Analysis Huperzia Crispata’s Endophytic Fungi RAPD And ISSR Molecular Fingerprintings Which Could Produce Hup A And Identification Theirs Genus

Huperzine A has the effect of treatment of Alzheimer’s disease(AD) which extracted from plant material Huperziaceae, but Huperziaceae plant natual resource is limited and its natural propagation, tissue culture are difficult. Hup A is difficult to meet the demand of the the AD patients which only extracted from Huperziaceae plant material.In this study, the metabolites of99endophytic fungi isolated from Huperzia crispata analysis by TLC and HPLC, Initial screening of38potential Hup A produced endophytic fungi. Using of improved benzyl choride method to extract the38fungus mycelium DNA. The good DNA extraction results with weigh about2.0g mycelium and ultrasound17minutes, So, benzyl choride method (ultrasound istead of quartz sand grinding) is a fast, efficient extraction of DNA from fungal mycelium. The genetic diversity analysis by ISSR and RAPD, According to the number of ISSR-PCR amplified bands and their distribution remove the duplicate samples. There are19potential Hup A produced endophytic fungi. Using cluster analysis (UPGMA) based on those polymorphic bands amplified with RAPD, ISSR and RAPD+ISSR primers, the relationships of19potential Hup A produced fungi species were constructed. The result show that the clustering results of RAPD and RAPD+ISSR markers were unanimously. The general clustering consequences indicated that19fungi species were classified into five groups when the similarity confficient was0.59. Therefore, RAPD and RAPD+ISSR is more suitable for the analysis of genetic diversity in endophytic fungi isolated from Huperzia crispata.In this study, indentify the19potential Hup A produced endophytic fungus by morphology and molecular methods. Based on the results of morphological classification, according to the BLAST results of ITS1-5.8S-ITS2sequence draw the NJ tree using MAGE5.0, Two classification results are consistency.19tested resources including eight penicillium fungi(11,12,15,19,28,38,51and87), two Paecilomyces fungi(37and40), four Aspergillus fungi(16,44,65and77), two Colletotrichum fungi(1and13), one Phomopsis fungus(88), one Cladosporium fungus(5) and one Gibberella fungus(57). The genetic distance analysis results show that the farthest genetic distance between endophytic fungus No.11and No.88(4.710), their phylogenetic relationship is farthest; the genetic distance between endophytic fungus No.87and No.38, No.15and No.28is0, their phylogenetic relationship is very close, may belong to one kind of different variants.