Enrich And Identify Candidate Genes Involved In Diosgenin Biosynthesis And Establishment Of Gene Function Analysis System

Diosgein is an important synthetic precursor of sterol drugs.More than three hundred sterol drugs can be produced by it,such as cortical hormone,sex hormone and so on.Therefore,it is named as "the gold of medicine".However,knowledge regarding how diosgenin is biosynthesized in plant remains scant.Fenugreek,which is one of the several plant sources that produce diosgenin,can be used to dissect the biosynthetic pathway of diosgenin.In this study,the fenugreek variety from Shanxi Province of China was employed as the material.The synthesis of diosgenin was induced by different concentrations of methyl jasmonate(MeJA)to enrich the genes related to the synthesis of diosgenin.Two fenugreek transcriptomes,which were derived from Fenugreek seedling treated with 0.01%MeJA and 0%MeJA,were constructed to identify candidate genes involved in diosgenin biosynthesis.Fenugreek hairy root system was also built,and the effect of two key factors on the transformation efficiency of fenugreek hairy roots has been investigated.The two key factors include the density of agrobacterium rhizogenes and the co-incubation supplemented with ultrasonic treatment.The results showed that:(1)Fenugreek seedlings were treated with different concentrations of MeJA(0%,0.005%,0.01%,0.02%,v/v).We found that 0.01%MeJA significantly increased the content of diosgenin,which is 13.38 mg/g.It was 1.76 times of that of control(0%MeJA).Cholesterol and ?-sitosterol were two possible synthetic precursor of diosgein.The changes of cholesterol and P-sitosterol contents in 0.01%MeJA-treated seedlings were detected by GC-MS.The contents of cholesterol and P-sitosterol in 0.01%MeJA-treated seedlings were 0.03 mg/g and 3.04 mg/g,respectively,which decreased by 1.41 and 1.65 times respectively.We hypothesized that both cholesterol and ?-sitosterol could be the biosynthetic precursors of diosgenin.(2)The content of diosgenin in Fenugreek was enriched significantly whentreated with 0.01%(v/v)MeJA.Two fenugreek transcriptomes were constructed,which were derived from Fenugreek seedling treated with 0.01%MeJA and 0%MeJA.A total of 86,291,104 high quality reads were obtained and assembled into 69669 unigenes.Total of 57579 unigenes were functionally annotated.Gene ontogeny enrichment analysis was performed by aligning all-unigenes with public databases.We obtained 13 enzymes including 36 Unigenes which take part in the downstreamof 2,3-oxidosqualene in the synthesis of cholesterol and ?-sitosterol.We found 14 Cytochrome P450 oxidases,12 glycosyltransferases,11 methyltransferases and 16 transcription factors expressed differentially.This laid a good foundation for dissecting the diosgenin biosynthesis pathway.(3)In order to provide a functional analysis platform for the synthesis of diosgenin,the fenugreek variety from Shanxi Province of China was employed as the experimental material.We investigated the effect of two key factors on the formation of fenugreek hairy root formation.The two key factors include the density of agrobacterium rhizogenes and the co-incubation supplemented with ultrasonic treatment.The hairy root numbers and its transformation frequency were improved by increasing the agrobacterium rhizogenes density with its OD600 value from 1.1 to 4.4.Compared to those measured at the OD600 value of 1.1,the incubation at the OD600 values of 2.2 and 4.4 led to better performance with the hairy root numbers being increased by 2.58 and 7.33 times,respectively,while the root transformation frequency being improved by 3.90 and 4.32 times,respectively.During the co-incubation of the agrobacterium rhizogenes with fenugreek explants,a 30s-ultrasonic treatment(Operating frequency 40 KHz,Ultrasonic power 180 W)slightly decreased the efficiency of the hairy root induction while a 60s-treatment increased the frequency of hairy root transformation by 2.48 times,in comparisons with the incubation without the ultrasonic treatment.