【作者】 梁超；

【导师】 王玮；

【作者基本信息】 山东农业大学 ， 植物学， 2007， 硕士

【摘要】 在盐渍、干旱等胁迫环境中,植物细胞内通常积累一些相容性物质,从而减轻环境胁迫对植物造成的伤害,甜菜碱是主要的相容性物质之一。甜菜碱醛脱氢酶（BADH）在植物体内甜菜碱生物合成过程中处于关键地位,BADH基因工程是提高植物体内甜菜碱含量的重要手段。本实验以一个野生型小麦（Triticum aestivum L.）株系和三个转菠菜BADH基因小麦株系为材料,200mmol/L NaCl溶液处理小麦幼苗（一叶一心期）2天和4天,模拟不同程度的盐胁迫条件,从水分状况的维持、抗氧化活性的改善以及蛋白功能保护等方面,研究了过量积累甜菜碱对小麦光合作用及抗盐性的改善机制。检测指标包括甜菜碱含量、相对含水量、渗透调节能力、有机渗透调节物质和无机离子含量、抗氧化酶活性、光合荧光参数、叶绿素含量、希尔反应活力、ATPase活性以及类囊体膜类脂组分等。主要结果如下:1.过量积累甜菜碱通过改善渗透调节和离子平衡调节促进了小麦从盐胁迫环境中持续吸水,维持了相对较好的水分状况。（1）外源BADH基因的导入明显诱导了转基因株系甜菜碱含量的增加。积累的甜菜碱除了本身是一种有效的渗透调节物质以外,还通过促进其它有机渗透调节物质的积累,如脯氨酸和可溶性糖,维持了盐胁迫条件下小麦叶片及根系较高水平的渗透调节能力,增强了吸水,从而维持转基因小麦株系较好的水分状况。（2）盐胁迫导致小麦体内有毒害作用的Na⁺和Cl^-含量增加,破坏了体内的离子平衡。过量积累甜菜碱缓解了Na⁺和Cl^-从根系到叶鞘和叶片的运输,维持相对较高的K⁺吸收、K⁺/Na⁺比值以及Ca²⁺、Mg²⁺含量的相对高水平,减轻了离子毒害作用,进而提高了转基因小麦株系的抗盐性。2.过量积累甜菜碱维持了主要抗氧化酶的高活性,有效保护了生物膜的完整性和有序性。盐胁迫破坏了生物膜的完整性和有序性,最终导致超氧阴离子产生速率增加,膜脂过氧化产物丙二醛（MDA）含量以及膜透性的增加。过量表达甜菜碱的转基因植株主要抗氧化酶（SOD、CAT、POD、APX）的活性相对较高,超氧阴离子产生速率、MDA含量以及电解质外渗量较低,最终降低了膜脂过氧化水平和盐胁迫对转基因小麦株系生物膜的破坏作用。3.过量积累甜菜碱通过保护类囊体膜上功能蛋白以及类脂组分和功能来维持相对高水平的光合作用能力。（1）盐胁迫导致小麦幼苗光合机构损伤,表现在叶绿素含量、PSⅡ光化学效率、希尔反应活力以及类囊体膜ATPase活性下降。相对于野生型小麦,过量积累甜菜碱的转基因植株碳同化速率、PSⅡ光化学效率以及气孔导度的下降得以缓解;叶绿素含量降解、希尔反应活力及类囊体膜ATPase活性的下降相对缓慢。（2）盐胁迫诱导形成的活性氧会进攻生物膜类脂中不饱和脂肪酸,促发膜脂过氧化,引起生物膜脂分子结构及组分的变化。过量积累甜菜碱的转基因植株类囊体膜脂组分和脂肪酸不饱和度相对稳定。这对于缓解色素降解、希尔反应活力以及类囊体膜ATPase活性下降,缓解盐胁迫对叶片光合机构的破坏作用,维持盐胁迫下小麦叶片较高的光合作用能力有利。综合分析认为,盐胁迫条件下,过量表达菠菜BADH基因积累甜菜碱的转基因小麦光合作用能力及耐盐性的提高可能与以下几个方面有关:（1）通过自身或者诱导其它渗透调节物质的积累,进行渗透调节作用,改善细胞的水分状况。这对于维持光合作用的气孔因素和非气孔因素都是有利的;（2）通过维持或提高抗氧化酶活性增强活性氧清除能力,减少活性氧的积累,减轻膜脂过氧化水平,维持光合膜的完整性和流动性;（3）甜菜碱直接或者间接地维持类囊体膜上光合蛋白复合体的功能,进而维持相对较高的光合作用能力。更多还原

【Abstract】 Under environmental stress such as salinity and drought, plant cell usually accumulates some compatible solutes (also regarded as osmolytes) in order to alleviate the injury derived from environmental stress. Glycinebetaine (GB) is regarded as an extremely effective compatible solute, therefore researchers pay much attention to the bio-engineering of GB. The BADH gene encoding BADH which catalyzes betaine aldehyde (BA) into GB stands the essential position in the synthesis of GB, and gene engineering of it has become an important means to raise the GB content in vivo of plants. One wild-type wheat cultivar (Triticum aestivum L.) Shi4185 and three transgenic lines with BADH gene from Atriplex hortensis L were used to study the improvement of over accumulating GB on salt tolerance of wheat and the underlying mechanisms involved in were discussed. Salt stress was imposed by Hoagland solution containing 200mmol/L NaCl for 2 and 4 days. Indexes determined included content of GB, relative water content, osmotic adjustment ability, content of compatible solutes, ions, and chlorophyll content, the activity of ATPase, Hill reaction, anti-oxidant enzymes activities, and the components of thylakoid membrane lipids. The main results are as follows:1. Over accumulated GB enhanced the water-absorbing from salt stress environment and also maintained well water status in wheat seedlings, which may due to amelioration of osmotic adjustment and ion homeostasis.(1)Introducing of foreign BADH gene visibly induced the accumulation of GB. Besides osmolyte itself, accumulated GB could sustain a higher level of osmotic adjustment (OA) via accumulating some other compatible solutes such as proline and soluble sugar, which enhances water-absorbing and consequently maintains a better water status. (2)Salt stress caused accumulation of toxic Na+ and Cl-, and destroyed the ion homeostasis of wheat plants. Over accumulating GB restrained the transport of Na+ and Cl- from roots to sheaths and leaves, maintained a higher level of content of K+, Ca2+, Mg2+ and rate of K+/Na+ in leaves, mitigated the toxic effect of ions, and ultimately ameliorated the salt tolerance of transgenic lines.2. Over accumulating GB maintained higher activity of the main antioxidant enzymes and effectively protected the membrane integrity and stability from salt stress.Salt stress caused increase in superoxide radical (O2.-) production and destroyed the membrane integrity and stability, and ultimately resulted in evident increase in malondialdehyde (MDA) content and electrolyte leakage. Some main antioxidant enzymes such as SOD, CAT, POD and APX of transgenic lines with over accumulating GB maintained higher activity under salt stress, which then inhibited increase of O2.- production, MDA content and electrolyte leakage caused by salt stress. And ultimately, over accumulating GB effectively decreased the peroxidant level and protected the membrane integrity and stability of transgenic lines from salt stress.3. Over accumulating GB maintained higher level of photosynthesis through protecting membrane lipid components and function of protein complexes from salt stress.(1)Compared with wild-type Shi4185, transgenic lines with over accumulating GB were ameliorated in many photosynthesis indexes, such as decrease in CO2 assimilation, photochemical efficiency of PSⅡand stomatal conductance alleviated. And what’s more, degradation of pigments, decrease in Hill-reaction and ATPase activity were all mitigated.(2)Reactive oxygen species induced by salt stress might attack the unsaturated fatty acid, caused peroxidantion of membrane lipid, and also induced changes in molecular structure and component of membrane lipids. Transgenic lines with over accumulating GB had a relatively stable status under salt stress in components and levels of thylakoid membrane lipids. And this is advantageous not only to moderate the pigment degradation, Hill-reaction and ATPase activity decline, destruction in photosynthetic apparatus, but also maintain a higher level of photosynthetic ability.From all the results above, we propose that, the enhancement in photosynthetic ability and salt tolerance in transgenic lines under salt stress might be relative to these aspects. Firstly, GB ameliorates water status of cell by enhancing OA, which functioning by GB itself and other osmoyltes induced by GB. This is favorable for stomatal and non-stamatal factors of photosynthesis. Secondly, through maintaining or enhancing the activity of antioxidant enzymes, the accumulation of ROS and peroxidant level of membrane lipids are mitigated, integrity and fluidity of thylakoid membrane are sustained. Functional proteins embed in thylakoid membrane exist in a relatively stable environment and their functions are also protected indirectly. Thirdly, GB can help to maintain higher photosynthetic ability by directly protecting the structure and function of protein complexes embed in thylakoid membrane.更多还原