【作者】 孙志宾；

【导师】 赵彦修； 张慧；

【作者基本信息】 山东师范大学 ， 植物学， 2009， 博士

【摘要】 盐芥(Thellungiella salsuginea)以其个体较小、生长周期短、自花授粉、种子数量多、自交结实、基因组小、易转化和易突变等特点,以及在cDNA水平与拟南芥高的同源性,并且具有极高的冷、干旱、高盐和低氮素耐受力,被作为研究植物非生物胁迫的新模式材料。目前关于盐芥的研究已经取得了一些进展,这些研究主要集中在离子运输、表达谱分析、单个基因的功能鉴定及个别生理性状研究等方面。通过这些研究对盐芥高的胁迫耐受能力的机制有了初步了解,与拟南芥相比,盐芥在正常条件下高表达一些与胁迫相关基因,在受到胁迫后采取较经济的调节方式;在离子选择性、保护性物质代谢及种类方面盐芥和拟南芥之间也存在差别,总之高的胁迫耐受力是由复杂调控系统控制的过程。蛋白是细胞功能的主要执行者,高通量的蛋白分析—蛋白质组学研究将会为我们对这些胁迫耐受差异提供更加直观和准确的信息。在蛋白水平将盐芥和拟南芥以及盐芥不同生态型间进行比较的工作未见报道。本实验以拟南芥、盐芥Shandong生态型及Xinjiang生态型为研究材料,对正常水培条件下拟南芥和盐芥叶片进行了蛋白质组学比较,鉴定分析了二者表达丰度较高的蛋白,目的在找出与盐芥耐逆相关的基础水平因素;对盐芥Shandong型和Xinjiang型的叶及根进行了比较蛋白质组学研究,并结合二者基本生理性状的差异对鉴定的差异蛋白进行了分析,目的在于寻找盐芥不同生态型间进化上的差异在蛋白水平的表现。本实验的主要结果如下:1、盐芥与拟南芥叶片蛋白质组分析水培装置中生长4周的拟南芥叶片及生长6周的盐芥Shandong生态型叶片提取全蛋白进行双向电泳,蛋白谱图显示盐芥、拟南芥在蛋白水平存在显著差异。本实验中将二者凝胶图中的蛋白点按体积值排序后,选取了表达量较高的蛋白点进行质谱鉴定,在选点的时候还要求二者凝胶图上可能会有对应关系的点也要选择,拟南芥中选择了87个蛋白点,成功鉴定出属于57个基因的70个蛋白点,盐芥中选择了84个蛋白点,成功鉴定出属于58个基因的67个蛋白点。将这些高丰度蛋白按功能分类后发现,盐芥和拟南芥之间还存在很多表达模式一致的蛋白,这些蛋白都属于生命活动必需的、至关重要的持家基因,如光合作用相关的光系统Ⅱ亚基O-2、光系统Ⅱ亚基P-1、磷酸核酮糖激酶、肽基脯氨酰异构酶/叶绿体基质亲环蛋白、类囊体膜结合蛋白、景天庚酮糖-1,7-二磷酸酶、叶绿体茎环结合蛋白、光系统Ⅱ稳定/装配因子及光呼吸系统中的线粒体甘氨酸脱羧酶P蛋白等,与糖酵解相关的果糖二磷酸醛缩酶1、果糖二磷酸醛缩酶3及磷酸丙糖异构酶等,与所用生命活动都相关的核苷二磷酸激酶、硫腺苷甲硫氨酸合成酶及腺苷激酶等,还有最近新报道的At1g13930蛋白等等。盐芥和拟南芥的光合碳固定及光呼吸相关酶类蛋白表达模式呈现多样性,RbcL、RbcS及RCA这些与植物光合能力直接相关的酶的表达模式不同;光呼吸中的重要酶类谷氨酸-乙醛酸转氨酶、甘氨酸脱羧酶复合体H蛋白在盐芥中表达量都比拟南芥高。这至少为解释盐芥生长速度比拟南芥慢找到了一个可能的着眼点,盐芥可能会有较高的光呼吸能力使得净光合低,生长较慢,高的光呼吸也是应对各种逆境胁迫的准备,可以减少光系统损伤,为其他保护性物质合成提供前体等。拟南芥中许多与逆境相关或者受各种胁迫诱导的蛋白在盐芥中的表达水平都比较高。如受氧化剂及其他胁迫诱导的抗氧化酶类抗坏血酸过氧化物酶1、铜锌超氧化物歧化酶2及几种过氧还蛋白在盐芥中都有很高的组成型表达;与系统防御相关的Jacalin lectin蛋白/JA诱导蛋白/黑芥子酶结合蛋白类似蛋白、MLP328蛋白-主乳胶蛋白类似蛋白及仙茅甜蛋白类植物凝集素家族蛋白在盐芥中表达量很高;与抗病相关的类甜味素病程相关蛋白在盐芥中表达量极高;与抵抗病菌侵染相关的β?1,3-葡聚糖酶1在盐芥中表达量很高;硫代葡萄糖苷-黑芥子酶系统在二者之间也存在差异,盐芥中高表达的黑芥子酶与拟南芥中高表达的该酶是同一个基因家族的不同基因编码的;分子伴侣类蛋白二硫键异构酶、热激蛋白70蛋白在盐芥中的表达量较高。这些差异可以概括为盐芥采取了一种“未雨绸缪”的生存方式,在牺牲生长速度的基础上,在正常生长条件下就储备一系列抗逆所需材料,以备不时之需,以免胁迫来临时产生胁迫恐慌,这种生存策略可能是生存环境对进化影响而成的,因为盐芥的生境是山东东营黄河入海口处盐碱地,时刻要面对高盐及沿海地区高紫外线辐射等非生物胁迫。2、盐芥Shandong生态型和Xinjiang生态型基础生理指标和叶片及根差异蛋白质组分析盐芥Shandong生态型和Xinjiang生态型在水培装置中生长6周后,整体植株表型差异较大。SD型叶色较深、叶片较短、叶的伸展比较平展;根呈现明显直根系模式,主根优势明显而长、侧根稀疏分布于主根上。XJ型叶色较浅、叶片较长、叶的伸展比较竖直;根系为直根系,主根优势不明显、侧根浓密而长、侧根密布于较短的主根上。叶片及根分别取材后,测量了最长叶长、根长、鲜重、干重及灰分重量,数据分析显示XJ型单株叶鲜重、灰分重、含水量及灰分含量都比SD型明显高,单株叶干重比SD型稍高,差异不显著;XJ型根单株鲜重、干重与SD型基本一致,根的含水量明显高于SD型,根灰分重、灰分含量都明显比SD型低。总之XJ型与SD型的整体区别为:XJ型整体含水量高、地上部分生物量大,根的生物量与SD型基本一致。无机离子分布(地上、地下部分灰分含量比)方面XJ型主要分布于地上部分,而SD型地上部分与根基本一致。将SD、XJ型叶片及根分别取材提取全蛋白进行双向电泳,蛋白谱图显示盐芥SD型、XJ型蛋白表达谱基本一致,叶片的差异蛋白占总蛋白点3.4%,根差异蛋白占2.2%。将差异蛋白点切胶回收后质谱鉴定,叶片差异蛋白的质谱鉴定成功率为比较低,仅为40.9%,而根差异蛋白的质谱鉴定成功率为85.3%,叶片与根质谱鉴定成功率差别的原因可能是地上部分行使的功能复杂、直接面对的选择压力大,不同物种间进化的特异性要强一些;而根功能相对较少,在根部表达的蛋白可能会比较保守一些。通过对盐芥SD、XJ生态型叶片及根蛋白表达谱的比较本实验找到了一些差异蛋白点,对这些差异蛋白点的生物功能特性及所涉及的生物过程进行综合分析使我们初步了解了生态型间表型差异背后的蛋白水平控制机制。盐芥XJ型叶片伸展比较竖直,主根优势不强侧根浓密,可能与激素水平或者激素应答元件含量不同有关。氨基环丙烷羧酸氧化酶是乙烯生物合成的最后关键酶,XJ型叶片中此酶蛋白表达量很高,叶片中可能会产生更多乙烯,高的乙烯释放量会刺激叶柄伸长和偏下性生长,致使叶片伸展比较竖直、老叶早衰。生长素可以诱导根系形成,受生长素影响的根系主根较短、侧根丰富。XJ型根中黄素氧还蛋白类醌还原酶1、甲基丙二酸半醛脱氢酶两个受生长素诱导蛋白表达量都比较高,而且黄素氧还蛋白类醌还原酶1还是生长素应答主反应蛋白,从受生长素诱导表达蛋白的表达水平看出XJ型根中生长素含量可能比较高,较高的生长素水平致使植物主根短小、侧根浓密发达。XJ型叶片中与生长密切相关的酮醇还原酶、蛋白正确折叠所需的分子伴侣表达量都比较高,而SD型叶片中与耐逆防御相关的质膜阳离子结合蛋白、类甜味素病程相关蛋白、几丁质酶及葡聚糖苷酶等表达量很高。SD型叶片将大量的能量用于合成耐逆防御相关蛋白必然会影响生长速度;XJ型叶片中乙烯含量可能会高些,乙烯也有刺激生长的作用。更重要的是XJ型根虽与SD型根生物量基本一致,但根中与呼吸产能相关的磷酸甘油醛歧化酶、丙酮酸激酶、线粒体NADH-辅酶Q氧化还原酶75kDa亚基及丙酮酸脱氢酶E1元件β亚基等表达量都很高,根中可能会产生大量的能量为无机离子的主动运输提供能量保障,使得地上部分(叶片)有足够的无机离子用于生长等生命活动。总之,生态型间激素水平及激素应答元件差异可能会是形态差异的原因,但需要进一步实验来检测相关激素在SD、XJ型的含量。根的能量产生差异及叶片的能量分配不同可能是XJ型较SD型具有较大的地上部分(叶片)生物量的原因。本研究的主要创新点:1、以拟南芥和盐芥为材料,利用蛋白质组学技术分离鉴定了大量高丰度蛋白,通过对高丰度蛋白的功能分析,揭示了正常生长条件下拟南芥和盐芥生长速度及胁迫耐受性差异的可能控制机制,净光合及物质能量分配差异影响了生长速度,物质能量分配偏向于防御耐逆相关蛋白致使盐芥在本底水平具有高胁迫耐受力。2、以盐芥Shandong、Xinjiang生态型为材料,对正常生长条件下二者的形态和生理特性进行了分析,明确了二者形态、生物量、含水量及根冠比等具体差异。3、对盐芥Shandong、Xinjiang生态型的叶片及根进行了比较蛋白质组学分析,从蛋白水平揭示了不同生态型盐芥的差异,鉴定出一些与盐芥Shandong、Xinjiang生态型形态、生理特性差异相关蛋白,发现生态型间形态差异可能由激素水平差异控制,叶片生物量不同可能由叶片物质能量流向及根部能量供给差异控制。更多还原

【Abstract】 Salt cress (Thellungiella salsuginea) is a kind of halophyte which belongs to the crucifer family (Brassicaceae), and it is very tolerant to cold, drought, salt and low nitrogen. Meanwhile, it is vey similar to the model species Arabidopsis such as small size, short life cycle, self-fertilization, copious seed production, small genome, and easy to transform and mutagenize. In cDNA level it has 90-95% identity to Arabidopsis, and has emerged as a new model system for abiotic stress study.Research of Thellungiella has made enormous progress over the last few years, and the studies were mainly focused on ion transport, expression profiling, gene identification, and some physiological analysis. These studies have got the preliminary knowledge of stress tolerance in Thellungiella. Compared with Arabidopsis, many stress-responsive genes in Thellungiella are constitutively expressed at higher level, ion transport mechanism is more selective to K+ than Na+, and the species and metabolism of protection materials are also different. So the higher stress tolerance of Thellungiella must be regulated by complex process.Proteins are the functional machine of cell, and the proteomic analysis can help us to get a further intuitionist and exact knowledge of stress tolerance variation. However, to the best of our knowledge, it has not been reparted that comparing Arabidopsis and Thellungiella at proteomic level, as well as the proteomic comparision between Thellungiella ecotypes.In this study, Arabidopsis Columbia ecotype, Thellungiella Shandong ecotype, and Thellungiella Xinjiang ecotype were selected as research materials, and all the materials were cultured in hydroponic system. In the first part of this study, the Arabidopsis and Thellungiella leaf proteome were compared, and many higher abundant protein spots in 2-D gel were selected and then identified by mass spectrum (MS). This part aimed to find the basal element for higher stress tolerance of Thellungiella. In the second part, the proteome of leaves and roots were compared between Shandong ecotype and Xinjiang ecotype. The varied expression protein spots were identified, and the differences were analyzed and compared at the optimal growth status, aiming to discover the evolution differentiation at protein level.The main results of this study were summarized below:1. Proteome analysis of Thellungiella and Arabidopsis leaves. After grown in hydroponic culture system for several weeks (4 weeks of Arabidopsis, 6 weeks of Thellungiella), leaves were harvested, and then undergone TCA/Acetone procession for total protein extraction. Total protein were separated by 2-D electrophoresis, 24 cm pH 4-7 IPG dry strips were used for IEF and 12.5% SDS-PAG were used for the second dimension. 2-D protein images showed a higher level difference between Arabidopsis and Thellungiella leaves proteome.Protein spots were arranged by their volume values. Spots with bigger vol. value were indentified by MS, some smaller value spots were also selected according to their similar relative position in the gel with other specie. 87 spots in Arabidopsis gel were selected to analyze by MS, and 70 spots belonging to 57 genes were successfully indentified. 84 spots in Thellungiella were analyzed by MS, and 67 spots belonging to 58 genes were successfully identified. Then these proteins were classified by their function.By comparing the function catalog and spots pattern, we found some proteins had similar expression pattern. All these proteins belong to key housekeep proteins, such as photosynthesis related proteins: PSBO2, PSBP1, Phosphoribulokinase, ROC4, thylakoid membrane binding protein, SBPase, CSP41A, HCF136, and ATGLDP1; glycolysis related proteins: FBA1, FBA3, and TPI; all life activity related proteins: NDPK1, SAMS1, and ADK1; and a new reported At1g13930 protein.Some proteins of photosynthesis and photorespiration had diversity between Arabidopsis and Thellungiella. Photosynthesis related several proteins had different expression pattern between these two species, such as RbcL, RbcS, and RCA. Photorespiration related enzymes, GGT1 and glycine cleavage system H protein 2, which were expressed higher in Thellungiella than in Arabidopsis. Based on these facts, at least, we could find a viewpoint to understand the mechanism of lower growth rate in Thellungiella compared with Arabidopsis. Thellungiella may have a higher level photorespiration, which limited the growth rate and reduce photosystem damage, and to provide substrate for protective molecule synthesis.Many stress related or stress induced proteins in Arabidopsis had higher expression level in Thellungiella in normal condition. Oxidative reagents or other stresses induced antioxidative enzymes in Arabidopsis, such as Apx1, CZSOD2, and several Prxs, had higher expression level in Thellungiella. System defense related proteins, such as Jacalin lectin family protein, MLP328 protein, and curculin-like lectin family protein, also were expressed at higher level in Thellungiella. Pathogenesis-related protein PR-5 had a much higher expression level in Thellungiella. Beta -1, 3-glucosidase 1, disulfide isomerase 1, and hsp70 were highly expressed in Thellungiella. Myrosinase was highly expressed in both Arabidopsis and Thellungiella, but their pI and sequence similarity were different.All these differences could be recapitulated as that Thellungiella took an“against a rainy day”surviving strategy. Based on limited growth rate in normal condition, Thellungiella accumulated many anti-stress materials to prevent stress scare. This strategy may be acquired during evolution according to their habitat2. Comparsion of basic physiological characteristics and proteomic analysis of leaves and roots between Shandong ecotype and Xinjiang ecotype.After grown in hydroponic culture system for 6 weeks, some differences were shown between Shandong (SD) ecotype and Xinjiang (XJ) ecotype. SD ecotype leaves were green and shorter; roots were tap root system with preponderant tap roots and lateral roots sparsely brached from tap root. In contrast, XJ ecotype leaves were jade-green and longer; roots were tap root system with less preponderant tap roots and very developed lateral roots. XJ leaves outspreaded more upright than SD leaves.Leaves and roots were harvested respectively, and then longest leave length, longest root length, fresh weight, dry weight, and ash weight were measured. Data showed that fresh weight, ash weight, water content, and ash content of XJ leaves were higher than that of SD leaves. Dry weight of XJ leaves was a bit higher than SD leaves, but not significantly. Fresh weight and dry weight of XJ roots were similar with SD roots; water content of XJ roots was higher than that of SD roots; ash weight and ash content of XJ roots were lower than that of SD roots. In conclusion, whole plant water content of XJ ecotype was higher than that of SD ecotype; XJ leaves biomass was bigger than SD leaves; roots biomass of XJ was similar with SD roots. Ion content of leaves was higher than that of roots in XJ ecotype, and ion content of leaves was similar with that of roots in SD ecotype.2-D protein images showed a higher level similarity between SD and XJ proteome. There were 3.4% of total leaves protein spots and 2.2% of total roots protein spots different between SD and XJ ecotype. These diversity protein spots were picked from the gel and then indentified by MS, using Arabidopsis protein MS database as reference. The successful MS identification ratios were 40.9% of leaves, and 85.3% of roots. The successful ration difference maybe due to that, upground tissues (leaves) must confront higher selective pression than underground organs (roots), thus leaf proteins maybe less conserved compared with Arabidopsis than root proteins. Comparing the 2-D maps of leaves and roots between SD and XJ ecotypes, several expression difference spots were found. According to these proteins functions, we got a glance of the mechanism controlling the phenotype diversity in protein level.Compared with SD ecotype, XJ leaves stretched more vertically, rap roots showed less predominant, and lateral roots were dense. These different phenotypes maybe caused by difference in phytohormone content or in phytohormone response elements. ACO, the key enzyme of the last step of ethylene synthesis, expressed higher in XJ leaves, and leaves may release more ethylene. Higher level ethylene may stimulate a more vertical orientation of the petioles (hyponasty), enhance elongation, and induce senescence. Auxin may induce root system formation, and plants had shorter rap root and thicker lateral roots with higher auxin level. In XJ roots, two auxin induced proteins, MMSDH and FQR1, had higher expression level, and FQR1 also was a fast and primary auxin response protein. According to these two auxin response proteins’expression pattern, the auxin level in XJ roots may be higher, and higher level may affect root system architecture.Some proteins closely related to growth, such as KARI and Cpn20, were highly expressed in XJ leaves; and some defense related proteins, PCAP1, PR-5, endochitinase, and beta-1, 3-glucanase 1, had higher expression level in SD leaves. More energy and material were used for defense proteins synthesis, which may reduce the growth rate. XJ leaves may have more ethylene, and ethylene could induce growth. The most significant difference was that, although they had similar biomass, XJ roots may produce more energy than SD roots. Some respiration related important proteins, such as PGMI, PK1, Complex I-75kD, and pdh-e1 beta, were highly expressed in XJ roots, which may provide more energy for root metabolism and ion uptake and transportation for upground tissue fast growth.In conclusion, differences in phytohormone content or phytohormone response elements may be the reason of different phenotype, and this needed to be further studied. The differences in roots energy production capability and leaves energy/materials distribution may be the explanation why XJ leaves had larger biomass than SD leaves.The main innovation points of this study were generalized as follows:1. Total protein of Arabidopsis and Thellungiella leaves were separated by 2-D electrophoresis, and some abundant protein spots were selected and identified by MS. According the function catalog and comparision, the mechanism controlling the growth rate and stress tolerance differences was uncovered. Net photosynthesis and energy/materials distribution differences limited Thellungiella growth rate; more energy and materials were used for defense and stress tolerance related protein synthesis, so Thellungiella could have higher stress tolerance in normal condition.2. Phenotype and some physiological characters of Shandong ecotype and Xinjiang ecotype, under normal condition, were analyzed and compared. Some different aspects of two ecotypes were quantitatively analyzed, such as biomass, water content, and root-crown ratio.3. Total protein of leaves and roots were separated, and comparision between Shandong ecotype and Xinjiang ecotype were conducted. Some differences were discovered for the two ecotypes in protein level, and some proteins were identified, which were related to phenotype and physiological difference. The phenotype differences may be determined by phytohormone level difference, and leaves biomass differences were caused by energy/materials distribution and energy production capability differences.更多还原