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

用γ射线辐照水稻品种金宝津种子，诱导出一个细长颖片突变基因slg。slg偶尔会恢复到野生型，推测其中包含活性转座因子。为了鉴定该因子的分子特征，我们尝试用定位克隆方法克隆slg。slg已经定位在两个YAC克隆Y1774和Y3356的重叠区域上。我们首先以YAC克隆Y1774为探针，从水稻品种日本晴的cDNA克隆文库中筛选出209个候选克隆，然后对其中的54个克隆进行了部分测序。基于序列数据，发现40个克隆彼此独立。以这40个克隆为探针，我们最终筛选出与两个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，表明发生了非特异性杂交。因此，很难鉴定除了27号以外真正位于两个YAC克隆上的克隆。利用Southern杂交技术分析了原始品种、突变株系和回复突变体之间的多态性带型。结果表明，在120号克隆中观察到多态性条带，表明该克隆与slg位点紧密连锁。由于slg的突变可能是由DNA甲基化引起的，因此还研究了slg对基因组甲基化的影响。突变株系MspI位点甲基胞嘧啶占总胞嘧啶的相对含量显著低于原始品种。这表明slg可能对基因组甲基化具有降低作用，导致slg的突变性。

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.