【作者】 许书军；

【导师】 何明； 王艳；

【作者基本信息】 上海交通大学 ， 环境工程， 2007， 博士

【摘要】 本文通过调查荒漠苔藓刺叶墙藓组织培养影响因素,探讨了荒漠苔藓及其生物结皮室内快速繁殖可能性,为大规模培养荒漠苔藓以及生物结皮提供基础研究数据;通过调查含水量、叶绿素荧光、电导率、色素含量、超微结构以及再生潜力等传统指标,评价刺叶墙藓对不同脱水速度、不同温度在形态学、生理学以及繁殖生物学等方面的响应,对荒漠地区苔藓生物结皮合理保护与修复具有指导意义;将扫描电镜(SEM)、显微红外(Micro-FTIR)及单次反射(ATR)技术、顺磁共振自选标记(EPR Spin Label)、气质连用(GC/MS)、液质连用(LC/MS)、离子色谱(IC)热动力测定工具(TGA和DSC)、飞行时间质谱(MALDI-TOF)等新技术运用在苔藓耐性研究方面,摸索了刺叶墙藓在表层结构与蜡质化学、细胞内化学成分与凝胶态-液晶态-玻璃态相互转变、细胞膜透性与细胞质分子流动性以及细胞膜界面结构等对不同脱水速度和温度响应,是苔藓生物学研究上新突破,更为研究相对低等且形态较小生物体开辟了一条新途径。同时,运用双向电泳技术,比较了脱水-复水过程中刺叶墙藓蛋白质表达差异,筛选与刺叶墙藓耐干相关功能蛋白,为荒漠苔藓耐干热基因分离及其功能基因在农作物改良中应用奠定基础。首先按照组织培养方法调查了几种常见影响因素如消毒方法、培养基、激素、培养温度、外植体、湿度及光照强度与时间等对刺叶墙藓再生的影响。最终确定刺叶墙藓快繁路线为:外植体离体叶片→温度25/15℃→营养源Knop→RH 65-80%→光周期16H→光照强度85-120 PPFD→培养周期2个月,材料无需经过消毒处理;在掌握刺叶墙藓生长影响因素后,尝试构建苔藓结皮:用离体绿叶在20/10℃诱导原丝体,继而转入河砂中,得到原始苔藓结皮,表明苔藓结皮存在人工培育可能。为了了解荒漠苔藓对沙漠常见水分和温度胁迫的抗性机制,研究了脱水和高温及联合处理下,刺叶墙藓在形态结构、生理和繁殖特征等方面的响应。形态上刺叶墙藓具有明显的生态优势:①双层细胞壁和脂肪粒含量丰富,其中老化组织比新生组织更具有耐性优势;②快速脱水处理后不同发育阶段的叶片细胞叶绿体变圆,没有出现细胞壁和细胞膜破损;③45℃热处理仅引起叶绿体结构疏松;④45℃干热处理引起叶绿体产生大量气泡且结构扭曲,部分细胞膜破损,表明45℃下脱水和温度对细胞膜存在协同效应;⑤快速脱水、45℃热处理、45℃干热处理后的叶片细胞复水后叶绿体能够恢复正常形态。生理上刺叶墙藓具有明显的抗干热响应:①几种胁迫都引起细胞溶质大量渗漏;②色素和可溶性糖含量仅对热和干热两种处理响应剧烈;③几种胁迫叶绿素荧光活性均降低,但脱水和干热处理后叶片光系统能够快速恢复至正常水平,高温处理(≥50℃)荧光活性丧失,表明极端高温对光系统有致命影响;④高温降低刺叶墙藓再生能力,但干热联合胁迫(<60℃)没有对再生能力产生显著影响;⑤脱水和高温联合处理对生理影响存在两种生态效应,即温度≤45℃二者存在协同作用,温度>45℃脱水对高温效应存在拮抗作用。为了进一步阐明刺叶墙藓耐干和耐热根本原因,从结构功能和结构化学方面对其进行了探讨。刺叶墙藓外被蜡纸层,有细胞壁瘤状凸起密布在表面,可以减少水分丧失和阻碍热空气影响,而且蜡质含量高达2.64mg g-1干重,90%以上为长链脂肪酸和烷烃,表明其细胞最外层具有明显生态优势。运用显微红外特征吸收峰揭示了刺叶墙藓化学成分变化与耐热机理存在相关关系。幼嫩组织增加亲水区脂肪和蛋白质含量、提高α-螺旋结构含量,使得细胞结构能够高温下稳定;老化组织通过提高碳氢化合物和疏水区内脂肪含量、稳定蛋白质结构等来维持细胞结构稳定,类似于糖类和蛋白质在脱水过程保护作用。运用顺磁共振自选标记方法研究了细胞膜、细胞质在脱水和干热过程中结构维持特性,刺叶墙藓细胞质稳定主要依靠高含量糖类和生物大分子,在脱水短期内实现玻璃化转变,钝化生理代谢和分子反应活性;依靠具有抗氧化功能两性分子参与膜内,减少或者消除氧自由基对细胞膜损伤,从而保证将脱水时细胞结构损伤降低在可修复范围。以上结果表明,刺叶墙藓耐干热除了形态学优势以外,细胞质玻璃化转变在脱水过程对细胞结构具有重要作用。那么再复水过程必然引起大规模细胞溶质渗漏,因此复水后启动损伤修复机制对于刺叶墙藓非常重要。先前研究过程已经证明复水后大量蛋白表达对于修复脱水以及复水造成细胞损伤至关重要。运用双向电泳技术,初步探讨了刺叶墙藓蛋白组学响应,结果发现再复水后新表达或者表达量增加蛋白有脱水素(13.1kDa)、复水蛋白(67.5 kDa)、热激蛋白(71.4 kDa和71.2 kDa)以及小分子热激蛋白(36.7 kDa),其中脱水素和复水蛋白已经在其它生物中证明与耐干性有关,热激蛋白已经证明与耐热和耐干有关,因此这些蛋白可能与刺叶墙藓修复机制相关。更多还原

【Abstract】 In this study, the factors influencing in the tissue culture of a typical desert moss Tortula desertorum were investigated and the possibility of macropropagating the desert moss was assessed. These works were the foundational data for scale of cultivation of desert mosses and for artificial construction of biological soil crusts (BSCs). Through investigation of the changes of water content, Chlorophyll fluorescence, electron conductivity of solution leakage, pigment content, ultrastructure and regeneration potential, some traditional responses of the desert moss was studied, including morphology and physiology and asexual reproduction to different dehydration rates and temperatures. Those indexes could help establish suitable methods for BSCs restoration in semi-arid and arid regions. Some new biophysical methods such as Scanning electron microscopy (SEM), Atomic force microscopy (AFM), Differentiate scanning calorimeter (DSC), Fourier Transform Infrared Spectroscopy (FTIR), Gas chromatography (GC) and Electron paramagnetic resonance (EPR) were firstly used in study of the desiccation tolerance and thermotolerance in desiccation tolerant mosses. The changes of surface structure and wax chemical characteristics, chemical components and state transition, membrane permeability and cytoplasmic molecular mobility under desiccation and heat stress were studied by those instruments. Those methods and results brought a new path for the study of lower and little size of biomaterial. Furthermore, the proteomics of the desert moss T. desertorum under different water content was studied by 2-demension gel electrophoresis (2-D gel). Some expressed proteins were found and identified, and the functions of these proteins were searched in protein database (NCBI and EBI). This step was crucial of choosing right genes relative with desiccation tolerance and thermotolerance and utilizing these genes for improve crops abiotic tolerances.At first, effects of media, explants and culture temperatures on regeneration potential of T. desertorum were investigated respectively in this study. The factors of relative humidity, light intensity and photoperiod were optimized through orthogonal test design. The life cycle of desert moss T. desertorum in cultivation was investigated according to the several factors on asexual reproduction. The protocol for macropropagation of T. desertorum according the following: detached leaves→day/night temperature 25/15℃→nutrient source Knop solid-agar medium→RH 65-80%→photoperiod 16h→light intensity 85-120 PPFD→2 months cultivation. No sterilized materials were used in cultivation. An incipient moss-dominated crust was constructed using detached green leaves cultivated at 20/10℃in Knop agar-solid medium for one month and transplanted in river sand under 25/15℃for other month. This process proved that it was feasibility to artificial construction of BSCs for desert restoration.In order to improve the abiotic tolerances and ecological functions of BSCs, the main component of BSCs, the desert moss T. desertorum, the responses in morphology, physiology and reproduction to the extreme environment, especially water and temperature, were investigated. The elder tissues had more advantages in cellular structure to cope with environmental stresses. In dehydration and thermostress and the combined stress test, cellular wall and membrane were not penetrated under fast dry. Only did chloroplasts change loose under 45℃treatment. Plenty of bladders emerged in chloroplasts and membranes were penetrated resulting in lipids transferred from cell inner after tissues were exposed to a combined stress of desiccation and thermostress. Increase of solution leakage, decrease of chlorophyll fluorescence activity, intense responses of pigment and sugars were observed when tissues were subjected to fast dry or higher temperatures. However, the fast recovery could be seen in tissues treated with desiccation or the combined stress after being re-irrigated. Meanwhile, higher temperature (> 30℃) could depressed the leaf regeneration potential but the combined stress (<60℃) have not significant effect on reproduction. These results indicated T. desertorum have desiccation tolerance and thermotolerance.To grasp the root cause of desiccation tolerance and thermotolerance of T. desertorum, the structure functions and structural chemistry were studied. There overloaded waxes in the surface between cell membrane and environment. Cell wall stretched outside and formed closed papilla, which could retard water loss and alleviate the damage of hot air. All these information proved the ecological advantages of T. desertorum. The relationship between the changes of chemical components in moss tissues and the mechanisms of thermotolerance were investigated by micro-FTIR. Increase of lipids in hydrophilic regions and proteins content, improvement the structure content ofα-helix in the younger was employed in coping with thermal stress and sustaining the stability of cell structures. The elder engaged with thermostress by promoting the content of carbohydrate and lipids in hydrophobic regions to stabilize the normal structures of proteins and cells. Using different spin labels, the changes of membrane permeability and fluidity and cytoplasmic molecular mobility under desiccation and heat were monitored by EPR. The results elucidated the mechanism of structure stability of cellular membrane and subcellular organelles. T. desertorum depended on high content of sugars and other bio-macromolecules to changes the cytoplasmic state from liquid crystalline to vitrification rapidly after cell lost majority of free water. The rate of metabolism and the activity among molecules were suppressed in glass state, and the substances in cytoplasm were sustained. The partitioning behaviors of amphiphiles could transfer some antioxidants into membrane and eliminated the damage of free radicals. Those processes could maintain the cellular damage within recovery regimes during fast dry.Because of vitrification, the cellular structure was stability and the solution leakage during rehydration became serious. There need powerful recovery mechanism to resume the normal function of cell. The former reports had proposed that the proteins expressed in rehydration were the key to repair the damage of moss in dehydration and rehydration. Some different expression proteins were found between dried and re-wet materials using 2-D gel. Especially, the content of dehydrin (13.1kDa), rehydrin (67.5 kDa), HSP 70 (71.4 kDa和71.2 kDa) and sHSP (36.7 kDa) were up regulation during rehydration and we believed those proteins and corresponding genes had a role in repair mechanism.更多还原