Study On Total Iridoid Glycosides And Related Synthase Genes Of Rehmannia Glutinosa

Rehmannia glutinosabelongs to ScrophulariaceaeRehmannia,a kind of perennial herb,which is one of the famous “four great medicine”.Terpenoidisone kind of important secondary metabolite of Rehmannia glutinosa,while the iridoid is an important monoterpene compound and one of the main components of the medicinal.DXR,GPPS,G10 H,10HGO are key enzymes for the synthesis of the iridoid terpenoids.In this research,the accumulation of total iridoid glycosides and the expression of related enzymes were studied in Rehmannia glutinosa,and the effects of variety,origin and methylation inhibitor on the synthesis of iridoid glycosides were analyzed,thenDXR gene and its promoter were cloned,which would laid the foundation for further validation of DXR gene function.The main results were as follows:1?The content of total iridoid glycosides increased with the growth and development of Rehmannia glutinosa,and the total iridoid glycoside content in leaves was higher than that in rootsduring the early stage of Rehmannia glutinosa,but was lower than that in roots after maturation period.The contents of total iridoid glycosides in different varieties of Rehmannia glutinosa were quite different,compared to that in Rehmannia glutinosa85-5 the content in leaves of Rehmannia glutinosa Jinjiuwas higher,and was lower in roots during the early stage of Rehmannia glutinosa.However,during the later stage of the growth of Rehmannia glutinosathe contents of total iridoid glycosides in roots of Jinjiu were higher,andwere slightly lower compared with that of Rehmannia glutinosa 85-5.2?The expressions of DXR,GPPS,G10 H and 10 HGO genethat was related to the synthesis of terpenoid compounds inRehmannia glutinosaappearedspecificity of tissue,time and space.The expressions of DXR gene in leaves were higher than that in roots and stems,and the peaks of DXR gene expression in leaves showed in ?-?growth period of Rehmannia glutinosa,which was slightly differently due to different species.The variation trend of GPPSexpression of Rehmannia glutinosa85-5was consistentand in different tissues,the expression level of GPPS gene reached the maximum at ? growth period,and was the highest in Jinjiu leaves during?growth period.The expressions of G10 H gene in leaves were higher than that in roots and stems,and reached the highest level in ? growth period,but the expressions of G10 H gene in roots and stems were different due to species of Rehmannia glutinosa.10 HGOas one kind of the multi-functional enzyme,its gene expression was more complex.3?The accumulation of iridoid and the expression of related enzymes were greatly affected by the origin,and the contents of total iridoid glycosides inRehmannia glutinosa were higher in Wenxianthan that in Xinxiang.The expression of related gene with irinoid glycosides was also differentin different regions,the expression of DXR2 gene indifferent tissues was all significantly higher in Wenxian areathan that in Xinxiang areaat the early growth period of Rehmannia glutinosa,andthe expression of DXR4 gene was similar to DXR2 gene.Compared to that in Xinxiang area the expression of GPPS in roots was higher in Wenxian duringthe growth processof Rehmannia glutinosa,but was higher in stems and leavesjust in the early growth period of Rehmannia glutinosa.Similarly,the expression of G10 H gene in different tissues was significantly higher than that in Xinxiang area during the early growth period of Rehmannia glutinosa.Compared to that in Xinxiang area,the expression of 10HGO6 in roots and stemsof Rehmannia glutinosa was significantly lower in Wenxian area,but the expression of 10HGO33 was higher than that of Xinxiang.4?5-azaC has a significant effect on the accumulation of terpenoid compounds and the expression of related enzyme gene.The accumulation of total iridoid glycosides in Rehmannia glutinosa was promoted by 5-azaC and increased with the increase of 5-azaC concentration,and 50?M 5-azaC had the greatest effect on the accumulation of total iridoid glycosides.However the effect of 5-aza C on the expression of related gene with the iridoid synthesis was very complicated,mostly for the promotion of gene expression,but also in the presence of inhibition,in which 30?M-50?M 5-azaC had the most extensive effect on gene expression.5?In this study,the full length of DXR gene was cloned,was composed of 205 bp 5'-UTR,1425 bp CDS and 356 bp 3'-UTR,and encodedone protein with 474 amino acids.The promoter of the DXR gene was cloned by modified hiTAIL-PCR method,and might contain CAAT box,CAT box,TATA box.In addition,the plant expression vector of DXRgene was constructed,which wouldlay the foundation for further study on the function of DXR gene.