| Ophiocordyceps sinensis is a kind of traditional Chinese medicines and food as well. It has been demonstrated there are great value for medical application. Gansu Province is one of the important place of origin for Ophiocordyceps sinensis in China. In order to investigate the resource of Ophiocordyceps sinensis on systematic development and genetic diversity in Gansu Province, resources from three main producing areas of Gansu Province was studied with molecular systematic analysis. The main works as following:1.18 strains isolated from three areas of Gansu Province was studied to select the best medium and performanced under different temperature. It was found the most suitable temperature for them is 18℃, they grow slowly in low temperature, and can't grow any more while the temperature was above 25℃. It was observed that hypha and conidiospore below optical microscope firstly by oblique cuting coverslip on medium. Stroma, which were same as wild Ophiocordyceps sinensis morphologically, produced after special treated in triangular flask. It can be concluded that the strains isolated are Hirsutella sinensis.2. The genomic DNA of the strains were extracted by improved CTAB method and then detected using ultraviolet spectrophotometer and agarose gel electrophoresis. Adding PVP andβ-Mercaptoethanol in extract buffer solution, while by combination of tris-balenced saturate phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol to extracte, adding 3mol/L NaAc, then subsiding in frozen absolute ethyl alcohol and washing several times in 70% ethyl alcohol. The products were proved being complete, High purity and qualified to meet the requirement of experiment further.3. The ISSR-PCR reaction system was optimized first time for Ophiocordyceps sinensis. The system are 25μL, for 10×PCR Buffer 2.5μL; Template 20ng; dNTP 0.12 mmol/L; Mg2+1.5 mmol/L; ISSR primer 0.9μmol/L; Taq DNA polymerases 0.5U.4.Using inter-simple sequence repeat(ISSR) interval augmentation polymorphisms analysis and 18S rDNA sequence analysis to mark and analysis system development of 18 strains. There are more different genetic and variety genetic diversity between samples that from different areas, and signifercant genetic differentiation between the sample from different sites within area but a little differentiation between samples from same sites within area using ISSR analysis. There were a little genetic differentiation between samples from different sites within the same area or the samples from same sites when 18S rDNA sequence analysis were used. There were no genetic differentiation between the samples isolated from same worm body but different position when above two methods were used.5.Part of the 18s rDNA genes of Ophiocordyceps sinensis were amplified and sequenced. Thel8s rDNA of eighteen strain species of isolated were 372-374bp in length. There was only one or two base pairs difference between them, according to comparing with Hirsutella sinensis, each of them has a similarity of 99% and the genetic distance under 0.01. which suggested their highly consistent on gene level. A molecule systems tree was constructed with Cordyceps militaris and Cordyceps bassiana as reference to compare the relationship between all of the strains isolated and H.sinesis. The results showed that our strains could be confirmed as Hirsutella sinensis.6.The contents of Polysaccharide in mycelium of Hirsutella sinensis was determined by phenol-sulphuric acid method, the content of the Mannitol was determined by Sodium periodate colorimetric method, RP-HPLC method for the determination of adenosine and 3'-Deoxyadenosine was established for the first time. The results show that there is 3'-Deoxyadenosine in natural Ophiocordyceps sinensis and the content of Cordyceps polysaccharide in strains isolated from different growing areas of Gansu is from 1.66% to 2.36%, the content of the Mannitol is about 2.04% to 5.72%, the 3'-Deoxyadenosine and adenosine are 47.213 to 76.927μg/g,249.778 to 439.797μg/g; and the content of Cordyceps polysaccharide from the same areas is from 1.79% to 2.00%, the content of the Mannitol is about 1.62% to 1.79%, the 3'-Deoxyadenosine and adenosine are 45.331-49.96μg/g,92.404 to 376.375μ/g. There were significant difference in content of active ingredients of strains between different area,and the same producing areas is not significant except adenosine by significance analysis. According to the content of polysaccharide and adenosine, the strains isolated from Gannan has higher content, it means that the Ophiocordyceps sinensis in Gannan is the best in three area, and the quality of Ophiocordyceps sinensis that from Jiacang of Gannan is the best. |
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