【作者】 高岩；

【导师】 韩梅；

【作者基本信息】 吉林农业大学 ， 生态学， 2019， 硕士

【摘要】 人参（Panax ginseng C.A.Meyer）为多年生草本植物,其干燥根及根茎入药,是传统名贵中药材,人参皂苷为人参主要药效成分,是评价其药材质量重要标准,具有补脾益肺、生津止渴、提高免疫力等功效。目前人参药材主要来自于栽培,吉林省是主要人工栽培地,人参栽培受外界环境条件的影响使其药材质量出现差异。相关研究发现,水分是影响人参质量的重要生态因子,通过调控其合成途径关键酶基因表达而影响皂苷的生物合成。本研究以3年生盆栽人参及其根源愈伤组织为试验材料,从植物生理及分子生态学的角度,开展不同水分调控下人参皂苷积累的动态变化规律研究,初步探讨人参药材质量形成的分子生态机制,对完善人工栽培生产过程中水分管理、实现人参质量调控具有重要意义和应用价值。本文对不同水分调控下人参皂苷积累规律进行研究,在此基础上,对人参皂苷合成过程中关键酶基因表达量、保护酶（SOD、POD、CAT）活性及丙二醛（MDA）含量进行测定,并进行相关性分析,进一步阐释人参皂苷积累及关键酶基因表达量、保护酶活性对水分调控的响应规律,最终为实现药材质量的生态调控提供理论依据。主要结果如下:1、通过水分调控对人参愈伤组织及盆栽人参生物量及皂苷含量的研究发现,高浓度PEG从15 d开始对人参愈伤组织鲜重产生抑制作用并达到显著水平10%、20%PEG分别促进单体皂苷Re和Rg₁的积累,并于20 d达最大值为CK组的1.93倍和3.05倍;盆栽人参土壤相对含水量为60%和80%时对人参生长具有显著促进作用,土壤相对含水量为40%显著抑制人参生长。盆栽试验中人参皂苷的积累对水分调控响应较为敏感,当土壤含水量为80%时在水分调控末期相对于40%的土壤相对含水量总皂苷提高了15%,60%土壤相对含水量对PPD和PPT型人参皂苷积累具有短时促进作用。2、水分调控对愈伤组织及盆栽人参中抗氧化酶（SOD、POD、CAT）活性产生不同程度影响。愈伤组织中SOD活性变化趋势与POD基本一致,呈现出降-升的变化趋势,CAT活性变化趋势与之相反,随培养时间的延长,抗氧化酶活性呈下降趋势,但MDA含量逐渐增加;盆栽试验中,CAT和SOD酶活性变化相似呈下降趋势随着处理时间的延长氧自由基过度累积超出保护酶的修复范围导致活性下降,细胞脂膜受损MDA含量增加,SOD、POD和CAT三者协同作用共同抵御不利条件。3、研究水分调控下人参皂苷生物合成途径中关键酶基因（HMGR、SS、β-AS、CYP716A47、CYP716A52v2、CYP716A53v2）表达量变化发现,人参皂苷合成关键酶基因在10%及20%PEG刺激下快速上调,其中HMGR、SS、CYP716A53v2在人参愈伤组织整个培养周期内活跃表达,β-AS、CYP716A47、CYP716A52v2在培养后期表达水平上调;盆栽试验中,相对于土壤相对含水量为40%时,60%和80%的土壤相对含水量促进了HMGR、β-AS、CYP716A47、CYP716A53v2基因的表达。因此,HMGR、SS、CYP716A47、CYP716A52v2、CYP716A53v2可作为人参质量调控的候选基因。4、对不同水分调控下人参皂苷含量、关键酶基因表达量、抗氧化酶活性及MDA含量进行相关性分析发现,10%PEG处理下Rg₁与CYP716A52v2基因表达量呈显著正相关（r=0.587,P<0.01）,20%PEG处理Re与CYP716A52v2基因呈显著负相关（r=0.574,P<0.05）;盆栽试验中,土壤相对含水量为60%时PPD型人参皂苷与HMGR、β-AS基因表达量呈极显著正相关（r=0.700、0.729,P<0.01）,土壤相对含水量为80%时PPT型人参皂苷与HMGR、β-AS表达量呈极显著正相关（r=0.701、0.679,P<0.01）,说明HMGR、β-AS、CYP716A47可能是人参皂苷合成响应水分调控的重要基因。更多还原

【Abstract】 Panax ginseng C.A.Mey is a traditional Chinese perennial herb with roots and rhizomes.Ginseng ginsenoside is an important criteria for evaluating the quality,with spleen lung,thirst,improve the immune system and other functions efficiency.At present,ginseng medicinal materials are mainly from cultivation,while Jilin Province is the main artificial cultivation area.Related studies have found that water has a great influence on the growth of ginseng,and affects the formation of saponin content by acting on the expression of key enzyme genes in the saponin synthesis pathway.The study used ginseng root callus and triennial potted ginseng as experimental materials,from the perspective of plant physiology and molecular ecology,the study on the response of ginsenosides under water regulation,explore the molecular mechanisms of ginseng herbs ecological quality of the formation has important significance and value to improve the artificial cultivation of the production process of water management,the quality of ginseng regulation.The study explore the effects of different ginsenosides on water,on this basis,respectively ginsenoside synthesis key enzyme gene expression,enzyme activity and MDA content protection study and correlation analysis,the medicinal ingredient is further illustrated Synthesis The response of the key enzyme gene expression and protective enzyme activity to the environment,and ultimately the regulation of the quality of the drug.The main results are as follows:1.Study of ginseng callus and potted ginseng saponin content and biomass,high concentration of PEG inhibited significantly the fresh weight of ginseng callus from 15 d,10%,20%PEG promote respectively of Re and Rg1.And reached maximum on 20d,it is1.93 times and 3.05 times of the CK;Ginseng potting soil water content of 60%and 80%is significantly promoted the growth of ginseng,the soil relative water content of 40%significantly inhibited the growth of ginseng.;The accumulation of ginsenosides in pot experiment is sensitive to water regulation,80%,the total saponin relative to 40%of the soil is increased by 15%at the end of the water regulation.60%soil relative water content has a short-term promoting effect on PPD and PPT ginsenoside accumulation.2.Water regulation has different effects on the activities of antioxidant enzymes（SOD,POD,CAT）in callus and potted ginseng.the change trend of SOD activity in callus is the same as POD,it showed down-up trend,the change of CAT activity was opposite.With the prolongation of culture time,the activity of antioxidant enzymes decreased,but the content of MDA increased gradually;In pot experiment,the activity of CAT and SOD decreased similarly.With the prolongation of time excessive accumulation of oxygen free radicals beyond the protection and restoration of the scope of enzyme resulted in decreased activity,Damage to cell lipid membrane and increased MDA content.It shows that SOD,POD and CAT work together to resist adverse conditions.3.Study the key enzymes gene expression changes（HMGR,SS,β-AS,CYP716A47,CYP716A52v2,CYP716A53v2）in ginsenoside biosynthetic pathway in the water regulation.The key enzyme gene for ginsenoside synthesis was up-regulated rapidly in 10%and 20%PEG.β-AS、CYP716A47、CYP716A52v2 were highly expressed in the end of culture;In pot experiment,the relative water content of soil was 40%.60%and 80%of soil relative water content promoted the expression of HMGR,β-AS,CYP716A47 and CYP716A53v2,HMGR,SS and CYP716A53v2 are actively expressed in the whole culture time of ginseng callus.Therefore,HMGR,SS,CYP716A47、CYP716A52v2 and CYP716A53v2 can be used as candidate enzymes genes for ginseng quality control.4.To analysis the water regulation of ginsenoside content,key enzyme gene expression,antioxidant enzyme activity and MDA content,found that under 10%PEG treatment there was a significant positive correlation（r=0.587,p<0.01）,between Rg₁ and CYP716A52v2 gene expression.Under 20%PEG Re and CYP716A52v2 showed significantly negative correlation（r=0.574,p<0.05）;In pot experiment,60%soil relative water,PPD ginsenoside was positively correlated with HMGR andβ-AS expression（r=0.700,0.729,p<0.01）.When the relative water content of soil was 80%,the expression of PPT ginsenosides was significantly positively correlated with the expression of HMGR andβ-AS（r=0.701,0.679,P<0.01）.These results suggest that HMGR,β-AS and CYP716A47 may be important genes of ginsenoside synthesis in response to water regulation.更多还原