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Mechanism of suppression of host-plant defense PR1a gene expression by the plant tumor-inducing 6b gene of Agrobacterium tumefaciens

根癌农杆菌植物肿瘤诱导6b基因抑制宿主植物防御PR1a基因表达的机制

基本信息

批准号：
13660055
负责人：
WABIKO Hiroetsu
金额：
$ 2.05万
依托单位：
Akita Prefectural University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

Expression of plant defense genes, termed Pathogenesis Related (PR) genes, is turned on upon infection of pathogens to plants. A soil bacterium Agrobacterium tumefaciens induces crown gall tumors on a number of plants. We have shown that the tumor-inducing 6b (AK-6b) gene obtained from a strain AKE10 enhanced the accumulation of several phenylpropanoids, which also possess anti-pathogenic activity. Since regulatory factor(s) responsible for PR gene overlaps with the biosynthesis of phenylpropanoid, we analyzed the effect of AK-6b gene on PR1a gene expression. We generated tobacco plants transgenic for the AK-6b gene under the control of the promoters of its own, CaMV35S, and dexamethazone inducible ones. Tissues were grown in the presence or absence of plant hormones, mRNA was extracted, and PR1a gene expression was analyzed by Northern hybridization. There was no obvious relationship between PR1a transcript levels and AK-6b transcript levels. However, when timorous tissues were cultivated on hormone-free medium and transferred to auxin-containing medium and followed time course of PR1a transcript accumulation, there was a clear difference. It was found that PR1a mRNA levels of transgenic tissues were reduced compared to those of wild type tobacco tissues until 6 days post transfer. At day 6, PR1a transcript of both tissues was comparable. We concluded, therefore, that both the AK-6b gene expression and presence of auxin were responsible for reduction of PR1a mRNA accumulation, and that the reduction was transient. Exogenous application of salicylic acid induced PR1a transcripts of both transgenic and wild type tissues, demonstrating transgenic tissues possess a normal potential to induce PR1a gene expression. We also purified modified AK-6b protein tagged with T7 and His, to near homogeneity by using affinity chromatography.

植物防御基因的表达被称为病原相关（PR）基因，其在病原体对植物的感染后被开启。土壤细菌根癌农杆菌在许多植物上诱导冠瘿瘤。我们已经表明，从菌株AKE 10获得的肿瘤诱导6 b（AK-6 b）基因增强了几种苯丙烷类化合物的积累，这些苯丙烷类化合物也具有抗病原体活性。由于PR基因的调控因子与苯丙素类的生物合成有重叠，我们分析了AK-6 b基因对PR 1a基因表达的影响。我们产生了烟草植物转基因的AK-6 b基因的控制下，其自身的启动子，CaMV 35 S，和地塞米松诱导的。在植物激素存在或不存在的情况下生长组织，提取mRNA，并通过北方杂交分析PR 1a基因表达。PR 1a转录水平与AK-6 b转录水平无明显相关性。然而，当在不含淀粉的培养基上培养茎组织并转移到含生长素的培养基上，并遵循PR 1a转录物积累的时间过程时，存在明显的差异。结果发现，与野生型烟草组织相比，转基因组织的PR 1a mRNA水平降低，直到转移后6天。在第6天，两种组织的PR 1a转录物相当。因此，我们的结论是，AK-6 b基因的表达和生长素的存在是负责减少PR 1a mRNA的积累，减少是短暂的。外源应用水杨酸诱导PR 1a转录的转基因和野生型组织，证明转基因组织具有正常的潜力，以诱导PR 1a基因的表达。我们还纯化修饰AK-6 b蛋白标记T7和His，通过使用亲和层析接近同质。