| Dogwood(Cornus officinalis Sizb.et Zucc.)belongs to the perennial tree in the Cornaceae family,which is distributed in the altitude of 400-1500 meters and mainly produced in shaanxi,zhejiang,henan and other places.Its dry and mature fruit(commonly known as Fructus corni)is a rare Chinese medicine in China,which has the functions to the liver and kidney and promoting astriction and immune regulation.The medicinal active ingredients of C.officinalis including iridoid glycosides(CIGs,such as loganin,morroniside and sweroside etc.),organic acids(such as gallic acid,protocatechuate and ursolic acid etc.)and polysaccharide etc.,it has good effects in the treatment of diabetes,antithrombotic,anti-inflammatory,anti-cancer and memory enhancement.In the early stage,systematic studies have been conducted on the evaluation and preservation of the germplasm resources of C.officinalis,genetic diversity analysis,extraction and content determination of medicinal ingredients,and molecular markers,etc.,but no relevant reports have been reported on the relationship between genomic DNA methylation and the accumulation of loganin and other medicinal active ingredients.Therefore,in this study,the germplasm of cornus officinalis(ZJ026)with good comprehensive traits and transcriptome sequencing in the early stage was selected in the laboratory as the experimental material,and treated with 5-azaC spray,while the control group was sprayed with distilled water,and samples were collected every 20 days.The contents of six medicinal active ingredients in fruits and leaves were determined by high performance liquid chromatography(HPLC),and the methylation rate and pattern of fruit genomic DNA were determined by methylation-sensitive amplification polymorphism(MSAP).The relative expression levels of the key enzyme genes G10H and SLS in the loganin synthesis pathway in fruits were determined by qPCR method.Through comparison and analysis of the control and different concentrations of 5-azaC treatment,the degree and site of cytosine methylation,The contents of 6 medicinal ingredients,such as loganin and mononoside,are used to explore the relationship between the level of genomic DNA methylation,the expression of key enzyme genes in the synthetic pathway and the accumulation of medicinal active ingredients,with a view to revealing the molecular mechanism of the accumulation and change of medicinal ingredients in dogwood fruits from the perspective of DNA methylation.The main results are as follows:1.Low concentration(10 ?M,50 ?M)5-azaC can significantly promote the accumulation of iridoid glycosides in fruits and leaves of Cornus officinalis(October)(P<0.05),and medium concentration(100 ?M)can significantly promote the growth period(August)accumulation of iridoid glycosides and organic acids in fruits.Treatment with high concentrations(500 ?M,1000 ?M)can significantly promote the accumulation of organic acids in fruits and leaves during the growing period,and most of them are inhibitory.From early August to mid-October,the content of total iridoid glycosides and 5-hydroxymethylfurfural(5-HMF)in the fruits of the control group gradually increased,and the content of mononoside gradually decreased.The content of loganin,sweroside,gallic acid and protocatechuate increased first and then gradually decreased.Within 60 days after treatment,the contents of the six medicinal ingredients were significantly lower than the control as a whole.After 60 days,the contents tended to the control level.In the same period,the contents of loganin and two organic acids in the leaves of the control group increased gradually and then decreased significantly.The content of mononoside reached the highest in mid-September,and no 5-HMF was detected in the leaves.Within 80 days after treatment,the contents of the three iridoid glycosides were significantly lower than the control as a whole,at the maturity stage,the contents of the two organic acids were significantly higher than the control.The correlations between the contents of the six medicinal ingredients are:between loganin and mononoside,sweroside,the two organic acids and mononoside,sweroside,protocatechuate and gallic acid there was a high positive correlation(P<0.001),white there was a high negative correlation between mononoside,argonin,protocatechuic acid and 5-hydroxymethylfurfural content(P<0.01).2.The expression levels of two key enzyme genes G10H and SLS in the loganin synthesis pathway were significantly higher than those of the control at 20 and 80 days after treatment with low concentration 5-azaC(P<0.01).The expression level of GI0H increased significantly(P<0.05),while the expression level of SLS decreased significantly at 20 days after treatment with medium and high concentration 5-azaC,20 d.Both expression levels decreased significantly at 40 days after treatment.The 80 days after medium concentration treatment,the expression levels of both expressions increased significantly compared with the control(P<0.05);The 60 days after high concentration treatment,the expression levels of the two were also significantly increased compared with the control.3.The genome hemimethylation rate and total methylation rate of the fruit of dogwood control group gradually decreased with the growth period.After 5-azaC treatment,both of them were generally lower than the control;the full methylation rate showed a decreasing trend with the growth period,20 d and 40 d after treatment,the full methylation rate was higher than the control,80 d after treatment(the maturity stage),it was significantly lower than the control.This indicates that 5-azaC treatment can increase the full methylation rate of the genome within a period of time(40 d),and significantly decreased the hemimethylation rate and the total methylation rate.On the other hand,after 5-azaC treatment,there were mutual transformation between,methylated and unmethylated sites,hemimethylation and full methylation sites and the higher the concentration of 5-azaC,the more demethylation sites,the effect of 5-azaC has a certain concentration range.4.Correlation analyses of genomic DNA methylation rate,key enzyme gene expression and medicinal active ingredient content after 5-azaC treatment,the results found that:(1)The fruit of control group and treated with a certain concentration(500?M)5-azaC there was a significant negative correlation between G10H expression level and full methylation rate(P<0.05),while a significant positive correlation was found between SLS expression level and total methylation rate(P<0.05).(2)There was a very significant positive correlation between the content of sweroside and the hemimethylation rate in the control group(P<0.01),when the 5-azaC concentration was 100 ?M,there was a significant positive correlation between the content of loganin and the full methylation rate,between content of sweroside and total methylation rate.(3)There was a positive correlation between the G10H expression level of the control group and the content of loganin and mononoside,but it was not significant,it is worth noting that the expression of G10H in the treatment group had a significantly negative correlation with the content of mononoside.After treatment with 5-azaC(100 ?M),the expression of SLS in fruits was significantly positively correlated with the content of loganin and mononoside,it shows that G10H and SLS gene has a certain regulation effect on the accumulation of content of both.The above correlation analyses results preliminarily explained that genomic DNA methylation affects the synthesis and accumulation of medicinal active ingredients such as loganin and mononoside in dogwood fruits by regulating the expression of key enzyme genes in the synthetic pathway.This study revealed preliminarily the regulation and accumulation of six medicinal active ingredients of Cornus officinalis from 5-azaC treatment,genomic DNA methylation level and the expression of key genes of synthesis pathway of loganin.The results of the above research will further reveal the molecular mechanism of the synthesis and regulation of medicinal active ingredients of the Cornus officinalis provides a certain theoretical basis. |
PDF Full Text Request