【作者】 苗青；

【导师】 郑成超；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 植物在生长发育的整个生命周期中,会面临各种不利的外部环境(如土壤盐害、干旱、涝害、低温、高温、病原菌侵袭、机械损伤等)。在长期的进化过程中,植物形成了响应胁迫和提高抗性的机制。植物感受到胁迫信号后,会自主启动防御机制:包括物理结构适应,如自身结构改变和器官功能特性对盐分和缺水的适应;胞内渗透保护物质(脯氨酸、甜菜碱和可溶性糖等)含量增加,如超氧化物歧化酶、过氧化氢酶、抗坏血酸酶等的合成水平上升;启动植物激素(赤霉素、脱落酸等)参与的适应性调节等,帮助植物体重新建立物质和能量代谢平衡,从而在胁迫环境下存活。伴随着多种生物及非生物胁迫的影响,呼吸作用和光合作用都会有所减弱,出现能量不足,从而引起一系列生理、代谢和分子水平上的事件发生,包括生物合成酶类活性的降低,分解代谢的活化以提供营养和植物生长的抑制等。线粒体是多种重要代谢过程发生的场所,例如呼吸作用、氧化磷酸化、控制植物细胞内ROS的平衡、参与脯氨酸、维生素C的合成等,因此线粒体是植物适应外界各种胁迫环境的重要细胞器。其中,线粒体磷酸转运体(Mitochondrial phosphate transporter/Phosphate Carrier, MPT/PiC)是位于线粒体内膜上的一种重要的功能蛋白,负责将重要代谢底物无机磷酸(Pi)通过线粒体内膜从细胞质转移到线粒体基质。但是人们对于植物MPT与逆境胁迫抗性的关系,仍不是很清楚。在模式植物拟南芥中,我们克隆得到了MPT基因家族的三个成员,分别命名为AtMPT1、AtMPT2和AtMPT3,对其表达模式和功能进行分析:(1)通过进化树分析表明,AtMPT蛋白进化保守,其中AtMPT2和AtMPT3同源性要高于两者与AtMPT1的同源性。(2)利用qRT-PCR和GUS染色分析表明,AtMPT1、AtMPT2及AtMPT3具有不同的组织表达模式,暗示着三个成员在拟南芥生长发育的不同阶段起作用。而且AtMPT3表达量明显的比AtMPT1和AtMPT2的表达量高,说明AtMPT3可能起主要作用。(3)AtMPT1、AtMPT2及AtMPT3突变体和超表达植株表现出对盐胁迫的敏感性。盐胁迫使WT中GAs的含量降低,而AtMPT1、AtMPT2及AtMPT3的突变体和超表达植株中GAs的含量有所提高;此外,外施GAs合成抑制剂UNI使其盐敏感的表型恢复。表明MPTs通过调节赤霉素使拟南芥对盐胁迫做出响应。(4)GAs代谢相关基因的表达明显受到了AtMPTs的调节。盐胁迫下,AtMPT1、AtMPT2及AtMPT3的突变体和超表达植株中GAs合成基因GA3ox1/2/3、GA20ox1/2/4表达上调,GAs失活基因GA2oxs表达下调。表明AtMPTs表达过高或过低都会破坏植物GA的正常代谢,造成GAs含量的紊乱,破坏GA介导的植物对盐胁迫的适应。(5)AtMPT3受到渗透胁迫的明显诱导。AtMPT3的突变体和超表达植株表现出对渗透胁迫的敏感性。350mM Mannitol处理24小时后WT、mpt3、OEMPT3体内ABA含量均有所上升。其中突变体和超表达株系分别是处理前的2.09倍和1.99倍,野生型仅为1.23倍。此外,外施ABA合成抑制剂钨酸钠能够恢复其敏感的表型。表明内源ABA含量的升高是MPT3突变体和超表达株系对渗透胁迫表现出超敏感的原因。(6)AtMPT3的突变体和超表达植株中ABA代谢相关基因表达受到影响。ABA合成基因NCEDs和AAOs表达量增加;ABA降解基因CPY707A3表达量降低,导致ABA过度合成。然而在突变体和超表达株系中CPY707A1表达量仍上调,但并未产生明显作用,可能由于表达部位不同而致。表明AtMPT3的异常表达会导致植株在幼苗生长阶段和成花诱导阶段对渗透胁迫的敏感。(7)GUS活性(GUS/LUC)的瞬时测定证明GT-1 box和双TM6的融合成功地提高了盐诱导启动子GhNHX1的活性。更多还原

【Abstract】 Plants are sessile organisms and hence they cannot escape unfavourable environmental conditions within their life cycle, such as high salinity, drought, waterlog, high or low temperature, pathogen attack and mechanical agitation. Among them, high salinity and drought are the main constraints in plant geographical distribution and crop productivity. Plants have evolved finely tuned high-salinity and osomotic signaling and resistance mechanisms. When exposed to salt or osomotic stress, plants respond rapidly and carry out protective processes, for example, physical adaptation (changes in cellular structure and organic functional adaptation), accumulation of osmoprotectants (soluble sugars, proline, betaine, etc.) in cytosol, increased contents of various antioxidants (superoxide dismutase, catalase, ascorbinase, etc.), phytohoromone (GA, ABA) regulated adaptatipon etc., which reprogram the biological activities and establish a new metabolism balance for stressed plants and help survive the salt or osomotic stress.Often associated with stress is a reduction in photosynthesis and/or respiration, which in turn results in energy deprivation and ultimately in growth arrest and cell death. Mitochondria can integrate numerous metabolic pathways that are important in adaptive responses to extreme environmental conditions, such as respiration and oxidative phosphorylation, the control of redox balance, and the metabolism of proline and ascorbate. The mitochondrial phosphate transporter/carrier (MPT/PiC) which is located in the mitochondrial inner membrane catalyzes phosphate transportation. Nevertheless, the relationship between MPT and environmental stress are still largely unknown.A total of three MPT genes have been cloned from the Arabidopsis genome, which were designed as AtMPT1 (AT2G17270), AtMPT2 (AT3G48850) and AtMPT3 (AT5G14040). Their expression modes and function are analyzed in detail:(1) The phylogenetic tree analysis showed that the evolution of MPTs family is conservertive in all specices. The data also indicate that AtMPT2 protein is more closely related with AtMPT3 than with AtMPT1.(2) By quantitative real-time reverse transcription-PCR (qRT-PCR) and promoter:β-glucouronidase (GUS) fusions, the expression patterns were different among three AtMPT genes during Arabidopsis development. Moreover, the expression level of AtMPT3 is much higher compared with the other two MPT genes, revealing that AtMPT3 plays a predominant role in Arabidopsis development.(3) Both the seed germination and seedling establishment of mpt mutants and OEMPTs were obviously inhibited by salt stress. Recent research proved that GAs contents had something to do with the adaptation to salt stress in Arabidopsis. And the levels of bioactive GAs were slightly reduced in WT, on the contrary evaluated in mpts mutants and OEMPTs after salt treatment compared to the control. Besides, the salt sensitivities of mpt mutants and OEMPTs were similar to WT after supplementing with UNI during the seeds germination and seedlings establishment stages. These results indicate that MPTs mediated early Arabidopsis responses to salt stress, most probably through bioactive GAs.(4) The expression of GAs metabolic-related genes were remarkably regulated by AtMPTs. The expressions of GA responsive and metabolic genes changed remarkably in mpt3 and OEMPT3 compared with WT plants under salt stress condition. In detail, the biosynthesis genes GA20ox1/2/3 and GA3ox1/2/4 in mpt3 and OEMPT3 were slightly higher than that in WT plants, whereas the induced transcriptional levels of most GAs-deactivation genes GA2oxs were lower than that in WT plants.(5) AtMPT3 was induced by osmotic stress. Both the seed germination and seedling establishment of mpt3 and OEMPT3 were obviously inhibited by osomotic stress. The levels of bioactive ABA were all elevated in WT, mpt3 mutants and OEMPT3 after osomotic treatment. However, the ABA concentration increased 2.09-fold in mpt3, 1.99-fold in OEMPT3 compared to untreated controls, only1.23-fold in WT. Moreover, the osomotic sensitivities of mpt3 and OEMPT3 were restored to normal after applicating with tungstate during the seeds germination and seedlings establishment stages. These results suggested that some increase in endogenous ABA levels during osomotic stress could contribute to the enhancement of stress sensitivity of the mpt3 and OEMPT3.(6) ABA metabolic genes changed remarkably in mpt3 and OEMPT3 compared with WT plants. The transcription levels of the genes involving in ABA metabolism were affected by variable transcription level of AtMPT3. In mpt3 mutant and OEMPT3, the transcriptional levels of ABA biosynthesis related genes NCEDs and AAOs was induced much higher than that of WT; moreover, the transcriptional level of ABA decompose genes CYP707As was induced much lower than that of WT, Which in turn leads to the increase of ABA levels in mpt3 and OEMPT3.(7) The relative GUS activity of the newly constructed P3 promoter showed that the induced levels of GhNHX1 promoter was largely enhanced by the repeated GT-1 box and the TM6s.更多还原