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番茄花叶病毒弱毒疫苗K(ToMV-K)的复制酶基因和运动蛋白基因分别具有乳石和赭石突变的特征. 为了对这一致弱特征进行扩展研究, 用PCR介导的定点突变技术, 对烟草花叶病毒普通株中国分离物(TMV-Cv)复制酶基因和运动蛋白基因分别进行乳石突变(UGA)和赭石突变(UAA), 构建了复制酶单突变体--TMV-Cvrase, 运动蛋白单突变体--TMV-Cvmp和复制酶与运动蛋白双突变体--TMV-Cvrase-mp. 烟草侵染试验发现, TMV-Cvmp和TMV-Cvrase-mp能侵染枯斑(Xanthi NC)和系统(K326)寄主, 后者侵染普通烟K326仅出现少量轻微的花叶症状; 子代病毒的电子显微镜观察、重复接种试验及其核酸的RNA斑点杂交、RT-PCR扩增与测序结果表明, TMV-Cvmp和TMV-Cvrase-mp子代病毒与TMV-Cv的大小和形态基本一致, 具有完整和稳定的侵染、复制和繁殖能力, 且其基因组仍保持着突变, 而复制酶单突变体TMV-Cvrase不能侵染烟草. 以上结果表明, 复制酶与运动蛋白的乳石突变与赭石突变协同致弱TMV-Cv对烟草的侵染症状, 从而暗示ToMV-K基因组的这种突变模式也可以用于其他植物病毒的致弱研究.
The tomavivus and ocher mutations of ToMV-K replicase gene and motilin gene of ToMV-K, respectively, were investigated in this study.In order to extend the study of this weakness feature, PCR-mediated site-directed mutagenesis Mucous mutation (UGA) and ocher mutation (UAA) were performed on the TMV-Cv replicase and motilin genes of Mosaic virus isolates (TMV-Cv), and the single mutant of replicase, TMV-Cvrase, TMV-Cvmp and TMV-Cvrase-mp were able to infect tobacco (Xanthi NC) and TMV-Cvrase-mp (K326) host, the latter infected with ordinary tobacco K326 only a small amount of slight mosaic symptoms; electron microscopy of progeny virus, repeated inoculation test and nucleic acid RNA dot blot hybridization, RT-PCR amplification and sequencing results show , TMV-Cvmp and TMV-Cvrase-mp progeny viruses are basically the same as the size and morphology of TMV-Cv, have intact and stable ability of infection, replication and reproduction, and their genomes still maintain the mutation, while the single copy of the replicase TMV-Cvrase can not infect tobacco Ming replicase proteins and milk stone movement with ocher mutations synergistic attenuated mutant TMV-Cv tobacco infestation symptoms, suggesting that this mutant gene model ToMV-K group can also be used other actuation weak study of plant viruses.