Study On The Precursor Origin Favored For Diosgenin Biosynthesis In Trigonella Foenum-graecum L.

Diosgenin serves as an important precursor to synthesize more than two hundred steroidal drugs.So far,the only to prepare diosgenin is the directly solvent extraction of plant materials,while this traditional approach has brought serious environmental pollutions and the government of China has no options but to shut down most of the diosgenin-producing factories.The recently fast development of the synthetic biology techniques has provided an alternative approach to produce diosgenin,but this strategy is dependent on the complete elucidation of the diosgenin biosynthetic pathway.Unfortunately,the diosgenin pathway is not completely understood so far,especially,the knowledge regarding that diosgenin is biosynthesized via cholesterol or from sitosterol remains in dispute.To address this point,using the fenugreek as the materials in this study,we performed experiments at either physiological or molecular level to investigate the contributions of cholesterol and sitosterol to the biosynthesis of diosgenin.From the physiological aspect,the upstream pathway up to cholesterol or sitosterol in fenugreek seedlings was inhibited by chemical treatments.The inhibited seedlings were then fed with cholesterol or sitosterol and the effect of these feedings on diosgenin production were analyzed.From the molecular aspect,the SMT1 gene specifically occurred in sitosterol biosynthetic pathway was cloned from the fenugreek plants,we made efforts in inactivating the SMT1 using the CRISPR Cas9 technique,which was planned for further investigating its effect on diosgenin biosynthesis.The results obtained from this study were as follows:(1)The accumulation pattern pf diosgenin in different tissues of fenugreek was investigated by GCMS analysis in this study,and the leaves were determined to be the major sites for biosynthesis of diosgenin in fenugreek.Using real-time PCRs,we also monitored the transcript profiles in the different tissues for the diosgenin biosynthesis genes.Interestingly,the upstream genes displayed distinctly different expression patterns from the downstream ones in the pathway steps toward diosgenin production.These data suggested that the tissue specificity for diosgenin biosynthesis genes depends on the stages at which they occur in the pathway.For example,the upstream genes are probably transcribed at relatively higher levels in the roots,whereas the leaves are the major sites for transcribing the downstream diosgenin pathway genes.(2)After treated with 200 ?M fosmidomycin,the MEP pathway was inhibited and the content of diosgenin in Fenugreek was increased.After treated with 100 ?M lovastatin,the MVA pathway was inhibited and the content of diosgenin in Fenugreek was significantly decreased.Therefore,it was concluded that diosgenin biosynthesis was mainly synthesized via the MVA pathway.(3)When the fenugreek seedlings were treated with lovastatin for 72 h,we started to feed 5 ?M cholesterol or 5?M sitosterol to the seedlings.The diosgenin content was obviously increased in both cholesterol-and sitosterol-fed plants with sitosterol inducing relatively higher levels of diosgenin.To further confirm the data,we also conducted another set of experiments,in which the 5 ?M cholesterol or 5 ?M sitosterol was fed to the plants at the same time as the chemical inhibitors from the beginning.The trend of the data was similar to that of the last feeding experiments,supporting that both cholesterol and sitosterol could be the precursors for diosgenin biosynthesis.(4)The gene expressions in the 72 h lovastatin-treated fenugreek seedlings were globally checked by RNA-sequencings.Although the diosgenin biosynthesis was dramatically decreased in the lovastatin-treated plants compared to the control seedlings,there were no significant difference in the expressions of diosgenin pathway genes between the lovastatin-treated and control plants.This data suggested that the chemical lovastatin could decrease the diosgenin biosynthesis through inhibiting the pathway enzyme activities while not by reducing the transcriptions of the pathway genes.Through the RNA-sequencing,a total of 43.29 G of the sequenced data were produced and about 50million clean reads were generated and assembled into 47953 unigenes,among which 37749 unigenes were annotated with public databases.(5)The SMT1 gene was isolated from the fenugreek plant and expressed in E.coli.cells,the in vitro enzyme assays were performed with the recombinant SMT1,but its activity was not confirmed by this assay,nevertheless,we continued to inactivate the SMT1 gene from the fenugreek genome using the CRISPR Cas9 technique,while the transgenic efforts did not allow us to obtain the positive transgenic plants,which prohibited the progress of the molecular part of the experiments in this study.