【作者】 刘丽艳；

【导师】 马凤鸣；

【作者基本信息】 东北农业大学 ， 作物栽培与耕作学， 2004， 博士

【摘要】 利用植物自身抗性建立一种无残毒、无污染、可持续地控制农业害虫的策略是人类孜孜以求的目标。为此就必须对植物自身的抗性反应机理进行深入的研究。 番茄(Lycopersicon eculentum)是重要的经济作物和分子生物学研究中的重要的模式植物。番茄对许多昆虫的天然抗性是由于具有组成型或诱导型化学物质在起作用，在植物与农业病、虫害相互作用关系的研究中，番茄历来被作为不可多得的模式植物。番茄也是植物诱导性抗性研究的理想模型系统。在此系统中可研究调控防御反应的各种信号途径。 本研究的基本思路是利用番茄这一模式植物的蛋白酶抑制剂(proteinase inhibitors，PIs)为抗性反应强弱的标记基因，筛选番茄昆虫抗性缺失突变体，在对突变体进行分析的基础上克隆相应的基因进而研究其生物学功能、调控机制和在抗性反应中所起的作用，尤其是在番茄诱导抗性模型系统所具有的功能。在此模型系统中，可以研究调控反应的各种途径和该途径在抵御刺吸式昆虫的作用。 本试验在前人研究的基础上，建立了一整套实验体系，解析信号传导途径在分子水平抵御昆虫的机制。主要研究结果如下： 1．雌性成虫二点叶螨侵袭两叶一心期(15天龄)野生型(WT)和昆虫抗性缺失突变体def1植株，结果显示：def1植株对二点叶螨捕食较WT植株表现了极显著的敏感性，同时，在def1植株叶片上比其在WT植株叶片上的虫卵数明显增加。这些结果指出：Def1基因发挥着重要作用--降低了番茄叶片作为二点叶螨食物来源和产卵基质的质量。 验证了脂肪酸途径调节着防御性化合物在植株叶片的合成。和未处理的对照植株相比，WT植株经二点叶螨捕食后，积累了高水平的PI-Ⅱ和JA。而在def1植株上，PI-Ⅱ水平仅是稍高于测定的检测限，JA水平没有受到二点叶螨侵袭的影响。 二点叶螨对番茄植株的捕食激活了脂肪酸途径，导致PI-Ⅱ基因的激活，从而诱导PI-Ⅱ蛋白的积累及相关基因的表达。由于def1突变体中脂肪酸合成途径受阻，组织的防御体系受到破坏，所以突变体受害严重。 外源施用茉莉酸(JA)诱导抗性试验结果表明：JA是恢复def1突变体的抗虫性必须的。足够量的JA可恢复突变体对二点叶螨的抵御能力。甲基茉莉酸(MeJA)处理也减少了二点叶螨对WT番茄的捕食量和在其植株上的产卵数。 在对番茄转基因品系35S∷prosystemin(35S∷prosys)transgene的实验观察了二点叶螨在番茄转基因品系35S∷prosys植株上的表现：35S∷prosys植株比def1和WT番茄植株都更抗二点叶螨危害。同时，二点叶螨产卵率在转基因品系上大量减少，证明了系统素在番茄系统抗性中起必不可少的作用。 苜蓿蓟马捕食诱导脂肪酸信号途径介导的寄主反应，通过原系统素的超表达，组成型地激活信号途径，因而提高该转基因番茄植株对多种刺吸式昆虫的抗性。 2．集群分离群体分析法(bulked segregant analysis，BSA)结合AFLP技术，鉴定了与Def1基因连锁的两个AFLP分子标记(EM-1和EM-2)。试验表明所有的受伤反应阳性东北农业大学农学博_J:学位论文(w十)对两个分子标记是杂合的。而所有的受伤反应阴性植株对两个分子标记(EM一l和EM一2)是纯合的。对由20个植株组成的BC，分离群体植株进行试验，未发现有重组体，说明分子标记EM一l和EM一2与Defi基因紧密连锁，它们位于第3染色体长臂端着丝粒远侧。证实D叨的位置在染色体3的末端，该位置距离含有Ds转座子T-DNA插入接近ZeM。3.嫁接试验表明:JA生物合成有缺陷的突变体d叨和JA反应有缺陷的突变体jai]‘丧失了受伤诱导的PI基因的系统表达。jai了突变体不影响嫁接传导系统信号在受伤位点产生，但它挠乱了未受伤叶片信号识别。系统受伤反应信号需要JA在受伤叶片的生物合成，而系统素不是系统受伤反应移动信号。JA是系统抗性长距离运输的信号分子。更多还原

【Abstract】 That Plant which made use of the " built in " defense system against mechanical wounding and herbivore established a kind of no remain toxin, no pollution could be continuously control agricultural pests’ strategies ,which was human being searching for objective continously and patiently. For this great propose, we must study further on the plant built-in mechanisms.Tomato was an important cash crop and a model plant in molecular biology field. Its natural resistance to herbivore attack was because of constitutional or induced phytochemicals taking function.Tomato had been thinking as one of the best model plants in study on the interactions between plants and agricultural pests. Tomato was also a kind of ideal model system for plant induced resistance. In this system, signal pathways that regulated defense responses could be studied.In this research, we follow this kind of basic technical route: tomato protainese inhibitors (Pis) as a molecular marker that could express the strength and weakness of the resistance of tomato to herbivore attack and mechanical wounding for screening the mutant deficient in resistance to insects , cloning corresponding gene based on analysis of the mutant and for further studing the biological function and action in the resistance response.A set of experimental systems had been established in this research on basis of previous study that analysed signaling transduction pathways for plants resistant to insects at molecular lever. The results were as follow:1. That defl and wild type tomato plants were attacked by female adult two spotted spider mite show: defl plant was significantly sensitive to two spotted spider mite, meanwhile the number of eggs per plant on defl in comparasion with that on wile type plant increased distinctly. These results indicated that Defl gene played an important role in reducing the quality of tomato leaves as a food source and oviposition substrate for two spotted spidermite.It had been demonstrated that octadecanoid signaling pathway mediated synthesis of defensive compounds in wild type tomato leaves. Wild type plants subjected to mite feeding accumulated high levels of PI-II and JA relative to untreated control plants. However, PI-II levels in defl plants were only slightly greater than the detection limit of the assay, and JA levels of defl plants were unaffected by herbivory.Two spotted spider mite feeding induced octadecanoid signaling pathway that resulted in activation of PI-II gene and expression of relative genes, further more PI-II protein wereaccumulated. Because Def1 mutation blocked the synthesis pathway of octadecanoid and damaged defensive systems, mutants were attacted seriously.The experiment of exogenous JA inducing resistance showed that JA was required for restoring resistance to insect. Sufficient JA restored defense of the mutant against two spotted spider mite. Wild type plants treated by MeJA reduced the feeding and eggs of two spotted spider mite.It had been demonstrated 35S::prosys constitutively express PI and others defensive genes in the absence of wounding and that 35S::prosys mediated signaling requires octadecanoid biosynthesis and percetion in transgenic experimental with 35S::prosysThrips feeding induced octadecanoid-mediated host responses and constitutive activation of the signal pathway by overexpression of prosystemin that enhanced resistance to multiple cell content feeding herbivores. Those results indicated that systemin was mecessary in systemic resistance of tomato.2. Bulk segregant analysis was used in combination with AFLP to identify markers that were linked to Def1.This experiment showed that W+ plants wre heterozygous for both marker (EM-1 and EM-2), whereas all W" plants were homozygous for the RFLP pattern. The absence of recombinants in the population of 20 BC1 plants demonstrated that EM-1 and EM-2 were linked to the Def1 locus tightly. EM-1 and EM-2 were located on the end of the long arm of chromosome 3. This finding confirms the locations of Def1 on the distal end of chromosome更多还原