Development of a new system for detecting mutation in higher plants and specificity of mutational spectra

活性氧、电离辐射、紫外线和各种化学物质损害DNA，并诱导基因表达、复制和突变的延迟。在植物中，持续暴露于太阳辐射中所含的UV中，遗传物质会产生各种类型的损伤。已知这些损伤会诱发突变。建立了一种新的拟南芥染色体DNA突变检测和分析系统。将携带诱变目的基因大肠杆菌rpsL基因的质粒pML 4插入穿梭载体中，构建了可用于植物转化的质粒。利用农杆菌介导法将pML 4基因导入拟南芥。从暴露于诱变剂的转基因拟南芥植物DNA中拯救pML 4 DNA，并将质粒导入大肠杆菌DH 10 B以检测突变体克隆。在该系统中，rpsL基因中的任何形式的失活突变都可以被阳性选择，因为它使大肠杆菌细胞对链霉素具有抗性。我们成功地建立了高等植物突变检测系统。我们研究了甲基磺酸乙酯（EMS）处理拟南芥的突变特异性。[结果] EMS处理植株的突变频率是背景突变的20倍。在背景突变中观察到移码、缺失和序列替换。在小鼠和鱼类中未发现序列替换。EMS诱发的突变以GC向AT的转变为主。与动物不同的是，几乎所有靶G侧翼的3 '-嘌呤都是A。

Reactive oxygen species, ionizing radiation, UV and various chemicals damage DNA and induce retardation of gene expression, replication and mutation. In plants continuously exposed to UV contained in solar radiation, various types of damages should be produced in genetic substances. These damages has been known to induce mutation. A new system was developed for detection and analysis of mutations occurring on chromosomal DNA in Arabidopsis. The plasmid pML4 carrying the Escherichia coli rpsL gene, a target gene for mutagenesis, was inserted into a shuttle vector to construct a plasmid which could be used for the transformation of plants. pML4 sequences were introduced into Arabidopsis thaliana mediated by Agrobacterium. The pML4 DNA was rescued from transgenic Arabidopsis plant DNA exposed to mutagens, and the plasmids were introduced into Escherichia coli DH10B to detect mutant clones. In this system, any form of inactivation mutation in the rpsL gene can be positively selected since it makes the E.coli cells resistant to streptomycin. We successfully established the assay system of mutation occurring in higher plants. We investigated the mutational specificity in Arabidopsis treated with ethtylmethanesulfonate (EMS).[Results]Mutant frequency of EMS treated plants was 20-fold higher than background mutation. Frameshifts, deletions and sequence substitutions were observed in background mutation. Sequence substitutions have not detected in mouse and fish. GC to AT transitions dominated in EMS-induced mutation. Almost all 3'-purine flanking to target G was A, which was different from animals.