Isolation Of Cycloartenol Synthase Gene And Its Effect On The Biosynthesis Of Diosgenin In Trigonella Foenum-graecum L.

Diosgenin is a naturally occurring steroid sapogenin.As a precursor,it can be used to synthesize hundreds of steroid drugs.Therefore,diosgenin is called "medicinal gold" by the medical community.The biosynthetic pathway of diosgenin is not completely elucidated,for example,it is not clear whether the diosgenin biosynthesis is via lanosterol or cycloartenol.A transcriptomic study was previously applied to the diosgenin-producing plant Trigonella foenum-graecum L.in our group.From the transcriptome data,only the cycloartenol synthase gene(CAS)was found while the lanosterol synthase gene was not detected,indicating that diosgenin is most likely to be biosynthesized from the cycloartenol.To test this hypothesis,the CAS gene was cloned from the Trigonella foenum-graecum in this study,and its biochemical function was confirmed by a yeast expression system.To further examine the effect of the CAS gene expression on the diosgenin biosynthesis,the CAS gene was over-expressed or downregulated by RNAi approach in the Trigonella foenum-graecum plant.After confirming the transgene expression,the relevance of the CAS gene expression to the diosgenin biosynthesis was investigated.The main findings of this study were shown as follows:(1)By searching the Trigonella foenum-graecum transcriptome that we previously constructed,the gene encoding cycloartenol synthase was isolated from the fenugreek.(2)The CAS gene was expressed in the yeast WAT11 strain,and its enzymatic product cycloartenol was produced in the CAS-expressed yeast cells while it was not found in the empty vector-transformed control yeast cells.The data from this experiment confirmed that the CAS gene that we isolated is functional in the biosynthetic step from 2,3-oxidosqualene to cycloartenol.(3)By transforming the CAS gene into the hairy roots of the fenugreek plant,we were successful in obtaining several positive transgenic lines.The real-time PCR data showed that the CAS gene expression was increased in about four times by the exogenous CAS transgene event.However,in comparisons with the empty vectortransformed control hairy roots,the overexpression of the CAS gene in the hairy roots led to only a slight increase in the production of diosgenin and ?-sitosterol,suggesting that the CAS is not a rate-limiting enzyme in the steps for diosgenin biosynthesis.