【作者】 王琪；

【导师】 王英平；

【作者基本信息】 吉林农业大学 ， 中药学， 2024， 博士

【摘要】 人参（Panax ginseng C.A.Meyer）为五加科人参属的多年生草本植物,是宝贵的中药资源。人参对生长环境要求严苛,极易受到极端温度、干旱和高盐等非生物胁迫因素的干扰,影响植株的正常生长发育,严重制约了人参产量和品质的提高。植物的进化历程漫长而复杂,其为适应外部环境变化,在抵抗非生物胁迫过程中,选择出一系列逆境应答基因,形成了复杂的逆境响应途径及基因调控网络,通过信号传导和逆境应答机制,维持物种正常发育、提高抗逆水平。挖掘人参抗逆基因,解析其逆境响应机制,提供人参分子育种理论依据,对抗逆人参新品种选育与开发具有十分重要的意义。胚胎发育晚期丰富蛋白（Late embriogenesis abundant protein,LEA）作为一类非生物胁迫应答蛋白,在提高植物对非生物胁迫的抵抗力和调控植物逆境生理中发挥了重要作用。本研究从全基因组水平挖掘鉴定了人参LEA基因家族,利用生物信息学分析了LEA理化性质、基因结构、表达调控、进化扩张等,利用公共转录组数据明确LEA基因在不同组织部位的表达模式以及对非生物胁迫和激素的响应情况,筛选并克隆在人参应答非生物胁迫进程中起关键作用的基因成员PgLEA2-50,基于烟草遗传转化进一步验证该基因在应答非生物胁迫中的功能。为了研究LEA蛋白应答非生物胁迫的作用机理,建立了人参原生质体介导的瞬时表达体系,通过免疫沉淀联合质谱分析（IP-MS）筛选出与PgLEA2-50相互作用的蛋白,并对关键靶蛋白展开分析。主要研究结论如下:1.人参全基因组中共鉴定出107个LEA成员（PgLEAs）,分布在8个亚族并以LEA2为主,不规律的分布在22条染色体,共有91个旁系同源基因对和6组串联重复基因。PgLEAs多为稳定性较高的碱性亲水蛋白,多定位于细胞膜、细胞质、叶绿体和细胞核等亚细胞结构,二级结构中富含α-螺旋和不规则卷曲。各亚族LEA基因的结构特征和保守基序具有组间差异性和组内保守性,而内含子数目普遍较少。基因本体论（Gene Ontology,GO）分析表明,PgLEAs参与非生物胁迫应答、植物信号转导、物质合成代谢和抗氧化等多个生物学过程。从PgLEAs上游调控区域主要鉴定出23类顺式作用元件,参与人参逆境应答、激素响应及生长发育等进程。对蛋白互作和转录因子调控的分析表明,PgLEAs与较多参与非生物胁迫的蛋白互作,受b ZIP、b HLH、ERF、NAC、HSF、CPP、BES1等多种与非生物胁迫或植物激素相关的转录因子调控。2.对PgLEAs在14个组织部位的表达模式分析显示,LEA蛋白在人参各个组织中均有分布,同时存在组织特异性,且在人参种子中特异表达的基因上游多分布着与种子发育相关的作用元件。部分PgLEAs的转录表达受激素的诱导和调控,但响应的类型和程度有所差异。进一步的差异分析表明,共有35个PgLEAs对激素处理有明显响应,其中21个PgLEAs受脱落酸（ABA）的调控,15个基因呈显著上调趋势;10个PgLEAs受赤霉素（GA）诱导而差异表达,5个为上调基因;吲哚乙酸（IAA）诱导下,有6个基因上调表达,2个基因下调表达;有的基因转录水平同时受多类激素的诱导而变化。PgLEAs对高温、低温、干旱和高盐胁迫均有响应,共有23个成员在非生物胁迫下差异表达,有10个LEA基因不止对某一种胁迫产生应答,如PgLEA2-41、PgLEA2-50、PgLEA2-51和PgLEA3-2以上调表达响应高温、低温和干旱3种胁迫。PgLEAs对ABA和干旱胁迫的响应尤为明显,对激素有明显响应的基因中,12个能同时响应非生物胁迫。其中,PgLEA2-50上游启动子调控区域广泛分布着对ABA、GA和SA等激素响应的元件,受非生物胁迫相关转录因子的调控,而在转录水平上PgLEA2-50在各组织部位高丰度表达,受GA的诱导而显著上调,对干旱、高温和低温均有明显响应,PgLEA2-50基因在人参对非生物胁迫的应答过程中较为关键。3.基于GUS表达的转录调控验证表明,PgLEA2-50启动子对下游基因有较高的驱动力,在各组织部位中均有转录活性。对PgLEA2-50过表达烟草株系的研究显示,PgLEA2-50行使功能的主要部位是细胞质,正向调控烟草对干旱和极端温度的抗逆水平。目的基因在烟草细胞的过表达不仅提高了烟草种子在干旱、高温和低温胁迫下的发芽率,转基因烟草植株在相应胁迫下比野生株系有更优的耐逆性和生理状态。对生理指标的检测结果显示,PgLEA2-50的大量表达有利于维持较高的总叶绿素水平,促进了非生物胁迫下的渗透物质积累,有效提高了超氧化物歧化酶（SOD）、抗坏血酸过氧化物酶（APX）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GSH-Px）的酶活性,显著降低了细胞中丙二醛（MDA）和过氧化氢（H₂O₂）的含量。人参LEA蛋白提高了烟草对活性氧（ROS）的防御水平和细胞抗氧化的能力,使其对干旱、高温和低温有更高的耐受性。4.通过优化对胚状体的解离条件和PEG介导的转化参数建立了人参的瞬时转化体系。体细胞胚在含2%（W/V）纤维素酶R-10和0.5%（W/V）果胶酶Y-23,0.7 M甘露醇的体系中解离效果较好,消化4 h时原生质体的数量超过10⁶/m L,酶解7 h时,产量超过了10⁷/m L,且活力处于较高水平（>90%）。使用40%PEG-4000转化质粒15 min时,基因的转化效率能达到48%。在此基础上的实时荧光定量PCR（q RT-PCR）和免疫印记（Western blot）检测、亚细胞定位等研究验证了该系统进行基因表达和蛋白互作研究的可行性。IP-MS实验中共检测到59个与PgLEA2-50潜在互作的蛋白质,涉及抗逆蛋白、转运蛋白、氧化还原酶、抗氧化酶和皂苷合成等多个类别。其中,PgLEA2-50和参与GA调控的途径蛋白有潜在的互作关系,如隶属于F-Box蛋白的SLEEPY1蛋白,GA通路的核心负调控因子DELLA蛋白等。PgLEA2-50与抗坏血酸过氧化物酶（APX）、过氧化氢酶（CAT）、谷胱甘肽S-转移酶（GST）的互作可能是转基因烟草株系中相关抗氧化酶活性提高的重要基础。此外,参与非生物胁迫的乳胶样蛋白（MLP）、ABC转运蛋白亚族的多向耐药性蛋白（PDR）和亚精胺合成酶等与PgLEA2-50间也可能存在相互作用。5.在GO和KEGG分析中,与PgLEA2-50互作的蛋白生物学功能呈现出多样化,涉及物质合成代谢、御逆抗逆、抗氧化活性调节等多个层次,并在不同代谢通路中行使功能,被富集至能量代谢、氧化磷酸化、糖酵解/糖异生、光合等生物学过程,同时参与植物激素信号转导和MAPK信号通路。对部分PgLEA2-50靶蛋白在干旱、低温、高温处理下的转录水平分析表明,肌动蛋白（Actin）、谷氨酸脱羧酶、MYB、CAT、APX、DELLA等至少能响应其中一种胁迫,这些基因的调控区域均具有应答非生物胁迫和激素诱导的作用元件。PgLEA2-50上游启动子序列中分布着MYB的结合位点,且PgMYB7与PgLEA2-50在几种非生物胁迫下的表达趋势相一致。PgMYB7与PgLEA2-50启动子的双荧光素酶实验表明,PgMYB7能结合PgLEA2-50启动子,并正向调控启动子活性。PgMYB7在原生质体中的过表达提高了PgLEA2-50的转录水平。构建DELLA基因PgRGA4与FLAG标签的融合表达载体,共转化至原生质体后,PgRGA4-FLAG与PgLEA2-50-GFP的CO-IP实验进一步证明二者存在相互作用。更多还原

【Abstract】 Panax ginseng C.A.Meyer,a herb of significant medicinal value,is a prized resource in traditional Chinese medicine and belongs to the genus Panax within the Araliaceae family.Ginseng,a plant species with strictly environmental requirements,is highly susceptible to abiotic stress factors,including extreme temperatures,drought,and high salinity,which affects the normal growth and development of plants and seriously restricts the improvement of ginseng yield and quality.The evolution process of plants is long and complex.In response to such challenges,plants have evolved to select specific stress response genes during their resistance to abiotic stress.These genes form intricate stress response pathways and gene regulation networks.Through intricate signal transduction and stress response mechanisms,plants maintain their normal growth and development while enhancing their stress resistance.Therefore,it is of great significance to excavate the stress-resistant genes of ginseng,analyze its stress response mechanism,provide the theoretical basis for molecular breeding of ginseng.This will facilitate the cultivation and development of new,stress-tolerant ginseng varieties.Late embriogenesis abundant protein(LEA),a type of abiotic stress response protein,plays a crucial role in enhancing plant resilience against abiotic stress and regulating plant stress physiology.In this study,the ginseng LEA gene family was identified from the whole genome level,and the physicochemical properties,gene structure,expression regulation,evolutionary expansion of LEA were analyzed by means of bioinformatics.Based on the public transcriptome data,the expression pattern of LEA gene family in different tissues and the response to abiotic stress and hormones were clarified.The gene member PgLEA2-50,which played a key role in the response of ginseng to abiotic stress,was screened and cloned.The important function of the gene in abiotic stress was further determined based on the genetic transformation of tobacco.To investigate the mechanism of LEA protein’s response to abiotic stress,the ginseng protoplast-based transient expression system was established initially.The proteins interacting with PgLEA2-50 were preliminarily screened by immunoprecipitation combined with mass spectrometry(IP-MS),and some target proteins were further analyzed.The main conclusions are as follows:1.A total of 107 LEA members(PgLEAs)were identified from the whole genome of Panax ginseng,which were distributed in eight subfamilies and dominated by LEA2,and irregularly distributed on 22 chromosomes.There were 91 paralogous gene pairs and six groups of tandem repeat genes.Most PgLEAs were alkaline hydrophilic proteins with high stability,which were mostly located in subcellular structures such as cell membrane,cytoplasm,chloroplast and nucleus.The secondary structure was rich inα-helix and irregular curl.The structural characteristics and conserved motifs of LEA genes in each subfamily had inter-group differences and intra-group conservation,while the number of introns was generally small.The Gene Ontology(Gene Ontology,GO)analysis showed that PgLEAs were involved in many biological processes such as abiotic stress response,plant signal transduction,anabolism and antioxidation.From the upstream regulatory region of PgLEAs,23 types of cis-acting elements were identified,which may be involved in the process of ginseng response to adversity,hormone treatment and growth or development.The analysis of protein interaction and transcription factor regulation showed that PgLEAs interacted with many proteins involved in abiotic stress and were regulated by b ZIP,b HLH,ERF,NAC,HSF,CPP,BES1 and other transcription factors related to abiotic stress or plant hormones.2.The expression pattern analysis of PgLEAs in 14 tissues showed that LEA proteins were widely distributed in ginseng and had tissue specificity.Some PgLEAs had strong tissue specificity,and the cis-acting elements related to seed development were mostly distributed in the upstream of the genes specifically expressed in ginseng seeds.The transcriptional expression of some PgLEAs were induced and regulated by hormones,but the type and degree of response were different.Further differential analysis showed that a total of 35 PgLEAs were significantly responsive to hormone treatment,of which 21 PgLEAs were regulated by abscisic acid(ABA),and 15 genes were significantly up-regulated.Ten PgLEAs were differentially expressed by gibberellin(GA)induction,and five genes were up-regulated.Under the induction of indoleacetic acid(IAA),six genes were up-regulated and two genes were down-regulated.The transcription level of some genes were induced by multiple hormones at the same time.PgLEAs possess the ability to respond to high and low temperatures,drought,and high salt stress.A total of 23 members were differentially expressed under abiotic stress,and ten LEA genes responded to more than one treatment,such as PgLEA2-41,PgLEA2-50,PgLEA2-51 and PgLEA3-2,which were significantly up-regulated in response to high temperature,low temperature and drought stress.The response of PgLEAs to ABA and drought stress was particularly obvious.Among the genes exhibiting a pronounced response to hormones,12 genes concurrently demonstrate the ability to respond to abiotic stresses.Aamong them,the upstream promoter regulatory region of PgLEA2-50 was widely distributed with elements responding to hormones such as ABA,GA and SA and regulated by abiotic stress-related transcription factors.From the transcription level,it was found that PgLEA2-50 was highly abundant in various tissues and significantly up-regulated by GA induction.In addition,it had obvious response to drought,high temperature and low temperature,so it was speculated that PgLEA2-50 gene may play a key role in the response of ginseng to abiotic stress.3.The transcriptional regulation verification based on GUS expression showed that the PgLEA2-50 promoter had a high driving force for downstream genes and had transcriptional activity in all tissues.After the construction of PgLEA2-50 overexpression vector,transgenic tobacco lines with PgLEA2-50 overexpression were cultivated based on the Agrobacterium-mediated transformation.The study of PgLEA2-50 overexpressing tobacco lines showed that the main functional site of PgLEA2-50 was the cytoplasm,and positively regulates the resistance level of tobacco to drought and extreme temperature.The overexpression of the target gene in tobacco cells not only increased the germination rate of tobacco seeds under drought,high temperature and low temperature,but also showed better stress tolerance and physiological status than wild strains under the corresponding stress.The results of physiological indexes showed that the high expression of PgLEA2-50 was conducive to maintaining a high total chlorophyll level,promoting the accumulation of osmotic substances under abiotic stresses,effectively improving the enzyme activities of superoxide dismutase(SOD),ascorbate peroxidase(APX),catalase(CAT)and glutathione peroxidase(GSH-Px),and significantly reducing the content of malondialdehyde(MDA)and hydrogen peroxide(H2O2)in cells.Ginseng LEA protein improved the defense level of reactive oxygen species(ROS)and the ability of cell antioxidant in tobacco,and made it more tolerant to drought,high temperature and low temperature.4.The transient transformation system for ginseng was developed by refining the dissociation conditions of embryoids and regulating the parameters of PEG-mediated transformation.The isolation effect of somatic embryo was better in the system containing 2%(W/V)cellulase and 0.5%(W/V)pectinase and 0.7 M mannitol.The number of protoplasts reached 10~6/m L at 4 h of digestion,and the yield exceeded 10~7/m L at 7 h of enzymolysis with the activity was always at a high level(>90%).When 40%PEG-4000 was used to transform the plasmid for 15 min,the transformation efficiency of the gene could reach 48%.On this basis,real-time fluorescence quantitative PCR(q RT-PCR),western blot(WB)detection,and subcellular localization studies also verified the feasibility of the system for gene expression and protein interaction studies.A total of 59 proteins potentially interacting with PgLEA2-50 were detected in IP-MS experiments,mainly involving stress-resistant proteins,transporters,oxidoreductases,antioxidant enzymes and saponin synthesis.Among them,PgLEA2-50 had a potential interaction with the pathway proteins involved in GA regulation,such as SLEEPY1protein belonging to F-Box protein,and DELLA protein,which are the core negative regulator of GA pathway.The interactions of PgLEA2-50 with APX,CAT and GST may be important basis for the improvement of related antioxidant enzyme activities in transgenic tobacco lines.Additionally,PgLEA2-50 may engage in interactions with multiple proteins,including the latex-like protein(MLP),the pleiotropic drug resistance transporter(PDR)from the ABC transporter subfamily,and spermidine synthase,all of which play crucial roles in abiotic stress responses.5.In GO and KEGG analysis,the biological functions of proteins interacting with PgLEA2-50 were diversified,involving multiple fields of substance synthesis and metabolism,stress resistance,antioxidant activity regulation,and performing functions in different metabolic pathways.It was enriched to the biological processes of energy metabolism,oxidative phosphorylation,glycolysis/gluconeogenesis,photosynthesis,and participated in plant hormone signal transduction and MAPK signaling pathway.The transcription analysis of some PgLEA2-50 target proteins under drought,low and high temperature treatments showed that the actin,glutamic acid decarboxylase,MYB,CAT,APX,and DELLA can respond to at least one of these stresses.The regulatory regions of these target genes contained response elements for abiotic stresses and hormone induction.The MYB binding sites were distributed in the upstream promoter sequence of PgLEA2-50,and the expression trends of PgMYB7 and PgLEA2-50 were consistent under several abiotic stresses.The dual-luciferase assay of PgMYB7 gene and PgLEA2-50 promoter showed that the PgMYB7 can bind to the PgLEA2-50 promoter and positively regulate its activity.Overexpression of PgMYB7 in ginseng protoplasts increased the transcription level of PgLEA2-50.A fusion expression vector was constructed by combining the DELLA gene PgRGA4 with a FLAG tag.After co-transformation to protoplasts,a CO-IP experiment was conducted to investigate the interaction between PgRGA4-FLAG and PgLEA2-50-GFP.更多还原