【作者】 刘万钱；

【导师】 王伯初； 段传人；

【作者基本信息】 重庆大学 ， 药物化学， 2004， 硕士

【摘要】 环境因子对植物生长的影响是植物生理学和生物物理学中的一个热点。但在环境对植物代谢的影响以及植物是如何感受应力信号的，应力信号又是如何诱导植物基因表达调控代谢等方面的研究极其有限。铁皮石斛是名贵中药材，资源稀少，具有滋阴清热、生津益胃、止咳润肺之功效；其主要成分多糖和总生物碱可提高机体免疫力、延缓衰老。有研究表明，随环境变化兼性景天酸代谢（CAM）植物铁皮石斛的光合碳多谢类型会发生根本性的变化，影响其多糖等成分的含量。本文以声波刺激为应力加载方式，铁皮石斛的组培苗为材料，从铁皮石斛代谢产物和基因表达水平上分析应力刺激对植物代谢的影响，初步探索应力刺激与植物代谢的关系，以期在理论上深入了解应力对植物代谢的影响，在应用上为植物优良品种的培育及品种改良寻求一种新的方法。本论文的具体研究工作和结论如下： （1）研究了声波刺激对铁皮石斛代谢产物含量的影响，结果表明当声波频率为1000Hz、强度为100dB时对铁皮石斛组培苗的多糖合成有明显的促进作用，刺激组多糖含量要明显高于对照组；而对总生物碱的含量影响却不大。 （2）研究了声波对光合速率、呼吸速率的影响。结果表明声波刺激对铁皮石斛正常的光合速率有显著影响、而对呼吸速率功能的影响甚微。提示声波刺激可能诱导铁皮石斛光合碳代谢在CAM方式和卡尔文代谢（C₃）方式间转变，提高二氧化碳（CO₂）的吸收和利用率，从而增加多糖的含量。 （3）研究了声波刺激下铁皮石斛特异性基因的表达。结果表明处理组的总RNA得率要明显高于对照组的总RNA得率，检测到特异性基因表达条带。用提取的刺激组和对照组总RNA为模板，运用mRNA逆转录差异显示技术（DDRT-PCR）筛选受声波诱导的差异表达基因片段，并对在变性聚丙烯酰胺凝胶上分离到差异片段进行重扩增，在琼脂糖凝胶上显示的结果表明：SA5、SA8、SG3、SC3、CC7均清晰地显示出基因表达条带（图5．4），为进一步确定是否为真正受声波诱导而表达的条带，对其进行了Northern杂交。结果有SA5、SG3和CC7与声波处理组总RNA杂交后在胶片上清晰显影，为阳性条带，分子量依次约为370bp、410bp和500bp左右，其中CC7是受到声波处理后而抑制性表达。而SA8对照组和刺激组都有显影，SC3则不显影，说明SA8、SC3为假阳性，提示声波刺激可能会通过基因调控方式影响铁皮石斛的代谢。更多还原

【Abstract】 Nowadays, the effect of environmental factors on the growth of plant has become one of the hot researches in plant physiology and biological physics. In terms of finding out some researches on. the process in which how environment affect botanic metabolism, how plant would receive the stress signals and how these signals induce the expression of genes of plant regulated metabolic pathway, we cannot receive some satisfactory outcomes. Dendrobium candidum is a rare and endangered Chinese herbal medicine. It mainly contains polysaccharide and total dendrobine that can improve the capacity of immunity and delay the cell aging. As the relevant research work indicated, with variations of environmental factors, the pathway of photosynthesis of Dendrobium candidumcovld change foundationally between CAM and C3 pathway which affect the content of polysaccharide etc. For the purpose of further exploring the influence of stress on the botanic metabolism theoretically and of finding a new way in the breeding and the improvement of plant varieties in application, in this paper, using sound wave stimulation as a loading manner and the young plant of Dendrobium candidum as materials, we analyzed the influence of stress on the botanic metabolism basing on the content of metabolism product and the differential gene expression of Dendrobium candidum under the stress, at the same time researched preliminary on the relationship between stress and botanic metabolism. Then the main contents and the results of the research are as follow:To begin with, the influence of sound wave stimulation on the content of metabolism product of Dendrobium candidum had been examined, and the results showed that, under the frequency of 1000Hz and the intensity of 100dB, the polysaccharide formation would get the most obvious acceleration, but small impact on the total dendrobine.Secondly, through investigation, we noted that the normal photosynthesis and respiration were notable influenced by sound wave. The result indicated that the changes between CAM and C3 pathway of photosynthesis of Dendrobium candidum could are possibly induced in the effects of the sound wave on the plant, the CO2 uptake rate are enhanced and improved the content of polysaccharide.Thirdly, we measured and analyzed the differential expression of the chrysanthemum gene responded to the sound wave. The results showed that the yieldrate of total RNA is larger in the treated group than that in the control group, and the differential expression gene can be detected. With the total RNA extracted from the treated group and the control group, mRNA reverse transcription differential display PCR(DDRT-PCR) is applied to screen out the differential expression gene fragments induced by the sound wave. Differential expression gene fragments are isolated by the polypropylene acyl gel electrophoresis and be re-amplified. The five bands (SA5, SA8, SC3, SC8, CC7) can be seen from the agarose gel. Northern dot hybridization is used to further confirm whether the screening bands are induced by the sound wave or not. The results revealed that after the SA5, SG3 and CC7 hybridized with total RNA of the treated group and the control group, they were developed clearly on the film in the treated group only, however, the SC3 was not, which indicted that the SC3 was fake positive. And SA8 were developed clearly on the film both treated group and the control group. CC7 was an inhabitation expression band. The molecular weight of SA5, SG3 and CC7 were approximately 370bp, 410bp and 500bp respectively. These imply the influence of stress on the metabolism of Dendrobium candidum are possibly controlled by the differential genes.更多还原