Fujita T, Kouchi H, Ichikawa T, Syöno K
Department of Pure and Applied Sciences, University of Tokyo, Japan.
Plant J. 1994 May;5(5):645-54. doi: 10.1111/j.1365-313x.1994.00645.x.
To identify genes involved in the formation of genetic tumors in interspecific hybrids (F1) between Nicotiana glauca and N. langsdorffii, genetic tumor-related cDNA probes were obtained by a subtractive hybridization procedure and used to screen libraries of genetic tumor cDNAs. As a result, 17 distinct cDNA clones were isolated for genes that are specifically or preferentially expressed in genetic tumor tissues but are not expressed at all or are barely expressed in normal F1 plants. Among the isolated clones were genes that encoded so-called stress proteins, such as glucan endo-1,3-beta-glucosidase, osmotin, pathogenesis-related proteins and proteinase inhibitor I. Transcripts corresponding to two of the isolated cDNA clones accumulated to a significant extent only in genetic tumor tissues and were not present in callus tissues from parental plants or in the stems and leaves from normal F1 plants. Analysis of genomic DNA revealed that four of these clones hybridized only to genomic sequences from N. langsdorffii and one hybridized only to a genomic sequence from N. glauca. Eight apparently novel clones were further analyzed to determine the kinetics of accumulation of the corresponding mRNAs during development of genetic tumors. The patterns of accumulation of the mRNAs after induction of tumors by cutting of F1 stems could be divided into three groups, an indication that at least three distinct regulatory mechanisms are operative at the transcriptional level to control the expression of these tumor-related genes during the formation of genetic tumors.
为了鉴定参与烟草（Nicotiana glauca）和郎氏烟草（N. langsdorffii）种间杂种（F1）中遗传性肿瘤形成的基因，通过消减杂交程序获得了与遗传性肿瘤相关的cDNA探针，并用于筛选遗传性肿瘤cDNA文库。结果，分离出17个不同的cDNA克隆，这些基因在遗传性肿瘤组织中特异性或优先表达，但在正常F1植株中根本不表达或几乎不表达。在分离出的克隆中，有编码所谓应激蛋白的基因，如葡聚糖内切-1,3-β-葡糖苷酶、渗透素、病程相关蛋白和蛋白酶抑制剂I。与两个分离出的cDNA克隆相对应的转录本仅在遗传性肿瘤组织中大量积累，在亲本植物的愈伤组织或正常F1植株的茎和叶中不存在。基因组DNA分析表明，其中四个克隆仅与郎氏烟草的基因组序列杂交，一个克隆仅与烟草的基因组序列杂交。对八个明显新的克隆进行了进一步分析，以确定遗传性肿瘤发育过程中相应mRNA积累的动力学。通过切割F1茎诱导肿瘤后，mRNA的积累模式可分为三组，这表明至少有三种不同的调控机制在转录水平上起作用，以控制这些肿瘤相关基因在遗传性肿瘤形成过程中的表达。
