【作者】 周燕；

【导师】 李凤兰； 高述民；

【作者基本信息】 北京林业大学 ， 植物学， 2009， 博士

【摘要】 胡杨（Populus euphratica Oliver）是我国及中亚腹地干旱荒漠区中特有的珍贵森林资源,具有耐盐碱、耐旱、抗寒、抗风沙等特性,对于维持和调控干旱荒漠地区脆弱的生态平衡、改善绿洲农业生态环境具有重要意义。由于胡杨实生繁殖有限且容易造成性状分离,无性繁殖日趋显得重要。但胡杨插条繁殖生根困难,成为制约胡杨产业发展的瓶颈问题,亟待解决。论文利用胡杨不定根再生调控体系,采用显微、气质联用（GC-MS）和cDNA克隆等技术,从细胞及分子水平研究了胡杨不定根发生前后的细胞组织结构及激素、同工酶等生理生化指标的变化规律;对克隆得到的cDNA差异片段进行了生物信息学分析。研究为深入探讨胡杨不定根发生发育的分子机制,实现人为调控胡杨不定根发生奠定重要的工作基础,对胡杨种质保存和遗传改良具有重要的理论和实践意义。论文的实验结果如下:1.首次用胡杨花序为外植体建立了稳定的胡杨组织培养快繁体系。胡杨雌、雄花序诱导愈伤组织在所涉及的培养基配方内最适培养基是以B₅为基本培养基,雌花序添加1.0 mg·L^-1 6-BA和0.5 mg·L^-1 NAA或雄花序添加1.0 mg·L^-1 6-BA和1.0mg·L^-1 NAA,诱导率达98.0%,绿色愈伤组织诱导率均在63.0%以上,绿色愈伤组织与芽的形成呈正相关。愈伤组织在B₅培养基上添加1.0 mg·L^-1 6-BA和1.25 mg·L^-1NAA或MS培养基上添加1.0 mg·L^-1 6-BA和0.5 mg·L^-1 NAA继代增殖较好。雌、雄花序以MS为基本培养基,添加0.5 mg·L^-1 6-BA和0.2 mg·L^-1 NAA,可诱导愈伤组织产生不定芽,长大的芽在1/2MS+0.5 mg·L^-1 IBA可诱导生根,生根率35.0%以上。采用花序作为外植体进行组织培养再生幼苗的周期短于其它外植体近2周。2.建立了除愈伤组织以外的胡杨实生苗插穗和芽诱导不定根发生的调控培养体系,可用作研究胡杨不定根发生与形态建成的良好实验系统。观察和分析了胡杨实生苗插穗不定根发生发育的细胞组织显微和超微结构特征。胡杨插穗培养在无IBA的培养基上,36 h在维管束周围有大核细胞发生;48 h有糊粉粒积累且富含糊粉粒的细胞相对集中;60 h根原基有维管束的形成,胡杨不定根原基发生在皮层维管组织邻近的薄壁细胞（相当于髓射线部位）处;72 h生成不定根,此时,核膜完整,核比较大,核仁多呈环状结构。而插穗培养在添加了0.5 mg·L^-1 IBA的培养基上,根原基的形成受到抑制,插穗基部细胞脱分化,形成愈伤组织。3.利用GC-MS技术,分析了与不定根发生相关的激素的变化规律。在不定根形成过程中,内源激素IAA和ABA可作为诱导根原基发生的重要信号分子。尤其是IAA,当浓度在一定范围内且处于较稳定状态时,有利于促进不定根的发生;如果IAA浓度超过一定的阈值且不稳定,则抑制不定根的发生;IAA与其他激素如GA₃和ABA等之间的比值也影响不定根的发生;当内源激素GA₃处于一个较稳定状态时,有利于不定根的形成。4.采用聚丙烯酰胺凝胶电泳（PAGE）技术检测到过氧化物酶（POD）2 b谱带的持续表达,它有利于根原基诱导,这与促进IBAβ氧化作用,激活抑制根生长的IAA,使其浓度达到有效的促根阈值有关。淀粉酶在60 h时表达增强,是根原基发育的标志。酯酶在根原基诱导时活性很低,而在根原基发育时则活性增强。5.采用Gene Fishing^TM等技术快速克隆了胡杨雌花序愈伤组织诱导不定根时应答IBA信号的差异cDNA。初步分析表明,在未加IBA的培养基上培养的愈伤组织中,克隆到可能编码水解酶的0-4基因,与抑制不定根生长密切相关。在未加IBA的培养基上培养的愈伤组织中,克隆到可能编码富含亮氨酸重复domain蛋白的0-5基因,此产物很可能参与抑制根有关的信号转导过程。在添加IBA的培养基上培养的愈伤组织中,克隆到可能编码Epidermal protein 140（RR-2家族）的1-34-1基因,此产物参与植物不定根的发育过程。6.通过对胡杨3个不定根发生调控培养体系的比较和与胡杨不定根发生可能相关的差异基因的克隆分析,初步建立IBA调控胡杨不定根发生的内源IAA阈值和激素间平衡的模型,即外源IBA通过插穗受体接收,转入细胞内并在POD酶作用下转化成具有活性的IAA,当IAA的水甲在促根的阈值内并保持相对平稳时,则有利于生根;当IAA的浓度增高超过阈值且不稳定时,则抑制不定根的发生。更多还原

【Abstract】 Populus euphratica Oliver is a rare but important natural forest tree species in the hinterlands of the deserts of central Asia,including China,with conditions of salinity,drought,cold,wind resistance and other characteristics of arid regions.It has a role play in conserve and regulation drought and desertification and help in keeping a fragile ecological balance in these regions,and as well improvement the ecological environment of oasis agriculture.As a result of the limit of sexual reproduction in a large-scale and easy separation of the genetic traits,the vegetative propagation became an important means of propagation.However,it is difficult for P.euphratica to induce roots from cuttings for propagation and become the bottleneck of industrial development of P.euphratica, need to be solved.Using the adventitious root formation（ARF） regulation system,and microscopy, gas chromatography-mass spectrometry（GC-MS） and cDNA cloning techniques in the cellular and molecular levels,our study describes a variation of cell,organization structure and several physiological and biochemical indicators such as hormones and isozyme during the process of the adventitious roots occurrence.Analysis the biological information of the difference cDNA clones. The research results will be the important basic works of identifying the molecular mechanism of adventitious root occurrence and developmen in-depth,and realizaed of human control of adventitious roots occurred.It has an important theoretical and practical significance for germplasm conservation and genetic improvement.The results are as follows:1.Rapid Propagation system of P.euphratica,were the first time establishment with inflorescence explants.The optimal medium for callus induction of male and female inflorescence on a B₅ medium, containing 1.0 mg·L^-1 6-BA and 0.5mg·L^-1 NAA for female inflorescence or containing 1.0 mg·L^-1 6-BA and 1.0mg·L^-1 NAA for male inflorescence,has an induction rate of 98.0%,which includes over 63%green calli with an affinity for a bud regeneration of both male and female inflorescence.The optimal medium for callus subculture were that in the B5 medium add 1.0 mg·L^-1 6-BA and 1.25mg·L^-1 NAA or MS medium add 1.0 mg·L^-1 6-BA and 0.5mg·L^-1 NAA.A bud induction on a MS medium,containing 0.5 mg·L^-1 6-BA and 0.2 mg·L^-1 NAA,has an induction rate of 31.2%.A LS medium containing 1.0 mg·L^-1 6-BA and 0.5 mg·L^-1 NAA has an induction rate of 33.3%,where the appropriate 6-BA/NAA ratio lies between 2.0 and 2.5.The optimal medium for rooting on a 1/2 MS medium,containing 0.5 mg·L^-1 IBA,has an induction rate of at least 35.0%.The seedlings propagation period of P.euphratica using inflorescence explants in vitro is shorter than the other explants nearly two weeks.2.In addition to the establishment of a callus of P.euphratica than seedlings stem cuttings and buds induction adventitious root occurred in regulation system can be used to study the occurrence and ARF as a good experimental system.The micro-and ultrastructural features of adventitious roots formation and development from seedlings stem cuttings were observed in detail.The stem cuttings in medium without IBA,has major nuclear cells around the vascular at 36 h.More aleurone grains accumulated in these cells and which distribute relative concentration at 48 h;Adventitious root primordial,formed vascular bundle,were original from vascular cambium and phloem organizations and pith ray at 60 h.The adventitious root formation was at 72 h,and meanwhile the nuclear membrane integrity,nuclear major,and mostly nuclear with ring nucleoli.The root primordia formation of stem cuttings in medium with 0.5 mg·L^-1 IBA were completely inhibited,and dedifferentiation organizations induced callus at the base of stem cuttings.3.The variation of hormone associated with occurrence of adventitious roots was more accurate analysis by using GC-MS technique.During the process of ARF,we noted that endogenous hormones IAA and ABA have an important role play in induced root primordium as an important signaling molecule.If the IAA concentration in particularly a certain extent and in a relatively stable state,that will promote the occurrence of adventitious roots.If the IAA concentration above a certain threshold and unstable,inhibit the occurrence of adventitious roots.Additional,the ratio between IAA and other hormones,such as GA₃ and ABA,etc.also affect the occurrence of adventitious roots.Endogenous hormone GA₃ status in a more stable and is conducive to the formation of adventitious roots. 4.Polyacrylamide gel electrophoresis（PAGE） was used to detect the action of peroxidase（POD）. The 2 b bands of POD continued expression related to the adventitious root primordium induction,it may be promotion IBAβ-oxidation and activated the IAA which inhibition of root growth,to achieve the effective concentration threshold of rooting.The amylase expression increase and be as a sign of root primordium development at 60 h.It does not in activation of esterase during root primordium induction,and does in activation during root primordia development.5.The different expression cDNA clones of female inflorescence callus response to the IBA signal during induced adventitious root were investigated by rapid cloning technology of Gene Fishing^TM and a preliminary analysis of the data.The cDNA clone,No.0-5,from the callus cultured in medium which without IBA,maybe encode leucine-rich repeat domain protein and participation the signal transduction-related with root inhibition processes;The clone of No.0-4,its protein sequences partly similar to the hydrolase,which maybe inhibit the primordium formation of adventitious root.The clone,No.1-34-1,from the callus cultured in medium with IBA,maybe encode epidermal protein 140 （RR-2 family） and involved in the process of ARF.6.Comparison of different types of stem cutting in rooting induced and analysis the cDNA clones which may be associated with the occurrence of adventitious roots of P.euphratica,the model of ARF were initial establishment of exogenous IBA controlling endogenous IAA occurred threshold and equilibrium,that is,exogenous IBA receiving by receptor of stem cutting,and transfer into the cells,then turn into active IAA by the effects of the POD enzyme.While the endogenous IAA concentration were in the range of rooting threshold value and remained relatively stable,it enhanced the ARF.However,while the IAA concentration were above the threshold and do not stability,it inhibited the ARF.更多还原