Construction And Screening Of Saccharopine Reductase Gene Disruption Mutant Of Embellisia Fungal Endophyte From Oxytropis Glabra

Oxytropis glabra is the one of toxic weed widely distributed in desert steppe in Inner Mongolia. It causes poisoning when consumed by grazing animals. The toxic substance is swainsonine, which inhibits activity of mannosidase in cells and leads to neurological disorder or even death. This causes heavy losses to animal husbandry in grassland. The toxicity is caused by Embellisia fungal endophyte that reside within Oxytropis glabra. For better governance and utilization of swainsonine, exploration of it’s biosynthesis pathway in this fungal endophyte has become necessary. A report(2012) of another swainsonine producing fungal endophyte indicated that disruption of saccharopine reductase gene lead to increased accumulation of swainsonine and it’s precursor along with decreased level of saccharopine and lysine, and partial biosynthesis pathway of swainsonine was also suggested.In this research the optimized conditions of protoplasts preparation and regeneration of Embellisia fungal endophyte from O. glabra were discussed. The purified protoplasts were transformed by targeted DNA fragments on a saccharopine reductase gene disruption vector, and regenerated on medium contained hygromycin B to screen for the transformants. Two sets of primers were designed according to the saccharopine reductase gene and hygromycin phosphtransferase gene. PCR was performed by taking the genomic DNA of wild type of Embellisia fungi and that of the transformants as samples using the two sets of primers to screen for saccharopine reductase gene disruption mutants. It laid foundation for investigating the role of saccharopine reductase gene in swainsonine metabolism from the Embellisia fungal endophyte. The main results were showed as follows:1. The effects of enzymes concentration、enzymolysis temperature and time、stabilizer and hypha culture time on protoplasts regeneration of Embellisia fungal endophyte from O. glabra were analyzed. The protoplasts concentration reached to 4.42×105 /mL, when mecilum mass was hydrolyzed by enzymes which consisted of 3% Driselase, 1% Lysing enzymes and 0.001% Chitinase. The culture time was for 10 days, and the enzymolysis temperature was set to 30℃. 1.2mol/L KCl solution was selected as osmotic stabilizer and the cultures were incubated in flat shakers(80rpm/min) for 3 hours. The protoplasts regeneration rate reached to 52%.2. The protoplasts were transformed by targeted DNA fragments on saccharopine reductase gene disruption vector mediated by PEG. The transformation plates were incubated at 25℃ for 10 days to keep the hypha growing. The colonies of the wild type could not grow on the media containing hygromycin. The Colonies of transformants were more compact and growing fast compared to that of wild type fungi.3. The PCR was performed using two sets of primers by taking the genomic DNA of wild type fungi and that of the transformants as templates. In these two reactions, no product was amplified in the wild type fungi sample, while the expecting product was amplified in transformant. The results ascertained that the transformant was saccharopine reductase gene disruption mutant.