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GENE TRANSFORMATION EXPERIMENT OF THE SELF-INCOMPATIBILITY RELATED GENES

自交不亲和性相关基因的基因转化实验

基本信息

批准号：
05556002
负责人：
HINATA Kokichi
金额：
$ 10.62万
依托单位：
TOHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

项目摘要

Objectives : To understand the molecular mechanism of self-incompatibility and to assess a possibility of molecular breeding of this character, molecular characterization and gene manipulation was conducted on the S-related genes in sporophytic self-incompatible plant : Brassica campestris and in gemetophytic self-incompatible plant : Pyrus pilifolia.Results : In B.campestris, SLG8, SLG9, SLG12, SRK8, SRK9, SRK12, SRA1, SRA2, SRA3, SRA4 and SRBs were isolated from cDNA or genomic DNA libraries and sequenced. Dendrograms constructed on the basis of synonymous and nonsynonymous substitutions indicated that SRA differentiated first, followed by SRB and SLG/SRK.When the SLG8 genomic clone was introduced into B.campestris some of transformed plants changed to self-compatible. Furthermore, the antisense SLG gene was transformed into the same species. Analysis of a transformant showed that the self-incompatibility was broken down by the action of the introduced antisense gene. These results i … More ndicated the self-incompatibility could be controlled, at least partly, by gene manipulation. The genes encoding 3 RNases were cloned from the style of a self-incompatible cultivar, Nijisseiki (S2S4), and its self-compatible mutant, Osa-Nijisseiki (S2S4sm, sm means stylar part mutant), of Japanese pear. For Nijisseiki, cDNAs coding for two S-RNases (S2-RNase and S4-RNase) and an RNase unrelated to self-incompatibility (non-S-RNase) were cloned from the stylar cDNA library. However, the cDNA coding for S4-RNase was neither amplified by PCR nor cloned from the library of Osa-Nijisseiki, suggesting that the mutation of self-incompatible Nijisseiki to self-compatible Osa-Nijisseiki is due to a failure of expression of S4-RNase. The S3-gene promoter of Petunia inflata was used in constructing the vectors for the Japanese pear S-genes. The binary vectors of the Agrobacterium were transformed in the petunia resulting in shoot regeneration. In vitro cultures of the Japanese pear were also extended. Less

目的：为了了解自交不亲和性的分子机制并评估该性状分子育种的可能性，对孢子体自交不亲和植物（Brassica Campestris）和分生体自交不亲和植物（Pyrus pilifolia）中的 S 相关基因进行分子表征和基因操作。结果：在 B.campestris、SLG8、SLG9、SLG12 中，从 cDNA 或基因组 DNA 文库中分离 SRK8、SRK9、SRK12、SRA1、SRA2、SRA3、SRA4 和 SRB 并进行测序。基于同义和非同义取代构建的树状图表明，SRA首先分化，其次是SRB和SLG/SRK。当将SLG8基因组克隆引入油菜中时，一些转化植物变为自交亲和。此外，反义SLG基因被转化到同一物种中。对转化体的分析表明，自交不亲和性被引入的反义基因的作用所打破。这些结果表明，自交不亲和性可以通过基因操作来控制，至少部分控制。编码 3 个 RNase 的基因是从日本梨的自交不亲和品种 Nijisseiki (S2S4) 及其自交亲和突变体 Osa-Nijisseiki (S2S4sm，sm 表示花柱部分突变体) 的花柱中克隆的。对于二十世纪，从花柱 cDNA 文库中克隆了编码两种 S-RNase（S2-RNase 和 S4-RNase）和一种与自交不亲和性无关的 RNase（非 S-RNase）的 cDNA。然而，编码S4-RNase的cDNA既没有通过PCR扩增，也没有从Osa-Nijisseiki文库中克隆，这表明自交不亲和的Nijisseiki突变为自亲和的Osa-Nijisseiki是由于S4-RNase表达失败所致。使用膨胀矮牵牛的S3基因启动子构建日本梨S基因的载体。将农杆菌的双元载体转化到矮牵牛中，导致芽再生。日本梨的体外培养也得到了扩展。较少的