Regulation of the plant defense gene expression

研究了苯丙氨酸解氨酶(PAL)和查尔酮合酶(CHS)基因的组织和结构，并通过基因枪法、农杆菌介导法和电穿孔法将含有PAL和CHS启动子片段的嵌合DNA转化烟草或豌豆植株，研究了PAL和CHS基因的激发子和抑制子的作用。PAL基因的分析。构建了以荧光素酶基因(Lux)、β-葡萄糖醛酸酶基因(GUS)或氯霉素乙酰转移酶基因(CAT)为报告基因的PAL启动子片段的嵌合DNA。利用农杆菌介导法将载体pBI101中的嵌合DNA(PAL-GUS)导入烟草和豌豆中。从愈伤组织中获得了转KM^R基因烟草植株。在部分转基因植株中，用病原菌或激发子处理可诱导GUS活性。从豌豆p…的再生系开始通过真菌诱导子处理和病原菌侵染，我们获得了KM、R愈伤组织和GUS活性的诱导。在一些愈伤组织中观察到相当大的GUS活性诱导。嵌合DNA整合到植物染色体中的研究目前正在进行中。使用粒子枪进行基因转移(杜邦公司)这些植物是在加州大学戴维斯分校进行的。细胞学研究表明，与35S启动子相连的LUX或GUS基因植株在可能携带嵌合DNA的细胞中表现出明显的GUS活性，而含有PAL启动子的植株即使用真菌激发子处理也没有表现出GUS或LUX活性。此外，将序列缺失的PSPAL1启动子片段连接到含有报告基因(CAT)的植物表达载体上，通过电穿孔将其导入豌豆原生质体，并检测了激发子的诱导和原钒酸盐的抑制作用。负责这些规则的顺式元件存在于PSPAL1.2的-340--140之间。CHS基因分析。从激发子处理的豌豆上胚轴组织中提取的38个CHS-cDNA中，确定了3个主要的CHS基因的结构和组织。豌豆CHS的两个主要基因(PSCHS1、PSCHS2)位于两个CHS基因的TATA盒的5‘上游，它们是一个带有间隔区的串联重复序列，以及一个相同的31bp序列，其中包含box I的共同基序。上述瞬时转化实验表明，在没有真菌激发子处理的情况下，BUTH在启动子-远端区域(PCHS1：-1493-+80，PCHS2：-1889-+83)中也有较高的表达，表明启动子-远端区域存在激活元件。病原菌信号物质的分离及信号转导途径的研究Kado教授于1991年12月来我实验室，对病原菌产生的信号物质进行了讨论。我们特别关注那些阻止防御反应诱导的物质。由于松突根结线虫抑制子特异性地抑制豌豆质膜ATPase，他从阻断PM-ATPase的角度研究了细菌的信号分子。较少

The organization and the structure of the genes encoding phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) were determined and the effects of elicitor and suppressor produced by a pea pathogen, Mycosphaerella pinodes, were examined by transforming a chimeric DNA containing the PAL- and CHS-promoter fragments connected to a reporter gene into tobacco or pea plant with particle gun, Agrobacterium-mediated transformation, or electroporation.1. Analysis of PAL genes. Chimeric DNA containing PAL promoter fragment connected to luciferase gene (lux), beta-glucuronidase (GUS) or chloramphenicol acetyltransferase (CAT) gene as a reporter were constructed. Chimeric DNA (PAL-GUS) in the vector, pBI101, were introduced into tobacco and pea by Agrobacterium mediated- transformation. Km^R-transgenic tobacco plants were regenerated from the callus. GUS activities were induced by the treatment with pathogen or elicitors in some of the transgenic plants. Since the regeneration line of pea p … More lant has not been established from the single cell lines, we obtained Km^R calli and the induction of GUS activities were examined by the treatment with fungal elicitor and the infection of pathogens. Considerable induction of GUS activity was observed in some of the callus. The integration of the chimeric DNA into the plant chromosomes are currently under investigation. Gene transfer by using a particle gun (Dupont Inc.) into these plants were performed at UCD, Davis. Cytological studies showed that plants with Lux or GUS gene connected to 35S promoter as a control exhibited the distinctive GUS atibity in the cells possibly carrying the chimeric DNA, however those with PAL promoter did not exhibit GUS or Lux activities even by the treatment with fungal elicitor. Furthermore, the sequentially deleted promoter fragment of PSPAL1 connected to a plant expression vector containing the reporter gene (CAT) was introduced into pea protoplasts by electroporation, and induction by elicitor and suppression by orthovanadate was examined. Cis-element responsible for these regulations exists between -340--140 of PSPAL1.2. Analysis of CHS gene. The structure and the organization of three major CHS genes were determined from 38 CHS-cDNAs prepared from elicitor treated pea epicotyl tissues. Two of the major pea CHS genes (PSCHS1, PSCHS2) from a cluster in a tandem repeat with a spacer, and an identical 31 bp sequence containing the consensus motif of box I located 5'-upstream from the putative TATA box of both CHS genes. Buth were highly expressed even without the treatment with fungal elicitor by transient transformation assay mentioned above, which indicate that the activator elements exist in the promoter-distal region (PCHS1 : -1493 - +80, and PCHS2 : - 1889 - +83).3. Isolation of signalling substances from the pathogen and study of the signal transduction pathway Prof. C. I. Kado visited our laboratory on December, 1991, and discussed about the signaling substances produced by the pathogens. We particularly focused on the substances that blocked the induction of defense reactions. Since M. pinodes suppressor specifically inhibits pea plasma membrane ATPase, he examined bacterial signalling molecules from the aspect of blocking PM-ATPase. Less