| Purpose:In this experiment,the seed of gentian and its annual seedling of Qingyuan County,Fushun,Liaoning Province,were used as the research object,and the primary metabolites of the annual Gentiana seedlings under the conditions of artificial simulated salt stress and drought stress were simulated.The key enzyme gene expression in the secondary metabolites and its biosynthetic pathway was studied,and the main factors and limiting factors were selected to influence the growth of gentian seeds and seedlings,and a certain theoretical basis for optimizing the method of gentian cultivation and the quality and yield of gentian medicinal materials was provided.Methods:1.Materials: The experimental materials of this project are three-year-old gentian seeds and annual Gentiana seedlings,all of which are collected in Yingkou Village,Yingmeng Town,Qingyuan County,Fushun City,Liaoning Province,and identified as gentiana scabra Bunge of Gentiana by Professor Yin Haibo,University of Traditional Chinese Medicine in Liaoning Province.The soil part of the experiment was collected in the cultivation base corresponding to the gentian seedling,and the part was purchased from the market.2.Method:2.1 A culture dish with a diameter of 9 cm was used,the paper bed was used as a germination bed,and different concentrations of pure Na Cl and PEG-6000 were used to create the salt stress and the drought stress environment,and the gentian seeds were cultured separately,and the samples were collected at the 2nd,4th,6th,8th,10 th,12th and 15 th day.2.2 The annual Gentiana seedlings were cultured under different light intensity and temperature,and the samples were collected on days 2,4,6,8,10,12 and 15.2.3 The content of soluble sugar and pyruvic acid of primary metabolite in the sample was determined by UV-spectrophotometry.The content of soluble sugar was determined by the method of one-to-one method,and the content of pyruvic acid was determined by the method of 2,4-dinitrobenzene.2.4 A high performance liquid chromatography was used to study the content of the components of the cycloalkenyl ether in the sample.As the content of effective components in the seeds and seedlings is small,the content of the bitter gourd of the gentian and the bitter gourd is determined.2.5 The three key enzymes,DXR,DXS and GPSPS,were found in the biosynthetic pathway of the effective components in gentian by reference to a large number of literature and KEGG,and the sequences of three key enzymes and internal reference genes were obtained by NCBI,and the primers were designed by NCBI Blast and Primer Premier 5.0.2.6 The expression of the three key enzyme genes was determined by RT-PCR,and the relative gene expression was calculated by using the method of 2-method.2.7 Using SPSS 20.0 to carry out the Spearman correlation analysis and multiple comparison,the data was analyzed by using Excel 2016.Results:1.The content of soluble sugar and pyruvic acid in gentian seeds was significantly affected by different concentration of Na Cl solution.There was a significant difference between the control group and the treatment group(P <0.05).There was no significant difference between the control group and the treatment group(P>0.05).There was no significant difference between 72 mmol/ L and 90 mmol/ L(P>0.05).No significant difference was found between the control group and the treatment group(P<0.05).There was a significant difference between the control group and the treatment group(P <0.05),and there was a significant difference between the control group and the other treatment groups(P<0.05).The expression of DXS,DXS and GPPS in the control group was significantly different from that of the control group(P<0.05).2.The content of soluble sugar in the seed of gentian under drought stress showed a decreasing trend,and the content of pyruvic acid showed a decreasing trend,and the content of pyruvic acid in the control group was significantly different from that of 3%,9% and 12%of the three treatment groups(P<0.05).The content of soluble sugar in the treatment group was significantly different from that of the 15% treatment group(P<0.01).There was no significant difference in the trend of the overall increase of the content of the Chinese gentian,12% and 15% of the two treatment groups.There was no significant difference(P>0.05)between the treatment group and the control group(P>0.05),there was no significant difference between the content of the gentian and the 9% treatment group(P>0.05),and there was no significant difference(P>0.05)between the treatment group and the other treatment group.There was a significant difference in the content of food and vegetable(P>0.05).The expression of DXS and DXR in the control group was significantly different from that of the control group(P<0.05),and there was a significant difference between the control group and the control group(P<0.01),and there was a significant difference in the expression of the GPPS gene in the 6% treatment group(P<0.05).3.The content of soluble sugar and pyruvic acid in the cultured Gentiana seedlings under different light intensity did not differ significantly(P>0.05).2000 Lx and 8000 Lx,There was a significant difference(P<0.05),there was a significant difference(P<0.05),and there was no significant difference(P<0.05).There was no significant difference in the expression of the10000 Lx DXS gene(P<0.05),and there was no significant difference between the other groups.The expression of DXR and GPSPS in the 10000 Lx treatment group was significantly higher than that of other treatment groups(P<0.01),and there was no significant difference between the other groups.There was no significant difference in the expression of GPS between the four treatment groups(P>0.05).4.There was a significant difference between the content of soluble sugar in the 10 ?treatment group and the content of soluble sugar at 15 ?(P<0.01)at different temperature conditions(P<0.01),and there was a significant difference(P<0.05)with the temperature of30 ?.The content of soluble sugar at the temperature of 15 ? and 30 ? was significantly different from the other groups(P<0.01).There was a significant difference in the content of pyruvic acid between each group(P<0.01).The content of the bitter gourd in the treatment group at 15 ? was significantly different from that of 20 ? and 30 ?(P<0.01),and there was a significant difference between 20 ? and 25 ?(P<0.01).There was no significant difference(P>0.05)at 10 ?,and there was no significant difference between 15 ? and20 ?(P<0.05).There was a significant difference(P<0.01)between the 30 ? and 30 ?treatment groups(P<0.01),and there was a significant difference between the two treatment groups at 10 ? and 15 ? and 20 ?(P<0.05),and there was a significant difference with25 ?(P<0.01).The relative gene expression of DXR was 10 ?,15 ?,20 ? and 25 ?,and there was a significant difference in the treatment group(P<0.05).There was a significant difference between 15 ? and 30 ?(P<0.01).There was a significant difference between25 ? and 30 ?(P<0.01).There was no significant difference between the treatment group at 20 ? and the other treatment groups(P>0.05).5.The grey correlation analysis results in that drought stress is the dominant factor that affects the gene expression of the metabolites and enzymes in gentian seeds and seedlings,and the salt stress is a limiting factor.The relationship between the gene expression of the enzyme and the drought stress is the most closely related to the stress,the temperature and the intensity of the light.Conclusions:1.When the concentration of salt reached 54 mmol/L,the metabolic process of gentian seed germination was strongly inhibited.In order to ensure that gentian seeds germinate in a good environment,it is necessary to prevent the salinization of soil in the process of seed germination of gentian.2.The content of the metabolite and the gene expression of the enzyme in the germination process of the gentian seed can be improved by the proper control of the soil moisture content,And provides the theoretical guidance for the cultivation of the gentian.3.When the illumination intensity is 8000Lx-1000 Lx,the content of the metabolite and the starting value of the gene expression of the enzyme are obviously higher than the other groups,and the average outdoor illumination intensity in the Qingyuan County in the summer is about40000 Lx,that is,the light-shielding net with the light transmittance of 20-25% during the period of the gentian seedling stage is more reasonable,And provides a theoretical basis for seedling culture of the gentian seedling.4.The optimum growth temperature of Gentiana is 25 ?,the temperature is too low to cause the plant to fall into the dormant state,and the temperature is too high to wither the plant.In order to ensure the accumulation of the metabolites of gentian seedling stage,the control temperature should be about 25 ? ~ 30 ?.5.The content of the product and the gene expression of the three enzymes in the seed and seedling stage of gentian were closely related to the degree of soil drought> salt content> temperature> light intensity.It is indicated that the degree of soil drought is the leading factor of the accumulation of the metabolism products of Gentiana and the expression of the key enzyme genes,while the salt content is the limiting factor.6.The main factor affecting the seed and seedling of Gentiana is the degree of soil drought,and the limiting factor is the soil salt content.in the cultivation process of the gentian seeds and the seedlings,reasonable irrigation is carried out on the gentian seeds and the seedlings,the soil moisture content is appropriately controlled,the salinization of the soil is strictly controlled,the growth and the physiological functions of the gentian seeds and the seedlings are limited after the soil salinization is prevented,The light-shielding net with light transmittance of 20-25% should be covered in the seedling stage of gentian,and the temperature can be properly controlled,so that the seed of the gentian can not germinate normally due to the low temperature,and the gentian seedling can not grow and grow normally,so as to provide a theoretical basis for further optimizing the method for cultivating the gentian. |
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