteristics identification, the strain CB37is identified asFusarium oxysporum。Conclusion: Through the study of the endophytic fungus in Rehmanniaglutinosa and its econdary metabolites, initially identified the populationstructure of Rehmannia glutinosa endophytic fungus, to provide aometheoretical basisi for understanding the diversitu of its endophyte; screened aactive strain which can produce catalpol and determine the optimalfermentation technology; get one more stable high-yield mutant strain bymutagenesis, which lay the foundation of raoid industrialization productioncataipol.