Role of Polyamine Oxidase in Apoptosis Studies with RNA Interference

描述（由申请人提供）：大量文献记录了植物和动物细胞中多胺、亚精胺和精胺的催化与程序性细胞死亡（细胞凋亡）过程相关的研究。多胺氧化酶（PAO）催化多胺的氧依赖性裂解以形成过氧化氢（H2 O2）等产物。许多研究表明，诱发细胞凋亡的病原体是由H2 O2介导的氧化应激。我们的项目假设是：由酶PAO催化的反应是导致H2 O2介导的真核细胞凋亡的细胞H2 O2的主要来源。我们已经获得了强有力的证据表明，PAO催化确实是H2 O2在植物模型系统进行过敏反应（HR）的主要来源。HR是由H2 O2介导的病原体引起的过程，其最终导致细胞凋亡。H2 O2的来源不明。在初步研究中，我们已经应用了RNA干扰（RNAi）方法，使用新发现的，快速的技术引入双链（dsRNA）到植物在HR，并已成功地敲低PAO酶的表达与同时废除的H2 O2爆发，通常伴随着HR。 本课题的具体目标是：（1）利用我们的dsRNA导入植物细胞的新技术，利用RNA干扰（RNAi）技术沉默（敲除）燕麦PAO基因的表达。(oat)幼苗这种敲除将显示导致五种细胞参数的抑制：PAO mRNA水平、PAO酶活性、HR中的H2 O2爆发、HR期间细胞多胺的分解代谢损失和进入细胞凋亡。(2)植物中存在NF-κ B样转录因子，这可能通过促凋亡、H2 O2激活的途径在调节PAO表达中发挥作用。我们在水稻PAO基因5 '端非翻译启动子区的两个位置发现了一段DNA核苷酸序列（gtgaatttcc）。该序列含有结合转录因子的识别位点，所述转录因子将PAO基因转录与NF-κ B样因子连接。我们将完成这种蛋白质的分离，以证实它在植物王国中的存在。(3)方法，植物种子股票，质粒载体将开发，以利用合成的铜（II）离子依赖的遗传启动子系统，由我们的实验室生产，用于控制植物和酵母宿主中的转基因的合成。

DESCRIPTION (provided by applicant): A voluminous literature documents studies that correlate the catabolism of the polyamines, spermidine and spermine, in plant and animal cells with the process of programmed cell death (apoptosis). Polyamine oxidase (PAO) catalyzes oxygen-dependent cleavage of the polyamines to form hydrogen peroxide (H2O2), among other products. Many studies indicate that the causative agent for eliciting apoptosis is oxidative stress mediated by H2O2. Our project hypothesis is: The reaction catalyzed by the enzyme PAO is the major source of cellular H2O2 that causes H2O2-mediated apoptosis in eukaryotic cells. We have gained strong evidence that PAO catalysis is indeed the major source of H2O2 in a plant model system undergoing the hypersensitive response (HR). HR is a pathogen-elicited process mediated by H2O2, which culminates in apoptosis. The origin of the H2O2 is not known. In preliminary studies, we have applied RNA interference (RNAi) methods, using a newly discovered, rapid technique for introducing double-stranded (dsRNA) into plants during HR, and have succeeded in knocking down PAO enzymatic expression with concurrent abolishment of the H2O2 burst that normally accompanies HR. The specific aims of this project are: (1) RNA interference (RNAi) methods that exploit our new technique for introducing dsRNA into plant cells will be used to silence (knockdown) PAO gene expression in Avena sativa L. (oat) seedlings. This knockdown will be shown to result in suppression of five cellular parameters: PAO mRNA levels, PAO enzymatic activity, the H2O2 burst in the HR, catabolic loss of cellular polyamines during the HR, and entry into apoptosis. (2) The existence of an NF-kB-like transcription factor in plants will be established, which may have a role in regulating the expression of PAO by pro-apoptotic, H2O2-activated pathways. The DNA nucleotide sequence (gtgaatttcc) was discovered by us in two locations of the 5'-untranslated promoter region of the PAO gene from Oryza sativa (rice). This sequence contains recognition sites for binding transcription factors that link PAO gene transcription to NF-kB-like factors. We will complete the isolation of this protein to confirm its existence in the plant kingdom. (3) Methodologies, plant seed stocks, and plasmid vectors will be developed in order to exploit a synthetic copper(ll) ion-dependent genetic promoter system, produced by our laboratory, for controlling the synthesis of trans genes in plant and yeast hosts.