The Influence Of Saccharopine Reductase Gene For Swainsonine Biosynthesis In Alternaria Oxytropis Endophytic Fungi From Oxytropis Glabra

Alternaria oxytropis endophytic fungi from Oxytropis Glabra synthesize swainsonine(SW),an indolizidine alkaloid which causes severe toxicosis in livestock after grazing SW containing locoweed plants.The SW synthesis pathway in fungi is unclear,and the reductase is one of the key enzymes in the pathway.The saccharopine reductase gene(sac)in Alternaria oxytropis plays an important role in the SW biosynthesis.The wild type strain(OW7.8)was cultured in vitro,and the sac gene disruption mutant(M1)was obtained,and a functional complementary vector(pBARGPE-Sac)was constructed in our previous studies.In this studay,the OW7.8 in Alternaria oxytropis was chosen as material,as well as the M1.Endophytic fungi were cultured in liquid medium and SW was extracted from both mycelium and fermentation broth.SWcontent was detected by High Performance Liquid Chromatography-Mass Spectrometry(HPLC-MS).The dynamic changes of SW contentthat in the experimental group(saccharopine added)and in the control group(no addition)at different culture times were explored and analyzed by statistical software.The complementary vector of sac gene was verified,and then transformed into protoplasts of M1.The complementary strains were obtained through regeneration and resistance screening.The sacsequence was detected by PCR,and the expression of sac and hph were detected by RT-PCR.SW content in the complementary strain was determined by HPLC-MS and then compared with that in OW7.8 and M1.The main results were showed as follows:1.In the liquid culture,the SW content of the mycelium was much higher than that in the fermentation broth.The SW content in the mycelium was 5.500μg/mg,whereas in the fermentation broth was only0.089 μg/ml.SW was mainly inside the fungal cells.2.The SW content of endophytic fungi was determined by HPLC-MS.SW content was first increased and then decreased in fungi.In the control group,the SW content in OW7.8 was higher than that in M1,the SW content in OW7.8 was the highest at day 29,while it was the highest at day 23 in M1.In the experimental group,the SW content in OW7.8 was the highest at day26,while it was the highest at day 20 in M1.For OW7.8,there was no significant difference in the SW content in the experimental group compared with the control group.For M1,there was significant difference in the SW content in the experimental group compared with the control group.In the experimental group,there was no significant difference in the SW content between OW7.8 and M1,while in the control group,the SW content between OW7.8 and M1 had a significant difference.It showedthat Sac promoted the SW synthesis,and the addition of saccharopine in the medium had a greater effect on the SW synthesis in M1 compare to OW7.8.3.The complementary vector pBARGPE-Sacwas transformed into M1 protoplasts,and the sac functional complementary strains were obtained through regeneration.The resistance of hygromycin and glufosinate were showed in the complementary strain.The sac was amplified by PCR in total DNA,and the expression of sac and hphwere detected by RT-PCR in the complementary strain.The SW content in sac functional complementary strains,wild type OW7.8 and sac knockout mutant M1 wereculyured for 14 days,and then SW in fungi wereextracted and determined by HPLC-MS.Results showed the content of SW was higher in complementary strain than that in OW7.8 and M1.