Study on development of a new transgenic tomato plant having self-incompatibility gene from wild species.

本研究旨在通过农杆菌介导的基因转移技术，建立一种新的转基因番茄分子育种技术，以获得具有野生番茄自交不亲和基因的转基因番茄，并用于植物繁殖的遗传控制。茄科植物（包括番茄的野生近缘种）具有一个单基因座多等位基因（S-基因）调控的配子体自交不亲和性。S基因编码一种在植物雌蕊组织中特异表达的核糖核酸酶（S-RNase）。已经报道了许多编码S-RNase的cDNA克隆和基因组克隆来自茄科植物，例如，烟草、矮牵牛和番茄。另一方面，栽培番茄植株是自交亲和的，导致其自交亲和性的遗传机制尚不清楚。本研究的研究结果表明，在通过农杆菌感染的叶盘获得的番茄栽培品种“Aki-Tama”的遗传转化体中，通过Southern杂交鉴定，导入的S-RNase基因在基因组中为单拷贝，并且如北方印迹分析所示，在花柱组织中特异性表达。然而，与野生种相比，导入基因的表达水平很低。这表明，用于构建Ti载体的启动子区（S-RNase ORF的5 ′上游区的1 kbp）足以用于组织特异性表达，但不足以增强基因表达水平。从花柱蛋白SDS-PAGE后的RNA酶活性染色来看，番茄品种中几乎没有检测到S-RNA酶活性，表明番茄自交亲和性是由于花柱中S-RNA酶活性较低所致。

The present study aims to develop a new technique of molecular breeding for production of transgenic tomato genotypes having self-incompatibility gene originated from wild species of tomato, and for genetical control of plant reproduction by means of Agrobacterium-mediated gene transfer method. Solanaceous plants including wild relatives of tomato possess a gemetophytic self-incompatibility regulated by a single locus with multiple alleles (S-gene). The S-gene encodes a ribonuclease (S-RNase) that specifically expresses in pistil tissues of plant. A lot of cDNA clones and genomic clones encoding S-RNase have been reported from solanaceous plant species, e.g., Nicotiana, Petunia and Lycopersicon. On the other hand, the cultivated tomato plants are self-compatible, and genetic mechanisms leading to it's self-compatibility are not known. From research results of the present study, it was shown that in genetic transformants of a tomato cultivar "Aki-Tama" obtained through Agrobacterium- infected leaf-disks, the introduced S-RNase gene was identified as a single copy in the genome by Southern hybridization, and expressed specifically in the style tissue, as shown by Northern blot a malysis. The expressin level of the introduced gene was, however, very low, when compared with the wild species. This suggests that promoter region (lkbp of 5'-upstream region from ORF of S-RNase) used for construction of the Ti-vector is sufficient for tissue-specific expression, but not enough for enhancement of gene expression level. From RNase-activity staining after SDS-PAGE of stylar proteins, almost no activity of S-RNase was detected in tomato cultivars, indicating that self-compatibility of tomato is due to a low level of S-RNase activity in the style.