【作者】 薛焱；

【导师】 王迎春；

【作者基本信息】 内蒙古大学 ， 植物学， 2008， 博士

【摘要】 植物的耐盐机制的研究一直是植物研究领域的一个热点问题。植物的耐盐机理相当复杂,不同的植物在适应环境的过程中,形成了自己独特的适应性特征。本实验对东阿拉善-西鄂尔多斯特有双子叶植物---长叶红砂（Reaumuria trigyna Maxim.）的耐盐机理进行了研究和探讨,所得结论如下:1.长叶红砂在非盐渍化土壤（盐分＜0.1%）、盐渍化土壤（盐分在0.1～0.3%之间）,重盐土中（盐分在0.5～1.0%之间）都能完成生活史,且长叶红砂的叶片和嫩茎上均具有盐腺,说明长叶红砂是一种泌盐盐生植物。2.长叶红砂的盐腺属典型的多细胞盐腺,由8个细胞组成,基部2个属收集细胞,外侧6个属分泌细胞,6个分泌细胞两两成对紧密排列,形成3列,内层的2个分泌细胞与2个收集细胞相连接,收集细胞从叶肉细胞中收集盐分并运送到分泌细胞,盐腺腺体除最内部细胞和基层下部细胞之间的渗入区域一小部分外,几乎全部被有一层厚的角质层。长叶红砂通过叶片的肉质化、叶表角质化、叶肉细胞排列为环形栅栏组织和茎、叶气孔下陷等形态结构的适应而降低蒸腾作用,通过茎、叶上的盐腺将侵入体内的多余的盐分排出体外,使其在盐渍化土壤或重盐土中生活。3.盐腺分泌特点表现为:盐腺分泌的离子种类与生存的土壤环境、植物体内离子种类一致,但Na⁺、Cl^-的分泌量大,K⁺、Mg²⁺等的分泌量小;分泌的日变化表现为上午高于下午,白天高于夜间,而且白天中受大气相对湿度的影响,从早到晚分泌量表现出降低的趋势。这些结果说明在外界环境因素的调节下,长叶红砂通过盐腺的选择性分泌使植物体内保持适当的盐分和养分浓度以适应盐渍生境。4.种子萌发实验显示:种子萌发的适宜温度为20～25℃。温度高于25℃时,温度越高,萌发率越低;在25℃下,浓度低于100 mM的NaCl对种子萌发的影响不大,但高于150 mM时,NaCl下种子的萌发率随NaCl浓度的增高而下降,直至0,说明长叶红砂种子萌发期间对土壤含盐量有一定要求,在土壤盐分含量高的生境中,植物的更新会受到一定的影响;NaCl溶液处理9 d的未萌发种子转移至蒸馏水中后,经高浓度NaCl胁迫（200～500 mM）处理的种子萌发恢复率约为90%,且在1～2 d内即迅速萌发。利用这个特点,先用高浓度的盐溶液处理种子,之后播种,可提高种子的萌发速度和整齐度。5.以不同浓度NaCl溶液胁迫30天的长叶红砂植株为材料,对其生长发育的研究结果显示:一定浓度的NaCl可以促进长叶红砂幼苗的牛长,200 mM浓度的NaCl是长叶红砂幼苗生长的最适浓度,比种子的耐盐半致死浓度148.107 mM要高,显示长叶红砂幼苗耐盐的能力比种子要强。说明盐渍环境对幼苗生长的影响不大,使长叶红砂成为残遗种、局限分布和更新速度缓慢主要与长叶红砂种子萌发阶段对盐分的要求低,而盐渍环境造成种子萌发困难有关。6.对不同浓度NaCl溶液胁迫30天的长叶红砂植株的抗氧化系统的活动和渗透调节能力的研究显示:（1）长叶红砂的相对耐盐力与高的抗氧化酶活性和谷胱甘肽含量,低的MDA含量、相对电导率相关。说明对长叶红砂的抗盐能力而言,限制氧化性的损害是重要的;（2）长叶红砂主要通过Na⁺和Cl^-、可溶性糖、脯氨酸及可溶性蛋白等进行渗透调节,而不依赖游离氨基酸、有机酸及K⁺、Ca²⁺、Mg²⁺等离子进行渗透调节。7.利用mRNA差异显示（DDRT-PCR）技术,将正常生长和用400mM NaCl处理48 h的长叶红砂的mRNA逆转录合成cDNA,然后在6%变性聚丙烯酰胺凝胶上分离。从凝胶上回收到了80个差异条带的克隆,经Reverse Northern斑点杂交验证,获得了5个阳性克隆,对其进行测序,使用blast工具将测序结果与GenBank数据库中的资料进行序列比对。初步结果为:得到5条长叶红砂特异表达基因的cDNA序列,长度分别为386bp,481bp,495bp,369bp和278bp,它们都与已知序列同源性低,可能是与长叶红砂耐盐相关的新基因,耐盐相关cDNA片段的获得,将为分离全长耐盐基因,搞清该基因表达调控的机理提供条件。根据以上研究结果认为长叶红砂体内具有多种耐盐途径,主要可以归纳为:1)盐腺的分泌活动;2)适应盐渍环境的茎、叶结构特征;3)良好的生理适应性;4)盐胁迫相关基因的表达。综合以上结果分析认为长叶红砂的耐盐绝不仅仅是盐腺的泌盐作用,而是多种耐盐途径综合作用而使长叶红砂能够适应荒漠盐渍的生存环境。更多还原

【Abstract】 Salt-tolerant mechanism of plants had been a hot issue in plant research field.Salt-tolerant mechanisms was rather complicated,different plants formed its own unique characteristics of adaptability in their adaptation to salt environment.Reaumuria trigyna Maxim,derived from Mediterranean Sea and endemic to Alashan-Erdos Desert of China,is an endangered shrubby perennial plant.This experiment aimed to study salt-tolerant mechanisms of Reaumuria trigyna. The main results were as follows:1.Reaumuria trigyna could complete life cycle in the non-saline soil（salt＜0.I%）,salinization of the soil（salt at 0.1～0.3%）,and in re-saline soil（salt at 0.5～1.0%）,has salt glands in the leaves and soft stem.Reaumuria trigyna is an Exo-reorto halophytes.2.Salt glands of Reaumuria trigyna belonged to typical multicellular salt gland.Salt gland originates in specialized epidermal cells,is composed of 8 cells.Among these cells,2 are collecting cells which are similar to the ordinary mesophyll cell with obvious vacuole,surrounding 6 cells are secretory cells which have thick cytoplasm,form 3 rows,2 secretion cells in the most outer layer are big,cells in the middle secretory and secretion cell in the inner layer connecting with 2 collecting cells are small.The collecting cell collects the salinity from the mesophyil cell and ships to the secretory cell,simultaneously may see a thick cuticle besides lower part of the internal cell and the basic unit between a cell’s permeating region small part.Reaumuria trigyna decreased transpiration and adapted to salt environment by succulent leaves,sunking stoma and salt glands which removed excessively absorbed ions and reduce the salt concentrations of active tissues.3.The types of icons was same in the soil solutions,plant tissues and excretion from salt gland,but the excreted ions amount from salt gland was obviously different.Among the excreted ions from salt gland,Na⁺ and Cl^- was higher,K⁺,Mg²⁺ was lower.The changes of salt gland secretion in the day showed decreasing trend which had very significantly or significantly positive correlation with the relative humidity of the atmosphere from morning to night.In addition,the excretion rate was higher during the day and in the morning than during the night and in the afternoon respectively.The above described findings showed that such high selectivity for Na⁺,Cl^-in absorption and excretion of Reaumuria trigyna is of high significance to the plant in balancing nutrition in Reaumuria trigyna and adapting to the wild habitat.4.The optimal temperature for germination was 20～25℃.When the temperature was over 25℃,the germination percentages decreased.NaCl at concentration below 100 mM had no effect on seed germination.When NaCl concentration was over 100 mM,the seed germination obviously decreased until 0.After treatment with NaCI solution for 9 days,then transferred in distilled water, recovery germination percentages of seed treated with high concentration of NaCl（200～500 mM） was about 90%.We can improve seed germination rate and uniformity by treating seed with high concentration of NaCl before sowing.5.Reaumuria trigyn was treated with NaCl of different concentrations（0,100,200,300 and 400 mM）.A study about its growth in the different conditions showed that the suitable NaCl concentration of Reaumuria trigyna seedlings growth was 200 mM which was higher than 148 mM --- the seed’s tolerance of half-lethal salt concentration.These results suggested that the reasons leading to Reaumuria trigyna for the endangered was saline environment which caused seed germination difficulties.6.A study on antioxidative activities and osmotic ability of Reaumuria trigyn with NaCl was conducted and its result demonstrated:（1） Under NaCl stress,the activities of SOD and POD in Reaumuria trigyna all increased significantly,CAT activity had no distinct difference（P＞0.05）. Glutathione and O₂ production rate significantly increased,ascorbic acid salt content decreased, increased significantly,while MDA and the relative conductivity appeared to decrease first and then increase as salinity increase.As a whole,these data suggest that the capacity to limit oxidative damage is important for salt tolerance of Reaumuria trigyna;（2） osmotic adjustments of Reaumuria trigyna mainly relied on Na⁺ and Cl^-,soluble sugar,free amino acid,proline and soluble protein, rather than relied on organic acids,K⁺,Ca²⁺ and Mg²⁺.7.In order to check out whether there exists specific expression in the Reaumuria trigyna treated by 400mM NaCl,mRNA was extracted the total RNA from the leaves of Reaumuria trigyna treated by 400raM NaCl and the control,cDNA fragments were generated by reverse transcription polymerase Chain reaction（RT-PCR）.80 differentially displayed fragments were separated on 6% polyacrylamide gel electrophoresis（PAGE）,and five of them were confirmed as positive clones by reverse northern blot hybridization.These fragments were then cloned into vectors for sequencing. The cDNA fragment sequences were compared with data in GenBank using basic local alignment search tool（blast）.Our preliminary results showed that five eDNA fragments were expression in Reaumuria trigyna treated by 400mM NaCl.The five cDNA fragments were 386,481,495,369 and 371base pairs,respectively.After searching in GenBank,there were no similar sequences reported about these fragments.In conclusion,salt-tolerant mechanism of Reaumuria trigyna can be summarized with the following features:（1） salt gland secretion;（2） stems,leaves structural characteristics adapting to saline environment;（3） physiological adaptation;（4） salt stress-related gene expression.All these study results showed that salt tolerance of Reaumuria trigyna can adapt to the desert and saline environment for the survival by above ways.更多还原