【作者】 岳宁；

【导师】 郑彩霞；

【作者基本信息】 北京林业大学 ， 植物学， 2009， 博士

【摘要】 胡杨是中亚和中国西北干旱荒漠地区风沙前沿唯一天然分布的乔木树种,具有较强的耐逆性,能够抵御风沙、遏制沙化,在维护区域生态平衡和保护生物多样性方面发挥着重要的生态作用。胡杨具有异形叶性,成年树上着生从披针形到锯齿阔卵形等多种形状的叶片,异形叶的形成,是对所在环境长期适应的结果。比较胡杨异形叶结构及生理特性的异同,研究异形叶产生的分子调控机制,能够从一定角度反应胡杨的生态适应性,对于全面研究胡杨的耐逆性具重要的理论和实践意义。本文以披针形、卵圆形及锯齿阔卵形3种胡杨成年树上典型形状的异形叶为研究对象,分别对叶片的形态、解剖结构、生理指标及蛋白质组进行了研究,为了解胡杨叶片发育的细胞与分子调控机制,揭示胡杨的生态适应性积累了有价值的信息。对胡杨异形叶的形态及解剖结构进行了观测统计,结果表明:随着叶片形状由披针形向锯齿阔卵形的变化,叶片宽度增加、表面积减小、肉质化程度提高、叶柄变长;同时锯齿阔卵形叶片的角质层、下皮层及栅栏组织厚度等反映叶片耐逆性的指标值都是最大的,披针形叶片则最小,且各叶形间差异非常显著。因此,锯齿阔卵形叶片的耐逆性结构特征最为明显,较其他两种形状的叶片更适合在逆境中生存。水势高低及束缚水、可溶性渗透调节物质、叶绿素的含量等生理指标,能够反映叶片的耐逆性及光合能力,通过测定这部分生理指标,发现异形叶间水分含量差异不大,但锯齿阔卵形叶片中束缚水含量最多且水势最低,渗透调节物质含量最高,叶绿素a的含量及总叶绿素含量均高于其他两种形状的叶片,说明其耐逆性及光合能力较强。建立了适合胡杨叶片可溶性蛋白质提取的Tris-HCl提取液配方:5%β-巯基乙醇,2%SDS,0.05mol/L Tris-HCl(pH8.8),10%甘油,1mmol/LPMSF。通过对异形叶可溶性蛋白质进行SDS-PAGE分离,发现不同形状叶片间的电泳图谱差异较大,说明随着叶片形状的变化,叶片蛋白质的表达也出现变化。进而通过优化裂解液的成分及配比,得到了适合胡杨叶蛋白质的裂解液配方:7mol/L尿素、2mol/L硫脲、2%CHAPS、60mmol/L DTT、0.2%载体两性电解质;并成功建立了合适的双向电泳条件,能够得到蛋白点分辨率高、干扰少、重复率好的高质量双向电泳图谱,为胡杨异形叶蛋白质组的研究奠定了良好基础。以同一小枝上成熟的披针形及锯齿阔卵形叶片为研究对象,对其叶片蛋白质进行双向电泳分离,对电泳结果进行软件分析,共得到73个差异点,其中在锯齿卵圆形叶片中表达上调的为51个,在披针形叶片中表达上调的为22个。并对其中24个差异最为显著的蛋白点进行串联质谱鉴定及数据库检索匹配,成功鉴定了其中13个蛋白点,代表了12种蛋白质,分别参与了叶片的光合作用、呼吸作用、氮代谢及提高叶片的耐逆性等。分析认为:披针形叶片具有较强的吸收利用光能的能力,能合成ATP等活跃的化学能;而锯齿阔卵形叶片的碳同化能力则较强,能够将活跃的化学能转化为糖类等可贮存的稳定的化学能,为植株的生长发育提供碳骨架及能量。同时,锯齿阔卵形叶片中耐逆相关的蛋白质表达量较高,在发生胁迫时能更快的建立起新的平衡体系,维持生命活动的正常运行,以满足成年胡杨树在干旱荒漠地区生存的需要。本文对胡杨异形叶之间解剖及生理学方面的差异进行了系统地研究,较好地解释了胡杨异形叶的形成与其生态适应性之间的关系:披针形叶片的光能吸收能力较强,但由于其光合碳同化能力及耐逆性较差,随着胡杨树体的生长及所处环境的恶化,无法满足植株的需要,所以光合碳同化能力及耐逆性较强的锯齿阔卵形叶片逐渐替代了幼树上占主体地位的披针形叶片,成为胡杨成年树上分布最多的叶片类型。胡杨的异形叶能通过形态、结构、生理代谢及蛋白质表达水平上的变化来更好的适应不同发育时期生存环境的改变,推测这就是其优于其他树种而能在荒漠地带生存至今,成为干旱荒漠地区唯一天然存在的高大乔木的原因。更多还原

【Abstract】 Populus euphratica Oliv is the only naturally-distributed tall arbor species in arid desert regions of northwestern China and Central Asia.The tolerance to severe drought and high salinity,high alkalinity soil makes it ecologically important in protecting species diversity and maintaining the ecological balance.The long term adaptation to such desert environment induces the formation of heteromorphic leaves of Populus euphratica,and a single adult tree commonly has ploymophic leaves,such as lanceolate,oval and serrated broad-oval leaves.Special research interest has arisen in understanding the eco-adaptability of Populus euphratica for several years.Comparing the structural and physiological characteristics of the heteromorphic leaves and studying on the relative molecular regulatory mechanism would have both theoretical and practical significance in this area.This paper studied three typical heteromorphic leaves of Populus euphratica,including lanceolate, ovate and serrated broad-oval leaves,and mainly focused on the leaves’ anatomical structures, physiological characteristics and proteomics.It will greatly help the understanding of cellular and molecular mechanism during leaves development and elucidating the mechanism of the eco-adaptability of Populus euphratica.Environmental conditions bave close relationships with leave growth and development.Different leaf shape and anatomical structure characteristics can reflect its adaptive capacity to its habitats. Experiments were conducted in leaf shape morphology and anatomical structure of Populus euphratica. The results showed that as the leaf shape changed from lanceolate to serrated broad-oval,the leaf width increased,surface area decreased,the level of carnification enhanced,and petiole extended;the serrated broad-oval leaves had the most well-developed subcortex and palisade tissue,as well as the thickest corneum,while lanceolate leaves’ xeromorphic structure was not as well-developed as serrated broad-oval leaves.This demonstrated that the serrated broad-oval leaves were more suitable for arid environments survival compared to the other two types of leaves.The capability of tolerating adverse environment and photosynthesis of leaves can be reflected by several physiological indexes,such as the levels of water potential,bound water,osmolyte,and chlorophyll.Those measurements showed that these three types of leaves had the similar amounts of water,but the serrated broad-oval leaves had lowest water potential,the highest level of bound water, osmolyte and chlorophyll,which explained why the serrated broad-oval leaves had strongest capability of photosynthesis and tolerating adverse environment.The soluble proteins of heteromorphic leaves were separated by SDS-PAGE.Established a suitable Tris-HCl buffer:5%β-mercaptoethanol,2%SDS,0.05mol/L Tris-HCl(pH8.8),10%glycerin, 1mmol/L PMSF。The results indicated that protein expressions were different among leaves in different shapes.The more intuitive and comprehensive understanding on the difference in protein expression can be obtained through proteomics research.Establishing a steady method is very crucial in proteomic study in order to assure the reproducibility of the results.In this paper,by optimizing the extraction method of Populus euphratica leaf proteins(7mol/L urea、2mol/Lthiourea、2%CHAPS、60mmol/L DTT、0.2%Bio-lyte) and the conditions of 2-DE,the electrophoretic techniques specific for proteins from leaves of Populus euphratica’s was successfully established and high quality 2-DE maps with less tailing,clearly and well distributed protein spots,and high repetition rate were generated,which laid a solid foundation for further proteomics research on Populus euphratica leaves.The lanceolate and serrated broad-oval mature leaves were collected from the same branch,leaf proteins were separated by 2-DE,and differential proteins were analyzed through tandem mass spectrum.Got 73 different protein spots,51 of them are expression more in the serrated broad-oval leave,and the other are expression more in the lanceolate leaves.Through tandem mass spectrum and Mascot search for 24 of them,successfully identified 13 spots,and representative 12 kinds of proteins, which participate in photosynthesis and respiration etc.The results suggested that lanceolate leaves had stronger capability in compounding active chemical energy from using solar energy,such as ATP etc; while serrated broad-oval leaves had stronger capability in carbon assimilation,and it can change active chemical energy into stable chemical energy,such as sucrose,and provide carbon skeleton and energy for plant growth and development.Meanwhile,the serrated broad-oval leaves had more types of proteins that can enhance its capability to tolerate adverse environment,which allowed them to be able to build a new balance system more efficiently and maintain the normal metabolism when stress occurred,and then satisfy the requirements for the adult Populus euphratica surviving in arid region.Through the studies on the differences in anatomy and physiology of heteromorphic leaves in Populus euphratica,this paper provided more comprehensive understanding in the association between the development and eco-adaptability in Populus euphratica heteromorphic leaves:lanceolate leaves had stronger capability in using solar energy,but it had weaker capability of tolerating adverse environment and carbon assimilation.With the trees growth and environment deterioration,the lanceolate leaves can’t meet the requirements for the adult trees.Consequently,the serrated broad-oval leaves,which had stronger capability of photosynthetic carbon assimilation and tolerating the adverse environment,took place of the lanceolate leaves and became the main leaf types in adult trees.Populus euphratica heteromorphic leaves can adapt different environment in different developmental stages through the changes of leaf shape,anatomical structure,physiological metabolism and protein expression,which can be presumed as the major reason for Populus euphratica survival in the arid and semi-arid desert region.更多还原