Studies On Relationship Of Endophyte Fungus Isolated From Huperzia Crispata (Ching) By ISSR Markers

The key pharmacological component in the Huperziaceae plants is Huperzine A, which is a special effect clinical drug to Alzheimer's disease, but Hup A is extremely low in the Huperziaceae plants, even in the high content plant--H. serrata, the content of Huperzia A only between 0.006%-0.120%, and Huperziaceae plant sources are limited, because of their special growth requirements and growth characteristics of the environment, and Huperziaceae plant is now in endangered status. Based we have found the endophytic fungus in the Taxus and Huperzia serratum can produce the same substance with the host plants, we have tried isolated endophytic fungus from Huperzia crispata which can produce huperzine A.we hope to slove the limited source of huperzine A in this way. Our study has isolated and purified 100 endophytic bacterias and fungus from Huperzia crispate,99 of them are endophytic fungus and 1 of them is endophytic bacteria, and we have found one of endophytic fungi can produce potassium tetroxalate. We have made a preliminary morphological identification to the endophytic fungus isolated and purified. However, morphological identification of fungus can not accurately identify species, so it cannot make the identification results completely credible. The key in the next work is to give them a more reliable classification by the molecular biology method and find their genetic relationship. Ultimately we hope to get endophytic fungus from Huperzia crispata which can produce Hup A, or other functional components of the strain, and find new resources of Hup A. This is not only has positive significance for the development of endophyte fungus from Huperzia crispata, and also privdes theoretical references to the study of endophytic fungus from the Huperziaceae plants.In the first part of this research we compared the DNA extraction result of endophytic fungus from Huperzia crispata by using the development CTAB method and the benzyl chloride method, the result is we can choose different methods to extract the DNA of fungus according to their different shapes, but in general the the benzyl chloride method has wider range in DNA extraction of fungus, it has a certain representation in the fungus' DNA extraction and is suitable for most DNA extract from fungus.The study also found that the DNA of endophytic fungus from Huperzia crispate have significant degradation after freezing and thawing several times even stored in TE buffe, so the DNA samples should be preserved in a few test tubes to prevent the DNA sample degrade and the follow-up tests can not be completed. We can choose whether purify the DNA sample or not by the next test, the DNA sample can do not undergo purification if the follow-up test has a low request in the purity of DNA samples. In this way, and the sample can be prevent lossing in the purification process.In the second part of the research we selected 41 ISSR primers which can amplify bands from the DNA of endophytic fungus from Huperzia crispata, and the 41 ISSR primers all from the 100 ISSR primers published by Columbia University. By setting the temperature gradient, the available primers' best annealing temperature are explored. The four factors of ISSR-PCR reaction system:the DNA template's amount, Mg2+ concentration, dNTPs concentration, Taq enzyme amount are all optimized by combining single factor experiments and orthogonal test method, then we establish the best ISSR-PCR reaction system of endophyte fungus from Huperzia crispata. The best ISSR-PCR reaction system of endophyte fungus from Huperzia crispata is 25μL reaction system containing the DNA template 30 ng, Mg2+ concentration is 2.0 mmol/L, Taq enzyme dosage is 1.5 U, dNTPs concentration is 0.6 mmol/L, 2μL primers(100 pmol/L) and 2.5μL 10×PCR Buffer.The seleted ISSR primers and their optimal annealing temperature is widespread and representative in the fungi's ISSR-PCR markers research, and is providing some reference in the study of ISSR molecular markers. The method of DNA extraction of fungus, ISSR primer and ISSR-PCR reaction system are widespread and representative for the rich species of the endophyte fungus from Huperzia crispata, and provide reference in ISSR research of fungus. We can use these real and reliable technical parameters in the next test.Using the optimized ISSR-PCR reaction system and choosing 10 ISSR primers which can amplified rich and clear bands from the 41 amplified primers amplified all the DNA of endophytic fungus from Huperzia crispate, The selected 10 primers amplified totally 3975 ISSR bands contained 3975 polymorphic bands, the polymorphic bands accounting for 100%, each ISSR primer amplified 397.5 bands on average. Ntsys-PC software is used to calculate the distance matrix and similarity coefficient of the 99 endophyte fungus form Huperzia crispata, the genetic distance is 0.9992-0.0429. Using the POPGENE 32 software, we get the percentage of poly morphic loci(P) is 100%, the Nei's genetic distance(H) is 0.2804 and the Shannon's information index (I) is 0.4392, we can know the endophyte fungus form Huperzia crispate have a largr varityThe results showed that all endophyte fungus can be broadly divided into 15 groups, compared with the the seven genus identified by morphological identification, the 15 groups are more abundant, it shows that we can't ascertain the species or genus of a fungi from its morphological features, the morphological identification is only a reference for the fungi's identificationand and the more precise identification is the molecular markers.And the resulr also revealed the similarity coefficients among endophyte fungus from Huperzia crispata are low, only nine pairs of fungus have a high similarity coefficients, while the remaining strains of the similarity coefficient is relatively low.The third part of the study also have a preliminary discussion about the relationship between the Huperzia crispata and endophyte fungus from Huperzia crispata by applied ISSR marker, the outcome shows that 5 of the choosed 10 amplification primers can amplified bands fome the DNA of Huperzia crispata. And we also found the same length bands from the amplification product of the DNA from fungus which are amplificted by the same primer. This result indicated the similar gene fragments exist between the Huperzia crispata and the endophyte fungus from Huperzia crispata, and the further reacher and explore is necessary. From this way we can find the endophyte fungus from Huperzia crispata which have the same function gene fragments with the Huperzia crispata.The study classified Huperzia crispata endophyte fungus by molecular markers, and further revealed the relationship among the endophytic fungus from Huperzia crispata, analyzed the relationship between the endophyte fungus from Huperzia crispata and plant. Preliminary ascertain the further work was not only researching the fermentation products of endophytic fungus, detecting the fermentation products, finding the function microorganisms, but also designing the primers which adapt to the endophyte fungus of Huperzia crispata and Huperzia crispata, establishing the best PCR reaction system, amplify the DNA of endogenous fungus and plants, useing the molecular biology method to find the same amplified fragments. In this way we want to find the same functional gene microbial from the fungus and plant, so then find the microbial which produce same material as the plant.