Expression Analysis Of Endophytic Fungal Saccharopine Reductase Gene And Cloning Of Its Promoter In Alternaria Oxytropis

Oxytropis glabra is a perennial herb.The endophytic fungus of Oxytropis glabra is isolated in vitro with toxic alkaloid swainsonine.Livestock was poisoned after feeding the plants to certain amount.The animal cell function is disorder,and severe poisoning causes the animals to die.Therefore,great loss in animal husbandry is caused.The saccharopine reductase plays a pivotal role in the SW metabolic pathways in the endophytic fungus.The OW7.8 strain of Alternaria oxytropis was chosen as material in this study.The qRT-PCR was performed with common housekeeping genes including ACTIN,TUB,GAPDH,APRT and 18S rRNA as candidates to screen the optimal stable reference gene in the fungus,and the qRT-PCR products were evaluated by GeNorm,NormFinder and BestKeeper softwares.The dynamic changes of SW level and the expression of sac in fungi were determined by HPLC-MS and qRT-PCR analysis under different culture time,different precursors addition（L-A,L-Lys,L-P）,and different precursor concentrations（0 mol/L,1×10^-33 mol/L,3×10^-44 mol/L,1×10^-44 mol/L and 1×10^-55 mol/L）in fungi.The sac promoter was cloned,and its expression vector（pBARGPEI-mCherry-sac）was constructed.The transcriptional activation was also detected.The Saccharopine reductase of the fungus was extracted and the basic enzymatic properties were analyzed including enzyme activity,optimum pH value,optimum temperature,enzyme kinetics,K_m and V_maxax values.The results which provides basic data for exploring the impacts of sac to SW biosynthesis in fungus were showed as follows:1.The expression stability of ACTIN was shown to be the best in Alternaria oxytropis endophytic fungi.2.The SW levels and sac expression in the experimental group（adding L-A,L-Lys and L-P）were higher than that in the control group.The higher of sac expression correlated with the higher of SW levels,while the lower of sac expression correlated with the lowed of SW levels.The highest levels of SW and sac expression were displayed in fungi cultured for 26d,23d and 32d with the same concentration of L-A,L-Lys and L-P（1×10^-44 mol/L）.The levels of SW and sac expression reached maximum at 26d with L-A concentration of3×10^-44 mol/L,23d with L-Lys concentration of 1×10^-44 mol/L,and 32d with L-P concentration of 1×10^-44 mol/L.The levels of SW and sac expression of fungi were higher in L-Lys supplementation groupthan those in L-A supplementation group and L-P supplementation group cultured at 23d,while the levels of SW and sac expression of fungi were higher in L-P supplementation group than those in L-A supplementation groupand L-Lys supplementation group cultured at 26d,29d and 32d.3.The sac promoter was cloned by TAIL-PCR,and its functional elements were predicted by online software PLACE and BDGP.The TATA-box,CAAT-box,G-box and seven stress respond elements were found.The promoter expression vector pBARGPEI-mCherry-sac was constructed,and the vector was transformed into OW7.8 protoplasts.The sac promoter transcriptional activated strain（glufosinate resistance）were obtained through regeneration.The fusion sequence was amplified by PCR,the transcription of mCherry was detected by RT-PCR,and the red fluorescence was observed under the fluorescence microscope in sac promoter transcriptional activated strain which showed the sac promoter realized transcriptional activation.However,no red fluorescence was detected in OW7.8 strain.4.The Saccharopine reductase in OW7.8 was extracted.The optimum pH value was set at 7.0,and the optimum temperature value was set at 24°C.The reaction time was determined at 10 min.The K_m and the V_max of Sac was0.118 mmol/L and 0.063μmol/min when the concentration of saccharopine was 1 mmol/L.The K_m and the V_max of Sac was 0.375 mmol/L and 0.213μmol/min when the concentration of NADP⁺was 2 mmol/L.