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Molecular and functional analyses of the induced mutable gene slg in rice.

水稻诱导突变基因slg的分子和功能分析。

基本信息

批准号：
11660003
负责人：
TANISAKA Takatoshi
金额：
$ 2.18万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660003/
关键词：
rice mutable gene YAC transposon slender glume gene 易変性遺伝子

项目摘要

A mutable slender glume gene slg was induced by gamma-ray irradiation to seeds of the rice variety Gimbozu. Occasionally reverting to its wild-type, slg is assumed to involve an active transposable element in it. To identify the molecular characteristics of this element, we attempted to clone slg with a positional cloning method. slg has already been mapped on the overlapping region of two YAC clones, Y1774 and Y3356. We first selected 209 candidate clones from the cDNA clone library of the rice variety Nipponbare using a YAC clone Y1774 as a probe, and then partially sequenced 54 clones. Based on the sequence data, 40 clones were found to be independent of each other. Using these 40 clones as probes, we finally selected out cDNA clones that hybridized with both YAC clones. But many of them were hybridized not only with both YAC clones but also with multiple regions of genomic DNA, indicating the occurrence of non-specific hybridization. Thus, it was difficult to identify the clones other than No.27 that was really located on both YAC clones. Southern analyses were also conducted to find out polymorphic bands among the original variety, mutant line (slg) and revertant (wild-type). Consequently, polymorphic bands were observed when using clone No.120, suggesting that this clone is tightly linked with slg locus. The effect of slg on the genomic methylation was also investigated because there was a possibility that the mutability of slg was caused by DNA methylation. The mutant line was significantly lower than the original variety in the relative amount of methylcytosine to total cytosine on MspI site. This indicates that slg may have a decreasing effect on the genomic methylation, resulting in the mutability of slg.

利用伽玛射线辐照诱导金伯祖种子获得了一个可变的细长颖片基因slg。有时会恢复到其原始类型，假设slg中包含一个活动转座元素。为了确定该元件的分子特征，我们尝试用定位克隆的方法克隆slg。slg已经被定位到两个YAC克隆Y1774和Y3356的重叠区域。首先以YAC克隆Y1774为探针，从水稻品种Nipponbare cDNA克隆文库中筛选出209个候选克隆，并对54个克隆进行了部分测序。根据序列数据，发现40个克隆相互独立。以这40个克隆为探针，我们最终筛选出了与两个YAC克隆杂交的cDNA克隆。但其中许多不仅与两个YAC克隆杂交，而且与基因组DNA的多个区域杂交，表明存在非特异性杂交。因此，除了27号之外，很难鉴定出真正位于两个YAC无性系上的无性系。南方分析还发现了原品种、突变系（slg）和回系（野生型）之间的多态性带。因此，在使用120号无性系时观察到多态性带，表明该无性系与slg位点紧密连锁。slg对基因组甲基化的影响也被研究，因为slg的易变性可能是由DNA甲基化引起的。突变系MspI位点甲基胞嘧啶与总胞嘧啶的相对含量显著低于原品种。这表明slg可能对基因组甲基化有减弱作用，从而导致slg的易变性。