【作者】 孙磊；

【导师】 钟秀丽；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 我国水资源严重短缺，干旱灾害日益严重，已经成为制约农业和社会发展的重要因素。发展节水农业，提高有限水资源的利用效率，是缓解水资源危机，实现农业可持续发展的必由之路。加强作物抗旱与高效用水机理的研究，发掘植物抗旱节水基因资源，增强作物自身的水分利用效率和抗旱性潜力，是生物性节水技术开发的核心目标。磷脂酶D（Phospholipase D，PLD），是植物中存在的一类重要的跨膜信号转导酶类，参与植株的生物与非生物逆境胁迫反应。本研究利用农杆菌介导法将PLD1基因的超表达载体和RNA干涉载体转化入84k杨（银白杨×腺毛杨），通过对转基因株系进行PCR检测及Southern blot分析，证明目的基因片段已经整合到84K杨的染色体中，成功获得PLD1基因超表达（OE）植株以及PLD1基因RNA干涉（RNAi）植株。将OE型植株、RNAi型植株及野生型（WT）植株三种材料在温室中利用盆栽实验，进行水分胁迫处理和表型抗旱性鉴定。结果发现，与WT相比，OE型植株抗旱性较提高，RNAi型植株抗旱性则下降，表明PLD1基因对植物的抗旱性有正调节作用。在水分胁迫处理后对三种基因型植株的渗透调节能力、气孔调节特性、细胞膜脂质组和信号离子流速进行测定，分析PLD1基因调控植物抗旱性的作用途径，主要结果如下：（1）水分胁迫处理后，三种基因型之间饱和渗透势与渗透调节能力没有显著差异，表明PLD1基因不是通过参与植物的渗透调节途径提高植株的抗旱性。（2）水分胁迫条件下，与WT相比，OE型植株的气孔导度下降幅度较大，蒸腾失水速率减小，RNAi型植株气孔导度下降幅度较小，蒸腾失水速率高。这一结果表明，PLD1基因通过参与水分胁迫条件下的气孔调节作用影响植物的抗旱性。（3）利用非损伤微测技术检测三种基因型植株根部分生区域Ca²⁺、H⁺、K⁺三种信号离子的流速，发现水分胁迫条件下，OE型植株的Ca²⁺流速明显高于RNAi型植株，同时OE型植株H⁺的内流流速也大于RNAi型植株，进一步证明PLD1基因参与干旱信号的转导途径。通过本研究，可以初步推定植物中存在这一信号转导途径：胞外水分胁迫信号—跨膜信号转导酶PLD1—Ca²⁺—气孔调节—调控抗旱性。抑制PLD1的表达，使其参与的信号转导途径部分受阻，气孔调节受到抑制，导致蒸腾失水量大，植株抗旱性减弱。（4）在水分胁迫条件下三种基因型细胞膜脂质组学分析发现，PLD1酶在活体条件下优先水解PC，导致OE型植株中PC含量较RNAi型植株显著降低，PA含量则明显提高,这种脂质组成变化不利于细胞膜的稳定性。但是，从植株生长量、净光合速率、气孔导度、蒸腾速率的变化来看，超表达PLD1增强了植株的抗旱性。PLD酶兼具脂质降解和信号转导双重功能。这一结果表明，在水分胁迫条件下，PLD1酶的信号转导功能发挥主导作用。更多还原

【Abstract】 With aggravating water resource crisis, drought has become an important factor of restrictingagricultural and social development in China. Developing water-saving agriculture to improve water useefficiency （WUE） is the only way to achieve sustainable agricultural development. Enhancing WUE anddrought tolerance of crop cultivars by exploring gene recourses on the basis of mechanism studies onhigh WUE and drought tolerance of plants is the main goal of biological water saving. Phospholipase D（PLD）, found widely in diverse plants, is a class of important transmembrane signaling enzymes, andhave been reported to be involed in many biotic and abiotic stress responses.In this research, Agrobacterium tumefaciens-mediated method was adopted to respectivelytransform the overexpression vector and RNAi vector of PLD1gene into Poplar84K（P.alba×P.glandulos）. The result of PCR test and southern blot analysis of transgenic plants showedthat the target gene fragment had been integrated into the chromosome of Poplar84K, and obtain thesurvival super-expressed plants and RNAi plants with PLD1gene.The three genotypes of OE, RNAi, and wild type （WT） were treated with drought stress in thegreenhouse to evaluate the droght resistance phenotypically. The results showed that, compared with WT,drought resistance was increased in OE type transgenic plants, while decreased in RNAi type transgenicplants, indicating that PLD1gene positively regulates drought resistance of palnts.To clarify the function pathways of PLD1gene in regulating plant drought resitance, osmoticadjustment ability, stomatal regulation trait, the signal ion flux, and the membrane lipidomics weremeasured and analyzed after the plants were treated with water stress. The main results are as follows.（1） No significant differences in saturated osmotic potential and osmotic adjustment ability wereshown among the three genotypes after water stress treatment, indicating that PLD1gene is notinvolved in the osmoregulation ways to improve plant drought resistance.（2） Under water stress, compared with WT plants, OE type transgenic plants shew increase in thestomatal conductance, while RNAi type transgenic plants shew decrease in stomatal conductance.Stomatal conductance reduction may lead to a decrease in transpiration rate under water stress, whichcontrol effectively water loss through transpiration. This suggests that PLD1gene regulate droghtresistance of plants through the pathway of stomatal regulation.（3） Ca²⁺, H⁺, and K⁺fluxes in roots of the three genotype after water stress treatment weresimultaneneously measured by using the Non-invasive Micro-test Technique. The results show that theefflux of Ca²⁺and influxes of H⁺in OE type transgenic plants were promoted, in contrast, that in RNAitype transgenic plants were inhibited. This further supports that PLD1gene is involved in droughtsignal transduction pathways. Through this study, one signal transduction pathway in plant may bespeculated as: extracellular water stress signal-transmembrane signal transduction enzymePLD1-Ca²⁺-stomatal regulation-Regulation drought resistance. Based on this, inhibition of PLD1expression blocked the signal transduction pathway to some extent, then the stomatal regulation was inhibited, which resulted in large amount of water loss through transpiration and decrease in droughtresistance of plants.（4） Lipidomic analysis of the cell membrane in two transfer genotypes under water stressconditions found that PLD1enzyme selectively hydrolyze PC in vivo, leading to a lower level of PCand a higher level of PA in OE type transgenic plant than that in RNAi type transgenic plants. Suchvariation in lipid composition can not stabilize cell membrane under stress. However, results of plantgrowth, net photosynthetic rate, stomatal conductance, transpiration rate indicated that overexpression ofPLD1enhanced the drought resistance of plants. PLD enzymes play both lipid degradation role andsignal transduction role in living organism. This result imlies that, under water stress conditions, thesignal transduction role PLD1enzymes plays contributed dominantly.更多还原