【作者】 李妹芳；

【导师】 杨兴洪；

【作者基本信息】 山东农业大学 ， 植物学， 2013， 博士

【摘要】 在非生物胁迫下，许多植物适应环境的重要机制就是积累一些低分子量的相容性物质，如甜菜碱（GB, Glycine betaine）、脯氨酸、可溶性糖等。其中甜菜碱是一种有特殊功效的相容性物质。甜菜碱在提高植物环境逆境（例如盐胁迫或高温胁迫）的抗性方面有重要作用，并且甜菜碱提高植物耐受性的同时伴随着热激蛋白（HSP, heat shockprotein）的积累。甜菜碱提高抗逆性与信号转导和离子稳态有关。本研究在前期工作基础上利用转BADH基因的烟草和番茄材料，结合外源甜菜碱处理的方法，分析甜菜碱的作用与不同的逆境胁迫及不同遗传背景的关系，探究转基因植物耐受性获得中与热激蛋白和钙-钙调蛋白信号途径的关系，甜菜碱提高光合性能以及甜菜碱对活性氧清除系统的作用。研究结果和主要结论如下：（1）利用非损伤微测技术，对外源甜菜碱处理和转BADH基因烟草（体内积累甜菜碱）进行钙离子（Ca2+）跨膜流速变化趋势的分析研究。实验结果表明：烟草根部的伸长区表皮细胞用NaCl处理后，产生一个瞬态Ca2+外流，高峰值在1-2分钟内。甜菜碱可降低这种外流的峰值。提高NaCl（50-100mM）浓度后，峰值不再升高，Ca2+-ATPase抑制剂（Eosin Y，Eryth-B和CPA）也没有显著影响Ca2+外流，表明这种瞬时的外流主要是细胞壁的阳离子交换；盐胁迫24小时后，Ca2+呈现内流趋势，与此同时低浓度的甜菜碱显著增加NaCl诱导的Ca2+内流值。药理学实验结果表明，胁迫条件下甜菜碱可通过LaCl3敏感的钙离子通道加强Ca2+内流。（2）甜菜碱升高了胁迫下细胞内自由钙离子浓度（[Ca2+]cyt）。激光共聚焦的实验结果证实，无论是外源还是内部合成甜菜碱都可适度升高胞质内自由钙离子浓度。非损伤微测技术和激光共聚焦的实验结果，从动态和静态两方面综合印证了盐胁迫条件下甜菜碱与钙离子之间的因果关系。（3）甜菜碱提高了非生物胁迫下的植物体内钙的积累。进一步研究甜菜碱对Ca2+吸收的重要作用，对植物中的Ca2+含量进行分析比较表明，在长时间盐和高温胁迫下，甜菜碱预处理的和转基因植物中Ca2+含量明显高于野生型。（4）甜菜碱增强钙调蛋白（CaM）和热激因子（HSF）基因的表达，从而诱导HSP的积累。实时定量PCR实验，从基因表达层面证实甜菜碱与钙-钙调蛋白（Ca2+-CaM）以及HSP的因果关系。药理实验证实Ca2+和CaM增加了HSFs和HSP基因表达水平，HSP70的Western杂交结果也证实了HSP的积累。Western杂交的药理学实验，从蛋白层面证明甜菜碱可以通过Ca2+-CaM信号途径诱导热激蛋白HSP70的合成积累。结果表明：甜菜碱作为辅助因子增强盐或热胁迫下Ca2+的吸收，并通过Ca2+-CaM的信号途径诱导HSP的转录和翻译。（5）甜菜碱降低活性氧（ROS）的积累。过氧化氢（H2O2）和超氧阴离子自由基（O2-）在热激下积累，但是转BADH基因植株中积累量少于野生型。另一重要发现是外源添加甜菜碱在活体外不能清除H2O2和O2-，但酶抽提物能降低ROS的浓度，并且转基因植物中的明显清除能力强。这些结果表明在活体内积累甜菜碱不能直接清除ROS，而是通过体内的ROS清除系统发挥作用。（6）甜菜碱提高植物的抗氧化酶活性，减轻膜伤害的。转BADH基因植物的抗氧化酶活性高于野生型，相对电导率（REC）和丙二醛（MDA）的含量低于野生型，表明甜菜碱积累可提高抗氧化酶活性，减轻膜伤害。（7）甜菜碱提高ROS清除酶的基因表达，药理学实验证实Ca2+和CaM增加了CuSOD、APX1和CAT1基因的表达水平，从基因表达层面证实甜菜碱甜菜碱可以通过Ca2+-CaM途径影响一系列基因的表达。（8）甜菜碱提高植物逆境胁迫下的光合能力，减轻光抑制。在42℃热激条件下观测叶绿素荧光表明转基因植物比野生型有较高的光合能力。表明积累甜菜碱可减轻热激引起的光抑制。在盐胁迫下也有类似的结果。同时D1蛋白的Western杂交结果表明甜菜碱提高了D1蛋白的含量，加快D1蛋白的周转，加速PSII的修复。盐和高温是两种普遍的非生物胁迫，利用外源甜菜碱处理及两种转BADH基因材料，在非生物胁迫（高温和盐胁迫）下，研究甜菜碱在不同植物的遗传背景下的抗逆性机制，排除了材料差异和转基因事件引起的其他影响，更好地证实了甜菜碱的生物学功能。从钙信号转导、活性氧清除、光合机构的保护以及热激蛋白等相关基因表达等方面综合阐明：非生物胁迫产生活性氧的积累，形成变性蛋白，伤害细胞的正常生命活动，引起光合能力下降，影响生物量的积累。胁迫可引起Ca2+内流，[Ca2+]cyt适度升高，可以加快下游的信号转导，从而诱导HSP的转录，获得HSP的积累，减少伤害，是植物细胞一种积极的防御措施。甜菜碱的积累，无论是外源处理还是内部合成，都可加强这一信号，诱导胞质内钙离子浓度适度升高，并通过Ca2+-CaM的信号途径诱导较多HSP的转录和翻译，提高防御能力。甜菜碱通过钙离子信号系统，增加HSP的积累，发挥分子伴侣作用，帮助变性蛋白复性，减少变性蛋白的积累，从而提高植物抗逆性。另外甜菜碱可诱导活性氧清除酶的表达，提高抗氧化酶活性，减少活性氧的积累，从而减轻胁迫造成的伤害。更多还原

【Abstract】 Glycine betaine (GB) is a substance with special efficiency of the compatibility. GBincrease plant tolerance at the same time accompanied by heat shock protein (HSP, heat shockprotein) accumulation in plants under abiotic stress. HSPs can act as chaperones of denaturedproteins and assist in the translocation and/or degradation of damaged proteins under variousstresses. GB improves salt resistance related to signal transduction and ion homeostasis.Based on the previous work, this study used these two kinds of genetically modified materialand processing method of exogenous betaine to analysis the role of GB and different adversitystress and the relationship between the different genetic background, to explore the transgenicplants for heat resistance and heat shock protein and calcium and calmodulin signalingpathways, the relationship between GB how to protect the photosynthetic mechanism andinfluence the expression of HSP and improve resistance and active oxygen removal system.(1) To investigate the mechanism of how GB influences the expression of HSP, both theaccumulation of GB in vivo and exogenously applied GB in WT seedlings was studied duringNaCl stress. The elongation zone in tobacco root epidermal cells treated with NaCl. Atransient Ca2+efflux was found after NaCl treatment for1-2min in the epidermal cells of theelongation zone of tobacco roots. But increasing the NaCl concentration (50-100mM) did notsignificantly increase the speed of the outflow of calcium ions. In addition, Ca2+-ATPasemetabolic inhibitors (Eosin Y; eryth-B and CPA) had not significantly effect on Ca2+efflux,which indicated the transient NaCl-induced Ca2+efflux were likely produced by the cell wallcation exchange. After24h of NaCl treatment, an influx of Ca2+was observed, meanwhile lowconcentrations of GB significantly increased NaCl-induced Ca2+influx. Pharmacologyexperiments showed that GB can enhance Ca2+influx through the LaCl3sensitive calcium ionchannel under stress condition.(2) GB increased intracellular free calcium ion concentration ([Ca2+]cyt) using LSCM,which proved whether foreign or internal composition GB can enhance [Ca2+]cyt.The resultsof non-invasive microelectrode ion flux measuring technique and LSCM proved there is the causal relationships between GB and calcium from two aspects of the static and dynamicunder salt stress and high temperature stress condition.(3) GB increased the calcium content of tobacco plants under salt or heat stress. Tofurther investigate whether GB plays a role in Ca2+uptake, the calcium content of WT plantspre-treated with GB (WT+GB) and transgenic plants (T) were compared with that of WTplants. GB affected Ca2+acquisition in shoots and roots during long-time NaCl stress, and thecalcium content of WT plants pretreated with GB and that of T plants was higher than that ofuntreated WT plants. The results were consistent with the calcium content of leaves underheat stress.(4) GB increased the intracellular free calcium ion concentration and enhanced theexpression of calmodulin (CaM) and heat shock factor (HSF) genes resulting in potentiatedlevels of heat shock proteins (HSPs). Pharmacological experiments confirmed that Ca2+andCaM increased the HSFs and HSPs gene expressions coincide with increased the levels ofHSP70accumulation. The causality of GB, calcium-calmodulin (Ca2+-CaM) and HSP isconfirmed from gene expression level by real-time quantitative PCR. It is proved that GB canenhance the levels of HSP70accumulation through the Ca2+-CaM signal pathway from thelevel of protein by Western blotting. A possible regulatory model of Ca2+-CaM in the signaltransduction pathway for induction of transcription and translation of the active HSPs is described.(5) Significant accumulation of hydrogen peroxide (H2O2) and superoxide radical (O2-)were observed in wild type plants under heat stress; however, these accumulations were muchless in transgenic plants. An important finding reported herein is that exogenous GB cannotdirectly reduce the content of ROS. However, enzyme extraction from the WT can slightlyreduce ROS. In particular, enzyme extraction from transgenic plants greatly decreased theROS compared to the WT plants. These results also indicate that GB indirectly scavengesROS through antioxidative defense in vivo.(6) GB increase ROS scavenging enzyme activities. Evidently, GB may enhance theactivities of these enzymes to quench ROS in vivo, resulting in a lower ROS concentration.The activities of antioxidant enzymes were also stronger in accordance with lower relativeelectrolyte conductivity (REC) and malondialdehyde (MDA) content in transgenic lines,indicating that the degree of membrane injury in transgenic plants was lower than that in wild type plants. In addition, GB enhanced the expression of antioxidant enzyme genes. The resultssuggested that the accumulation of GB in vivo cannot directly eliminate reactive oxygenspecies (ROS), rather, through maintaining higher activities of antioxidant enzymes to lessenthe accumulation of ROS in transgenic plants and decrease the degree of membrane injury.(7) GB increase ROS scavenging enzyme (CuSOD, APX1and CAT1) gene expression.Pharmacological experiments confirmed that Ca2+and CaM increased these genes expressions.It is confirmed that GB can affect a series of gene expression through Ca2+-CaM pathway byreal-time quantitative PCR.(8) The chlorophyll fluorescence analysis of wild type and transgenic plants exposed toheat treatment (42°C) showed that transgenic plants exhibited higher photosynthetic capacitythan wild type plants. This result suggests that the accumulation of GB increased the toleranceto heat-enhanced photoinhibition. This increased tolerance is associated with theimprovement of D1protein content, which accelerated the repair of photosystem II (PSII)from heat-enhanced photoinhibition. GB also increased the tolerance to NaCl-enhancedphotoinhibition.GB is researched in different plant genetic background for salt stress resistance and hightemperature mechanism, to confirm biological function of GB. The relation of GB, HSP andCa2+-CaM signal transduction pathways is clarified from the heat shock protein accumulation,signal transduction, active oxygen removal, photosynthetic mechanism as well as theprotection of related gene expression. Environmental stresses generate ROS accumulation toformate denatured protein to affect normal metabolic activity. These results suggests thatstress signals are perceived by an unidentified receptor and GB applied exogenously oraccumulated in vivo in BADH-transgenic plants may act as a cofactor to activate Ca2+channels in the plasma membrane or intracellular Ca2+store membrane resulting in anincrease in [Ca2+]cyt. GB accumulation can strengthen this signal, This moderately elevatedlevel of [Ca2+]cytpromoted the expression of CaM1, which increased the DNA-bindingactivity of HSF. Activation of HSF initiated transcription and translation of HSP genes. Theelevated HSP, as molecular chaperone, assist in modified protein renaturation, reducedenatured protein accumulation, to improve plant resistance. In addition, GB induced theexpression of ROS scavenging enzyme, also reduce the accumulation of ROS to alleviate the damage caused by stress. Other pathways are possible including the regulation of HSFphosphorylation by regulation of CaM-dependent kinase, CDPK, MAPK activity, etc.; futurestudies may determine definitively which pathways are playing a role.更多还原