Regulation of the plant defense gene expression

采用颗粒枪、农杆菌介导转化或电穿孔等方法，将含有与报告基因连接的苯丙氨酸解氨酶（PAL）和查尔酮合成酶（CHS）启动子片段的嵌合DNA转化到烟草或豌豆植株中，研究了豌豆病原菌pinodes Mycosphaerella产生的激发子和抑制子的组织和结构。PAL基因分析。构建了以荧光素酶基因（lux）、β -葡萄糖醛酸酶（GUS）或氯霉素乙酰转移酶（CAT）基因为报告基因的PAL启动子片段为嵌合DNA。通过农杆菌介导转化，将pBI101载体中的PAL-GUS嵌合DNA （Chimeric DNA）导入烟草和豌豆中。从愈伤组织中再生出转Km^ r基因烟草植株。在部分转基因植株中，病原菌或激发子处理可诱导GUS活性。由于单细胞系尚未建立起豌豆再生系，我们获得了Km^R愈伤组织，并通过真菌激发子处理和病原菌侵染检测了GUS活性的诱导作用。在一些愈伤组织中观察到GUS活性的显著诱导。嵌合DNA与植物染色体的整合目前正在研究中。在加州大学戴维斯分校，使用粒子枪（杜邦公司）将基因转移到这些植物中。细胞学研究表明，与35S启动子连接的Lux或GUS基因作为对照的植物在可能携带嵌合DNA的细胞中表现出独特的GUS活性，而与PAL启动子连接的植物即使经过真菌激发子处理也没有表现出GUS或Lux活性。将PSPAL1序列缺失的启动子片段与含有该报告基因（CAT）的植物表达载体连接，通过电穿孔法导入豌豆原生质体，并对激发子的诱导作用和正钒酸盐的抑制作用进行了研究。负责这些规定的顺式元件存在于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制备的38个CHS- cdna中，测定了3个主要CHS基因的结构和组织。两个主要的豌豆CHS基因（PSCHS1, PSCHS2）来自一个带有间隔的串联重复序列簇，以及一个相同的31 bp序列，该序列包含box 1的共识基序，位于两个CHS基因的推定TATA box的上游5'处。2 .瞬时转化实验表明，激活元件存在于启动子远端区（PCHS1: -1493 ~ +80, PCHS2: - 1889 ~ +83）。Kado教授于1991年12月访问了我们的实验室，并对病原体产生的信号物质进行了讨论。我们特别关注了阻断防御反应诱导的物质。由于M. pinodes抑制因子特异性抑制豌豆质膜atp酶，他从阻断pm - atp酶的角度研究了细菌信号分子。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