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

Oxytropis glabra harbours endophytic Embellisia fungi,which produces a toxic alkaloid,swainsonine.Animals take some of the plants that leads to poisoning and even death.Therefore,serious loss to animal husbandry is caused.Little is known about the biosynthetic pathway of swainsonine in this endophytic Embellisia.Saccharopine reductase is a key enzyme for saccharopine.Saccharopine is also a key intermediate for the synthesis of swainsonine.That is why the saccharopine reductase gene plays an import role in swainsonine biosynthesis.The saccharopine reductase gene disruption mutant of the endophytic Embellisia from Oxytropis glabra was obtained in previous research.In this study,the wild type strain OW7.8 of Embellisia fungus was chosen as material,as well as the saccharopine reductase gene disruption mutant strain.The dynamic changes of swainsonine content were explored by HPLC through addition of three precusors(?-aminoadipicacid,L-lysine,picolinic acid)and at different culture time,then a statistical analysis was made.Detection of wild and mutant strains by Southern blot was performed.The regulatory sequences of saccharopine reductase gene were cloned and sequenced in the Embellisia fungus.The complementary vector of saccharopine reductase gene in fungus was constructed,and then the vector was transformed into E.coli for preliminary functional validation.The main results were showed as follows:1.The content of swainsonine in the wild type strain OW7.8 of Embellisia fungus in Oxytropis glabra was generally higher than the content of it in the saccharopine reductase gene disruption mutant M1.In the control group,the content of swainsonine in the wild strains OW7.8 reached the highest(3.678 ?g/mg)at 29 th day while the content of swainsonine in the mutant strains M1 reached the highest(0.041 ?g/mg)at 23 th day.2.The content of swainsonine in wild and mutant strains was higher than that in the control group after three kinds of precursor compounds were added into the culture medium.Moreover,the effects of precursor addition toswainsonine synthesis in wild OW7.8 strains was more significant than that in the mutant M1.Compared to the other two precursors,the addition of picolinic acid had the greatest effect on the production of swainsonine.The content of swainsonine in wild OW7.8 strains reached the highest(19.547?g/mg)at 32 th day.The content of swainsonine in the mutant M1 reached the highest(8.837 ?g/mg)at 26 th day.The content of swainsonine in wild OW7.8strains reached the highest(5.819 ?g/mg)at 26 th day,and the content of swainsonine in the mutant M1 reached the highest(0.082 ?g/mg)at 17 th day after ?-aminoadipicacid were added into the culture medium.The content of swainsonine in wild OW7.8 strains reached the highest(7.381 ?g/mg)at 23 th day,and the content of swainsonine in the mutant M1 reached the highest(7.979 ?g/mg)at 20 th day after L-lysine were added into the culture medium.It was showed that the addition of lysine promoted the synthesis of swainsonine in the mutant even more.3.Southern blot results showed that the intermediate sequence of saccharopine reductase gene was detected in the wild OW7.8 strain while the hygromycin phosphotransferase gene(hph)was detected in the mutant M1 strain.However,no intermediate sequence of saccharopine reductase gene was detected in the mutant M1 strain,which proved that the saccharopine reductase gene was knocked out in the mutant M1.4.Including 227 bp of ORF,the 5? of 1230 bp partial regulatory sequences of saccharopine reductase gene were cloned respectively by using genome walking technique.And including 223 bp of ORF,the 3? of 1500 bp partial regulatory sequences of saccharopine reductase gene were cloned respectively by using genome walking technique.The conserved sequences(TATAAT,CAAT,CCGCCC)were found at 5' of ATG,and the conserved sequence of AAAAA was also found at the 3' of TAA as well.5.The complementary vector of saccharopine reductase gene(pBARGPE-Sac)in Embellisia fungus was constructed,in which there were a promoter of gpdA,a terminator of trpC.The cDNA sequence of saccharopine reductase(1406 bp)was directional cloned into the vector.The resultingcomplementary vector was screened with Amp in E.coli and with Cremart in fungi.The vector was successfully transformed into E.coli for preliminary functional validation.