| Huperzine A is one of the most important medicine on Alzheimer’s disease for it’s powerful selective and low toxicity inhibitor of acetylcholinesterase. The main source is extracted from Huperziaceae plant and its concentration is very low, the content is about0.2mg/g. Huperziaceae plant have2genera, about150species, scattered distribution in the world, China has28species,4varieties. It general10-30cm high, major growth in the elevation of300-2600m’s in original secondary forest, bamboo, pine, was accompanied by moss, rubrum and other plants around the roots, the growth conditions is dark and damp, mostly grow slowly and demand of especial living conditions and environments. The search for new sources of HupA to alleviate the need for medication has attracted widespread attention. It’s a research focus to discover the new active compounds from endophytic fungi for its easy culture, easy control, low cost and short cycle, because of the large scale industrial production, the application of endophytic fungi prospect is very broad. Based on earlier researches, the studies applied Microbiology, Biotechnology and Bioinformatics knowledges, focus on five aspects in this papers as follows:rapid and effective method for isolating total DNA from Huperzia crispata’s endophyte fungi, genetic diversity analyzed by ISSR markers, endophyte fungi species classification and identification, chromatographic analysis of fermentation liquid, high purity HupA preparation by HSCCC and fermentation process optimization target.(1) A time-saving, simple and efficient total DNA extraction method for Huperzia crispata’s endophyte fungi mycelia has been developed, In this study, used benzyl chloride method to isolate total DNA of endophyte fungi and comparied quartz sand grind method with ultrasonic break method. Results showed that the DNA extraction efficiency and quality obtained by ultrasonic break method was superior to quartz sand grind method, and it only needed10minutes. The genomic DNA extracted by ultrasonic break treatment was digested by RNaseA and amplified with ITS primers, agarose gel electrophoresis checking results showed that this treatment could obtain good quality PCR effects. It can use for large scale samples a specially for firm texture fungi colony. (2) The suitable optimal reaction system was established for ISSR-PCR of endophytic fungi of Huperzia crispata, namely25μL reation system containing IO×PCR buffer (2.0mmol/L Mg2+)2.5μL, dNTPs(10mmol/L)0.6μL, primer(10pmol/μL)2.0μL, Taq E(1U/μL)1.5μL, templet DNA(10ng/μL)3μL, aseptic ddH2O15.4μL.10reliable ISSR primers of endophytic fungi of Huperzia crispata analyzed by ISSR markers, based on10primers,99endophytic fungi generated3795clear and reproducible polymorphic bands, of which accounting for100%(PPB=100%). The genetic similarity (GS) among the tested genotypes ranged from0.59to0.96, the test strains were classified into11groups in the GS of0.64. At the GS was0.67, No. I group could be classified into5subgroups. Results showed that higher genetic distance and wider genetic base were existed among endophytic fungi of Huperzia crispata. With primer UBC868to amplify DNA of No.13strain, unambiguous bands were appeared at500bp and200bp. The fermentations products of No.13strain were detected and analyzed by TLC/HPLC and found that No.13strain could produce HupA with the same as host plant Huperzia crispata, the same ISSR category fungi as No.13strain were the most important potential screening and induced mutation strains.(3) TLC and HPLC analyses were developed on endophytic fungi fermentation liquid of Huperzia crispata, the target product (HupA) was preparated by HSCCC After TLC detection, it can be preliminary estimated that38strains (No.1, NO.4and et al.) fermentation liquid may have HupA or its similar structural of chemical composition. HPLC detection results showed that No.13and No.87endophytic fungi strain have fermentation production of HupA and its similar compounds, the output is199.6μg/L and18.64μg/L. The products of No.13and No.87strain liquid fermentation isolated by HSCCC were high purity HupA. HSCCC method:with a two-phase solvent system composed of hexane-butyl alcoho-water(3:1:2, V/V). The upper phase was used as the stationary phase and the lower phase was used as the mobile phase at a flow rate of2mL/min. The experimental conditions were controlled at800r/min and the sample volume was5mL. The NKA-2macroporous adsorption resin was selected as the best resin for static adsorption, it can be used as large scale HupA industrial production. (4) On the TLC detection to infer the possible production of HupA and its similar structure of the chemical composition, further ISSR analysesd, after removed duplicate samples obtained19strains, the morphological identification based on rDNA ITS DNA sequence identification, www.ncbi.nlm.nih.gov online BLAST, homology compared with GenBank, EMBL DDBJ and PDB multiple nucleic acid database, then analysesd NJ phylogenetic tree. Used p-distance model to calculate genetic distance, used neighbor joining method for evolutionary distance analysis.19strains of the genus were classified and identified, No.13strain is Colletotrichum gloeosporioides, No.87strain is Penicillium oxalicum.No.5,11,12,13,15,16,19,28,37,38,40,44,51,57,65,77,87,88and No.1strains of ITS GeneBank Accession No. sequences are presented, respectively JX230994, JX230995, JX230996, JX230997, JX230998, JX230999, JX231000, JX231001, JX231002, JX231003, JX231004, JX231005, JX231006, JX231007, JX231008, JX231009, JX231010, JX231011and JX231012.(5) High purity HupA preparation and fermentation process were optimizated by using No.13strain, the best fermentation process condition is follow:1L potato liquid culture system with20g maltose, The experimental conditions were controlled at pH(5.0), shaking speed800r/min and28℃Incubation for84hours, the output by HPLC detection was0.1996μg/mL... |
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