| Purpose:In this experiment,the seed of Dioscorea nipponica Makino was taken as the main research object.The effects of environmental factors on the germination and metabolism of Dioscorea nipponica Makino seeds were studied by dynamic analysis of the gene expression of primary,secondary metabolites and key enzymes in the metabolism process of Dioscorea nipponica Makino seeds.In order to improve the quality and uniformity of medicinal materials to provide the basis.At the same time,the classification methods of Dioscorea nipponica Makino seeds from different producing areas in China were studied,which provided the basis for establishing the classification standard of Dioscorea nipponica Makino seeds.Materials and methods:1 Material:The experimental materials were 27 batches of Dioscorea nipponica Makino seeds from different areas,which were identified by Professor Yin Haibo of Liaoning University of traditional Chinese Medicine as Dioscorea(Dioscorea nipponica Makino)seeds.2 Methods:2.1 The medicinal resources of Dioscorea were studied by reviewing the ancient books of the genus Dioscorea.2.2 The germination test was carried out on the paper of culture dish,and the samples were collected at the corresponding time point.Two kinds of primary metabolites of soluble sugar and pyruvate at different stages,prodiosaponin,protoslender diosgenin,methyl protodiosaponin and pseudo-protodiosaponin,were measured by using artificial climate box to simulate different temperature environment under the corresponding temperature conditions,and two primary metabolites of soluble sugar and pyruvic acid at different stages were determined under the corresponding temperature conditions.Seven growth and physiological indexes,such as the content of five secondary metabolites of diosgenin,root diameter and stem diameter,as well as the expression of three key enzymes of HMGR,FPS,CAS,were observed.Thecorrelation between each component and index and the change of each component under different temperature conditions were compared.2.3 The content of primary metabolites in seeds of Dioscorea nipponica Makino was measured by ultraviolet spectrophotometry and the content of secondary metabolites was measured by high performance liquid chromatography(HPLC)based on different photoperiod conditions.The gene expression of key enzymes was measured by realtime fluorescence quantitative PCR amplification,and the growth and physiological indexes were measured by Vernier caliper and chlorophyll analyzer.The correlation analysis of the results was carried out.2.4 Based on different light quality conditions,the content of primary metabolites in seeds of Dioscorea nipponica Makino was measured by ultraviolet spectrophotometry,and the content of secondary metabolites was measured by high performance liquid chromatography(HPLC).The gene expression of key enzymes was measured by real-time fluorescence quantitative PCR amplification,and the growth and physiological indexes were measured by Vernier caliper and chlorophyll analyzer.The correlation analysis of the results was carried out.2.5 The data were collected and compared horizontally among different types of environment by grey correlation method,and the main indexes,namely primary metabolite,secondary metabolite and key enzyme,were selected and analyzed to explore the indexes and temperature,light cycle,and so on,in order to find out the main indexes,such as primary metabolite,secondary metabolite and key enzyme.The degree of closeness between light and quality.2.6 According to the rules for the Inspection of crop Seeds,the indexes of seed purity,water content,100-seed weight,germination rate and vitality of Dioscorea nipponica Makino were determined by principal component analysis and cluster analysis.The seeds of Dioscorea nipponica Makino were divided into different grades by discriminant analysis,and the classification discriminant of seeds of Dioscorea nipponica Makino was obtained.Results:1 By consulting the ancient books,the research on the different name,origin,origin and efficacy of Pangolin was completed,and the research on the efficacy of its related medicinal plants was completed.2 The results of correlation analysis showed that the content of primary metabolite and secondary metabolite had a significant positive correlation with the expression of HMGR,FPS gene,indicating that the activity of key enzymes increased with the increase of primary metabolite of Dioscorea nipponica Makino seedlings.The secondary metabolites were also increased.It was further verified that the secondary metabolites were transformed from soluble sugar,pyruvate,etc.Rich primary products were beneficial to the accumulation of secondary metabolites,and under the influence of different temperatures,The content of secondary metabolites was closely related to the gene expression of HMGR,FPS,that is,the production of secondary metabolites was regulated by regulating the gene expression of HMGR,FPS under different temperature conditions.At the same time,through the line chart,we can see that the optimum temperature for the accumulation of the original diosgenin is 20 ° C,the optimum temperature is 25 °C for the protoposaponin,and the optimum temperature for the methyl diosgenin is 30 ° C,and the optimum temperature for the original diosgenin is 25°C.The optimum temperature of diosgenin was 15°C.3 The experimental results showed that under the influence of different photoperiod,the secondary metabolites increased most rapidly under the condition of day and night illumination,and the accumulative amount was significantly higher than that of other groups.It is proved that long-term illumination is more conducive to the formation of the quality of medicinal materials.There was a positive correlation between soluble sugar and five secondary metabolites,and a significant positive correlation between pyruvate and diosgenin.There was a positive correlation between FPS,CAS and secondary metabolites,indicating that under the influence of light cycle,there was a significant positive correlation between pyruvate and diosgenin.The content of secondary metabolites was closely related to the gene expression of FPSand CAS,that is,the generation of secondary metabolites was regulated by regulating the gene expression of FPS,CAS in different periods of illumination.4 The results showed that the accumulation of soluble sugar,diosgenin,pyruvate and other secondary metabolites were the most in white light,diosgenin,pyruvate and other secondary metabolites in white light.There was a positive correlation between soluble sugar and proto-diosgenin,protoslender diosgenin and pseudo-protodiosaponin,but no significant correlation was found between pyruvate and several products.FPS was positively correlated with proto-slender diosgenin and diosgenin,and there was no significant correlation between FPS and proto-diosgenin and diosgenin.There was a positive correlation between CAS and proto-fine diosgenin and pseudoprotodiosaponin.5 The content of the soluble sugar,the pyruvic acid,the original fine sweet potato soap,the methyl original potato,the soap powder,the pseudo-original sweet potato,the soap and the soap is the most closely related to the daily light time(i.e.,the light period),and the close degree of the soluble sugar,the pyruvic acid,the original fine sweet potato and the sweet potato soap is the closest to the daily light time(i.e.,the light period),and the close degree with the light quality and the temperature is reduced in turn,The above components can be controlled to the greatest extent during the cultivation and planting of the medicinal materials by controlling the illumination period.The expression of HMGR,FPS and CAS gene was the most closely related to the photoperiod and temperature,and the effect of adjusting light quality on the above indexes was described.6 Seed purity,water content,100-seed weight,germination rate and vitality can be used as reference indexes for seed classification of Dioscorea nipponica Makino.According to these indexes,27 seeds from different regions of China were divided into two grades,and the discriminant formula of seed grade of Dioscorea nipponica Makino was obtained.Conclusions:1 By looking up ancient books for a different name,the textual research on the primordial plants,the origin and the efficacy,we found that Dangshan has a history of drug use for nearly 200 years now,although the history of medicine is not very long.However,it is widely used all over the country,and there are many folk applications.Therefore,it is very important to make clear the medicinal name of Dioscorea nipponica Makino in various areas,which provides the basis for the extensive use of Pangolosaurus,and lays a foundation for the further development of Dioscorea nipponica Makino in the future.Through the study of its related drugs,many plants of Dioscorea contain a large number of diosgenins,which can be used as important raw materials for anti-tumor,antithrombotic,antiallergic,antiviral and anti-shock drugs.2 Seed quality is an important prerequisite to ensure the quality of its original medicinal materials,seeds are the embryonic form of individual plants,germination is considered to be the most important period of plant life,so,It is of great significance to explore the physiological,biochemical and metabolic conditions of seed germination for the growth and quality of Dioscorea nipponica Makino.In this study,the expression levels of primary,secondary metabolites and key enzyme genes during the germination of Dioscorea nipponica Makino were determined for the first time,and the results of correlation among the components were obtained during the germination of Dioscorea nipponica Makino.The results showed that primary metabolites were transformed into secondary metabolites under the action of key enzymes during plant growth and development.By comparing the effects of different temperatures on secondary metabolites,it is concluded that the accumulations of each component are different at different temperatures.It is suggested that we can control the activity of key enzymes by controlling the temperature,and then control the content of some secondary metabolites and the quality of medicinal materials.3 The active components of medicinal plants are usually secondary metabolites accumulated by them.The effects of light cycle on the germination and metabolism of Dioscorea nipponica Makino seeds show that long-term illumination is beneficial tothe accumulation of secondary metabolites,and the effect of light on the seed germination and metabolism of Dioscorea nipponica Makino L.That is to say,longterm illumination is beneficial to the improvement of the quality of medicinal materials.This conclusion can be applied to the cultivation and planting industry of Chinese herbal medicine,increase the illumination time and improve the quality of medicinal materials through artificial intervention.4 It was found that the accumulation of diosgenin was the most under red light and the other four saponins were the most under the action of white light.In order to control the quality of medicinal materials,the contents of different saponins can be controlled by artificial intervention of different light sources in agricultural production.It is of great significance to improve the quality of medicinal materials and to control the homogenization of the quality of medicinal materials.5 The results of grey correlation analysis showed that the content of each component in the seeds of Dioscorea nipponica Makino during germination was most closely related to the light time(that is,photoperiod),but decreased in turn with the light quality and temperature.It is suggested that the content of each component can be controlled by controlling the light cycle in the process of cultivation and planting of medicinal materials.This is of great significance to the cultivation and planting of Dioscorea nipponica Makino.6 High quality seeds of Chinese medicinal materials are not only the important guarantee of the yield and quality of Chinese medicinal materials,but also the important source of standardized production of Chinese medicinal materials.In this study,systematic clustering,principal component analysis and discriminant analysis were used to classify the seeds of Dioscorea nipponica Makino for the first time,and the discriminant formula of seed quality grade was obtained in order to improve the quality of medicinal materials.The implementation of the standardized production of Chinese medicinal materials laid the foundation. |
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