Culture, Identification And Analysis Of Swainsonine Assay Of Endophytic Fungi From Oxytropis Glabra DC.

Leaves and stems of individual plant of Oxytropis glabra DC. were cut as explantation to isolate and culture the endophytic fungi in vitro.The morphologic characteristics of colonies and the conidiophores were investigated. The 5.8SrDNA/ITS of the in vitro cultured endophytic fungi were analyzed, and then the sequence data of the endophytic fungi were submitted to GenBank and analyzed by homologous blast.The phylogenetic tree was constructed by MegAlign program of DNASTAR software package.The molecular biological and microbiological characters were investigated to presume the classification of the endophytic fungi. The genomic DNA of Oxytropis glabra indoor cultured was extracted, then the 5.8SrDNA/ITS region was amplified and sequenced.Meanwhile, temporary slide of leaf epidermal cell and paraffin section of leaf and stem were observed;The swainsonine of endophytic fungi from Oxytropis glabra DC. were extracted by chemical method,the molecular structure of swainsonine was identified by TLC and MS methods. Later the swansonine contents were determined by GC. The results indicated as follows:1.The morphologic characteristics of the colonies and conidiophores of this 11 endophytic fungi were the same as that of Embellisia sp.L12.The sequences of ITS1 and 5.8SrDNA of endophytic fungi from Oxytropis glabra DC. were totally the same. Compared to the sequence of 5.8SrDNA/ITS of Embellisia sp.L12,there was only one viriation site which was in the 5.8SrDNA region in each sequence.In addition, two variation sites were showed in the ITS2 region.These results implied that the endophytic fungi had a close relationship to Embellisia, but the classification of them needs further research.2.The 5.8SrDNA/ITS of endophytic fungi were amplified from genomic DNA of 4 Oxytropis glabra cultrued indoor.All the PCR products were sequenced and the results showed that these sequences were the same as the other 5.8SrDNA/ITS from endophytic fungi cultured in vitro.The structure of the myceliums were observed in temporary slide at lower epidermis of the leaf.The results of paraffin section of leaf also indicated that the myceliums were distributed in the leaf cuticular layer.3.The results of TLC indicated that, the spots and relative mobilities of swainsonine of endophytic fungi from Oxytropis glabra DC. and standard swainsonine were close to each other, and the results of MS were further detected that the M/S of swanisonine in all samples and standerd swainsonine were 174.Swainsonine contents of 11 endophytic fungi were determined by GC.The results were showed parallelism to endophytic fungi and their parasitifer plant.