【作者】 李敏；

【导师】 韩梅；

【作者基本信息】 吉林农业大学 ， 药用植物， 2021， 博士

【摘要】 桔梗[Platycodon grandiflorum(Jacq.)A.DC.]为桔梗科桔梗属多年生草本植物,该属全世界仅1种1变种,广布于我国东部温带至亚热带的湿润半湿润地区,是我国大宗常用中药材,也是典型的药食同源植物。桔梗以其干燥根入药,具有宣肺、散寒、祛痰、消肿、镇咳、排脓等功效。表征其药材质量的主要药效成分为桔梗皂苷类成分,其中桔梗皂苷D含量是《中华人民共和国药典》规定的质量标准成分。药材质量的形成与控制研究是其生产技术创新的重要基础。水是植物光合作用的基础物质,也是植物各种生理代谢过程的重要介质和参与者,在农业生产中水分调控也是提高作物产量和质量的重要农艺措施,因此,研究水因子对药材产量和质量形成的影响,特别是干旱逆境对药材质量的影响具有重要意义。有关水及其调控对桔梗药材产量和质量影响的研究较少,其对干旱胁迫的生理与分子生态响应的研究尚属空白,为此,我们采用盆栽试验,模拟自然环境,对同一种源、相同年限桔梗进行水分调控,通过对不同生长时期(营养期、花期和果期)的桔梗进行干旱胁迫,分析研究了桔梗的光合生理、抗逆生理、皂苷含量及其生物合成基因表达等变化对干旱胁迫的响应,通过转录组学分析揭示其对干旱响应的分子机制(基因调控网络),阐释桔梗皂苷合成对干旱逆境响应的分子生态过程,旨在为桔梗药材生产实施水分生态调控提供科学依据。主要研究结论如下:(1)不同生长时期干旱胁迫下,桔梗初生代谢产物(12种氨基酸和8种核苷类成分)对水分信号响应机制不同。DS(干旱胁迫最重)阶段,营养期(6种)和果期(11种)桔梗的初生代谢产物受负面影响较大;花期桔梗仅4种核苷类成分受到负面影响。AT(秋季)阶段,营养期桔梗中仅ALA含量显著提高;花期和果期桔梗分别有7种和4种初生代谢产物积累显著增加。另外,DS阶段,营养期和花期桔梗中ALA、次黄嘌呤、别嘌醇含量显著降低,而果期桔梗中ALA、次黄嘌呤、别嘌醇却分别提高了28.04%、31.09%、22.39%。总体来说,当干旱发生时,由于桔梗所处的生育状况不同,初生代谢产物对水分响应方式存在差异,从直接影响(DS)和长期影响(AT)来看,干旱胁迫对营养期和果期桔梗初生代谢产物的积累多为负面,而对花期桔梗则多为正向促进。同时,对于干旱胁迫,同一生长时期桔梗的氨基酸或核苷类成分的生物合成调控表现出促进和抑制相互转换的双重性分子生态响应策略。(2)干旱胁迫对果期桔梗的调控更有利于桔梗药材质量的提高。DS阶段,营养期、花期、果期桔梗PD3、DPD积累具有一致性,均显著高于CK(p<0.05)。桔梗可能通过次生代谢产物的积累来积极以抵御短期干旱逆境。AT阶段,营养期桔梗仅PD3和SPD含量提高,花期桔梗皂苷(PD、PD3、DPD、SPD)均未显著积累,而果期桔梗三种单体皂苷(PD、PD3、DPD)和桔梗总皂苷(SPD)含量分别提高了8.95%、11.67%、12.54%、21.67%。次黄嘌呤是影响营养期桔梗皂苷PD、PD3的生物合成的重要初生代谢产物;多种氨基酸及核苷对花期桔梗中PD的积累有显著的促进作用;鸟嘌呤、ALA对果期桔梗皂苷的积累有显著的促进作用,而VAL对果期桔梗皂苷积累多为负面的,说明果期桔梗中鸟嘌呤、ALA和VAL是影响其桔梗皂苷积累的重要因素,是调控果期桔梗质量的重要初生代谢产物。从干旱胁迫长期影响(AT阶段)来看,与营养期和花期相比,干旱胁迫诱导果期桔梗中桔梗皂苷类成分大量合成,是把控桔梗药材质量的关键时期。(3)不同生长时期桔梗的光合生理、抗性生理、次生代谢对干旱胁迫响应机制研究表明:DS阶段,三个生长时期桔梗因体内积累大量的活性氧,使其光合生理特性减弱,桔梗通过提高保护酶T-SOD、POD和CAT活性的方式抵御干旱带来的细胞氧化伤害。DS阶段,花期桔梗SS、PRO含量分别为CK的2.37倍和1.53倍,其SP含量显著降低,营养期桔梗PRO、SP均没有显著增加,而果期SP、PRO显著增加。不同生长时期干旱胁迫下,桔梗不同生理应激指标间相互促进和制约,对干旱胁迫做出协同反应,维持桔梗体内生理平衡,但不同生长时期桔梗对干旱胁迫响应并不一致,揭示桔梗适应逆境生理变化的复杂性。营养期桔梗中PD、PD3与T-SOD,DPD与CAT、POD,SPD与SS显著正相关(p<0.05);花期桔梗DPD和PD3与根干重、PRO、φPSII显著正相关(p<0.05);果期桔梗PD与SS、φPSⅡ显著正相关(p<0.05),PD、PD3与CAT和WUE均极显著正相关。揭示桔梗皂苷的合成与光合作用、叶绿素荧光、渗透调节物质和保护酶之间存在相关性。(4)不同生长时期桔梗皂苷生物合成关键酶对水分调控响应过程分析表明:受干旱胁迫影响,三个生长时期DS阶段上调基因数目的差异(8个、6个、3个)是不同生长时期桔梗对水分的区别响应,也是影响此阶段桔梗皂苷合成差异的主要因素。岭回归分析显示,营养期桔梗HMGR、IPPI、MVD、PMK、SS、β-A28O的显著上调,花期桔梗HMGR、UGT1的显著上调,果期桔梗PMK的显著上调,是各时期桔梗皂苷在DS阶段积累的重要影响因素。同时β-A28O和β-AS在AT阶段的显著上调是果期桔梗皂苷在秋季显著积累的主要关键酶基因。(5)果期桔梗不同阶段(F＿DS、F＿RW、F＿AT)转录组分析表明:分别在F＿DS、F＿RW、F＿AT中差异表达基因(DEGs)有2087、1453、1447个,说明大部分DEGs是在F＿DS阶段被诱导表达的。萜类骨架和三萜类化合物合成途径中有5个DGEs(TRINITY＿DN21285＿c0＿g9、TRINITY＿DN22095＿c1＿g3、TRINITY＿DN24750＿c0＿g3、TRINITY＿DN22394＿c1＿g10、TRINITY＿DN27385＿c1＿g4)仅在F＿DS阶段表达模式发生变化,这些基因可能是果期桔梗皂苷合成途径中参与干旱胁迫应答的重要调控基因。干旱胁迫对果期桔梗NAC转录因子中Group1和Group 3基因有明显抑制作用,而对Group 4基因有明显诱导作用;对C3H转录因子中Group1和Group 3基因有明显抑制作用。推测和NAC C3H转录因子可能与果期桔梗抵御干旱逆境密切相关。更多还原

【Abstract】 Platycodon grandiflorum is a perennial herb of Platycodon genus in Platycodon family.This genus has only one species and one variety in the world.Platycodon grandiflorum is widely distributed in humid and semi-humid areas from temperate to subtropical zone in eastern China,which is a large number of commonly used Chinese medicinal materials and a typical medicinal and food homologous plant in China.The root of Platycodon grandiflorum is a famous Chinese medicinal material,which has the functions of promoting lung,removing phlegm,dispersing cold,relieving cough,reducing swelling and discharging pus.An essential component to characterize the quality of Platycodon grandiflorum are the platycodin,and platycodin D is the standard component stipulated in the Pharmacopoeia of the people’s Republic of China.The technological innovation of medicinal materials production is based on the formation and control of medicinal materials quality.Water is the basic substance of plant photosynthesis,and which is also an important medium and participant in various physiological metabolic processes of plants.Water regulation is also an important agronomic measure to improve crop yield and quality in agricultural production.Therefore,it is important to study the influence of water factors on the yield and quality of medicinal materials,especially the effect of drought stress on the quality of medicinal materials.The effects of water regulation on yield and quality of Platycodon grandiflorum were less studied.The physiological and molecular ecological response of water to drought stress is still blank.To this end,we used pot experiments to simulate the natural environment,and Platycodon grandiflorum of the same source and same age were used for water regulationthe.Drought stress of Platycodon grandiflorum during different growth periods(vegetative,flowering and fruiting)was observed.The responses of photosynthetic physiology,stress resistance physiology,platycodin content and biosynthesis gene expression of Platycodon grandiflorum to drought stress were studied.The molecular mechanism of drought response(gene regulation network)was revealed by transcriptome analysis,and the molecular ecological process of platycodin synthesis in Platycodon grandiflorum was explained.The aim is to provide scientific basis for water ecological regulation in the production of Platycodon grandiflorum.The main conclusions are as follows:(1)The response mechanism of primary metabolites(12 amino acids and 8 nucleosides)to water signal was different under drought stress during different growth periods of Platycodon grandiflorum.During the DS,the primary metabolites of Platycodon grandiflorum(6 species)and fruit stage(11 species)were negatively affected by drought stress.Only 4 nucleoside components of Platycodon grandiflorum were negatively affected by flower period drought stress.Only the ALA content increased significantly in AT(autumn)stage of vegetative period of Platycodon grandiflorum,while the accumulation of 7 and 4 primary metabolites increased significantly in flower and fruit periods,respectively.The contents of ALA,hypoxanthine and allopurinol,decreased significantly in the DS stage of vegetative and flower periods Platycodon grandiflorum,while that of ALA,hypoxanthine and allopurinol increased 28.04 %,31.09 %and 22.39 % in fruit periods Platycodon grandiflorum.In general,when drought occurs,the response mode of primary metabolites to water was different due to the different fertility conditions of Platycodon grandiflorum.Judging from the direct effects(DS)and long-term effects(AT),drought stress had a negative effect on the accumulation of primary metabolites of vegetative and fruit periods Platycodon grandiflorum,but had mostly positive effect on the flower period of Platycodon grandiflorum.At the same time,for drought stress,the biosynthesis regulation of amino acids or nucleoside components of Platycodon grandiflorum during the same growth period showed dual molecular ecological response strategies to promote and inhibit mutual transformation.(2)The regulation of fruit Platycodon grandiflorum by drought stress may be more beneficial to the improvement of the quality of Platycodon grandiflorum.The PD3、DPD accumulation in the DS stage of Platycodon grandiflorum in the three growth periods was consistent,all of them were significantly higher than that in CK(p <0.05).It can be seen that Platycodon grandiflorum may actively cope with adverse environmental conditions through the accumulation of secondary metabolites,which may be a means for Platycodon grandiflorum to resist short-term drought stress.During the AT stage,the PD3 and SPD contents increased only in flower period Platycodon grandiflorum,and the platycodin(PD、PD3、DPD、SPD)did not accumulate significantly in flower period Platycodon grandiflorum,while the contents of platycodin(PD 、 PD3 、 DPD)and total saponins(SPD)of fruit period Platycodon grandiflorum increased by 8.95 %,11.67 %,12.54 % and 21.67 %,respectively.Hypoxanthine is an important primary metabolite that affects the biosynthesis of PD and PD3 in platycodon grandiflorum during vegetative period.A variety of amino acids and nucleosides significantly promoted the accumulation of PD in platycodon grandiflorum.Guanine and ALA significantly promoted the accumulation of platycodin at fruit stage,while VAL was mostly negative on the accumulation of platycodon at fruit stage,indicating that guanine,ALA and Val were important factors affecting the accumulation of platycodon grandiflorin at fruit stage and important primary metabolites regulating the quality of platycodon grandiflorin at fruit stage.From the long-term effect of drought stress,compared with the vegetative stage and flowering stage,drought stress induced the synthesis of a large number of platycodon in Platycodon grandiflorum at fruit stage,which was the key period to control the quality of Platycodon grandiflorum.(3)Studies on the response mechanism of photosynthetic physiology,resistance physiology and secondary metabolism of Platycodon grandiflorum in different growth stages to drought stress showed: The three growth periods of Platycodon grandiflorum accumulated a large amount of reactive oxygen species in the DS stage,which affected photosynthetic physiology.They all resist the oxidative damage caused by drought stress,by increasing the TSOD、POD and CAT activity of protective enzymes.In DS stage,the SS、PRO content in flower period of Platycodon grandiflorum was 2.37 times and 1.53 times of the CK,and the SP content decreased significantly,the content of PRO、SP did not increase significantly in vegetative period of Platycodon grandiflorum,but the SP、PRO increased significantly in fruit period of Platycodon grandiflorum.It is speculated that different osmotic regulators may respond to drought stress by synergistic or antagonistic action in Platycodon grandiflorum.The response of the same osmotic adjustment to drought stress in Platycodon grandiflorum was not consistent.There were obvious positive correlation between PD、PD3 and T-SOD,DPD and CAT、POD,SPD and SS,in vegetative period of Platycodon grandiflorum(p < 0.05).In flower period of Platycodon grandiflorum,the DPD and PD3 were positively correlated with the root dry weight、PRO and φPSII,respectively(p < 0.05).The PD was positively correlated with SS、φPSⅡ,PD、PD3 were positively correlated with CAT and WUE in fruit period of Platycodon grandiflorum(p < 0.05).To sum up,there was some correlation between the secondary metabolite(platycodin)accumulation and photosynthesis,chlorophyll fluorescence,osmotic regulator,protective enzyme in Platycodon grandiflorum.(4)Analysis of the key enzymes in the biosynthesis of platycodin during different growth periods showed that: The difference in the number of up-regulated genes(8,6,3)in DS stages of the three growth periods of Platycodon grandiflorum,which was the differential response to water in different growth periods,and also the main factor affecting the difference in the synthesis of platycodin at this stage by drought stress.Ridge regression analysis showed that,the significant up-regulation of HMGR,IPPI,MVD,PMK,SS and β-A28 O in Platycodon grandiflorum at the vegetative period,HMGR and UGT1 in Platycodon grandiflorum at the flowering period,and PMK in the fruit period were the important influencing factors for the accumulation of platycodin at each stage in DS stage.At the same time,β-A28 O and β-AS were significantly up-regulated at the AT stage,which were the key enzyme genes for the significant accumulation of platycodin in fruit period in autumn.(5)At different stages(F＿DS、F＿RW、F＿AT)of fruit period Platycodon grandiflorum,transcriptome analysis of showed: In the stages of F＿DS、F＿RW、F＿AT,contained DEGs2087、1453、1447,respectively,which indicated that most of the DEGs were induced in the F＿DS stage.The five DGEs in the biosynthesis pathway of triterpenoids showed significantly down-regulated in the stages of F＿DS 、 F＿RW 、 F＿AT(TRINITY＿DN18371＿c0＿g1 、TRINITY＿DN20252＿c1＿g4 、 TRINITY＿DN22757＿c0＿g2 、 TRINITY＿DN21641＿c0＿g5 、TRINITY＿DN24994＿c0＿g2).These genes may be important regulatory genes involved in drought stress response in the synthesis pathway of platycodin in fruit period Platycodon grandiflorum.Drought stress had obvious inhibitory effect on genes of Group1 and Group 3 in NAC transcription factors of Platycodon grandiflorum,but had obvious induction effect on genes of Group 4,and on Group1 and Group 3 genes in transcription factors.And drought stress had obvious inhibitory effect on genes of Group1 and Group 3 in C3 H transcription factors of Platycodon grandiflorum.NAC and C3 H transcription factors may be closely related to the resistance of Platycodon grandiflorum to drought stress.更多还原