Molecular cloning and analysis of mutable genes in Japanese Morning Glory.

日本牵牛花突变基因的分子克隆与分析。

• 批准号: 60480002
• 负责人: KOMEDA Yoshibumi
• 金额: $ 4.35万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1985
• 资助国家: 日本
• 起止时间: 1985 至 1986
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-60480002/
• 关键词: mutability of Japanese Morning Glory; transposon; DNA断片の多型

Many higher plants have phenotypic variegations. The genetic mutability is included in this class of phenotypes. Japanese scientists have studied extensively with Japanese Morning Glory and the four o'clock plant. In this study, the author is aiming to isolate and analyse possible mutable genes from Japanese Morning Glory. The first step was to purify higher molecular weight DNA from the plant. Two methods were emplyed for isolation of the genes; one is to examine genetic polymorphisms among strains of Morning Glory. The other is to detect DNA fragments carrying the structure of stem and loop. I found many polymorphisms among DNA samples of various horticultural strains by Southern hybridization. However, no correlation was detected between the mutability and the polymorphic pattern. one polymorphic pattern was difficult to be concluded by single insertional event.Next, I tried to isolate transposon-like DNA fragments by the detection of stem and loop structures which are characteristic of transposons. The DNA of Morning Glory mutants was digested by DNase I and was denatured. The single stranded DNA was reannealed under the condition that makes intramolecular double strands. The fraction of single stranded DNA was digested by single strand specific nuclease. The resulted double stranded DNA was cloned into dG-tailed plasmid pUC9. A pool consisted of chimeric plasmids carrying cloned trasposon-like DNA. After the examination of the length and copy number of the inserts, representative five clones were sequenced. I detected an inverted repetitive sequence and the fragments that showed polymorphism among strains and that had about 60% homology to the stem of Tam1 a transposon of snap dragon).Accordingly, the latter method was shown to be effective. I will further this study from two points; polymorphism and movability.

许多高等植物具有表型变异。遗传突变性包括在这类表型中。日本科学家对日本牵牛花和四点钟植物进行了广泛研究。在这项研究中，作者的目的是分离和分析日本牵牛花可能的突变基因。第一步是从植物中纯化较高分子量的DNA。采用两种方法分离基因：一种是检测牵牛花品系间的遗传多态性。另一种是检测携带茎环结构的DNA片段。通过Southern杂交，我在不同园艺品种的DNA样品中发现了许多多态性。然而，没有检测到的变异性和多态性模式之间的相关性。一个多态性模式很难通过单一的插入事件来推断。接下来，我试图通过检测转座子特有的茎和环结构来分离转座子样DNA片段。牵牛花突变体的DNA用DNase I消化并变性。单链DNA在形成分子内双链的条件下再退火。用单链特异性核酸酶消化单链DNA部分。将得到的双链DNA克隆到dG加尾质粒pUC 9中。由携带克隆的转座子样DNA的嵌合质粒组成的池。在检查插入片段的长度和拷贝数后，对代表性的五个克隆进行测序。通过对转座子Tam 1基因的同源性分析，发现了一个反向重复序列和一个在菌株间表现出多态性的片段，该片段与转座子Tam 1基因的茎同源性约为60%，证明了后一种方法的有效性。我将从多态性和可移动性两个方面来进一步研究。