Isolation and functional analysis of the self-incompatibility gene in the grasses (Poaceae)

禾本科植物自交不亲和基因的分离及功能分析

• 批准号: 15580006
• 负责人: KAKEDA Katsuyuki
• 金额: $ 2.37万
• 依托单位: Mie University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15580006/
• 关键词: Self-incompatibility; S gene; Hordeum bulbosum; mRNA fingerprinting; Poaceae; Linkage analysis; Barley; 2D-gel analysis; 形質転換; 二次元電気泳動; AFLP; Poaceae; 連鎖地図

The AFLP-based mRNA, fingerprinting (AME) analysis was conducted to find S genotype-specific transcripts from pistils and anthers at anthesis in Wild barley, Hordeum bulbosum (2x) that possesses the gametophytic self-incompatibility (SI). A detailed genetic linkage map around the S locus was constructed using several clones obtained by the AMF analysis. Comparison of the linkage map with a physical map of barley choromosome 1 indicated that the S locus of H. bulbosum was located in a large chromosomal region across the centromere, where recombination is severely suppressed. In the linkage analysis, we have obtained five clones that showed no recombination with the S locus. Two of those clones showed pistil- or anther-specific expression. From sequence analysis of, the full length cDNA, the pistil-specific clone (HPS10) was shown to encode a small unknown protein that seems to be a putative secreted signaling molecule. Together with a high degree of allelic polymorphism, this clone turned out to be a promising candidate for the pistil S gene. Thus, we have been trying, to transform H. bulbosum calli with its RNAi construct, but so far we have obtained no transformants. For the anther-specific clone (122-7), sequence analysis revealed that it would encode a protein carrying an F-box motif. This finding is notable because the pollen S factor recently identified in the other gametophytic SI species (Solanaceae, etc.) is found to be an F-box protein. Further investigation on allelic polymorphism of the clone is needed. In the final experiment, we conducted 2D-gel analysis of pistil proteins in H. bulbosum and identified an S genotype-specific protein by LC-MS/MS. A cDNA clone coding for the protein was isolated and revealed to be tightly linked to the S locus.

采用基于aflp的mRNA指纹图谱（AME）分析方法，从具有配子体自交不亲和（SI）特征的野生大麦Hordeum bulbosum （2x）花期雌蕊和花药中找到了S基因型特异性转录本。利用AMF分析获得的多个克隆，构建了S位点周围的详细遗传连锁图谱。将该连锁图谱与大麦1号染色体物理图谱进行比较，发现大泡大麦S位点位于横跨着丝粒的大片染色体区域，该区域的重组受到严重抑制。在连锁分析中，我们获得了5个与S位点没有重组的克隆。其中两个克隆显示雌蕊或花药特异性表达。从全长cDNA的序列分析，雌蕊特异性克隆（HPS10）被证明编码一个小的未知蛋白，似乎是一个假定的分泌信号分子。加上高度的等位基因多态性，该克隆被证明是雌蕊S基因的一个有希望的候选者。因此，我们一直在尝试用其RNAi构建体转化球茎愈伤组织，但到目前为止我们还没有获得转化子。对于花药特异性克隆（122-7），序列分析显示它将编码一个携带F-box基序的蛋白质。这一发现值得注意，因为最近在其他配子体SI物种（茄科等）中发现的花粉S因子是一个F-box蛋白。该克隆的等位基因多态性有待进一步研究。在最后的实验中，我们对球芽草雌蕊蛋白进行了2d凝胶分析，并通过LC-MS/MS鉴定了一个S基因型特异性蛋白。分离到一个编码该蛋白的cDNA克隆，发现该蛋白与S位点紧密相连。

项目成果

掛田克行: "オオムギ野生種の自家不和合性遣伝子座周辺における組換え抑制"育種学研究. 6(別冊1)(印刷中). (2004)

Katsuyuki Kakeda：“野生大麦物种自交不亲和基因座周围重组的抑制”育种研究 6（特刊 1）（出版中）。

Differential expression proteomics in barley and its wild relatives

大麦及其野生近缘种的差异表达蛋白质组学

• 发表时间: 2004
• 期刊: Proc. 9th Int. Barley Genetics Symp.
• 作者: 掛田克行;K.Kakeda
• 通讯作者: K.Kakeda

Molecular characterization of a 313 kb genomic region containing the self incompatibility locus of Ipomoea trifida, a diploid relative of sweet potato

含有三裂蓼（甘薯的二倍体近缘种）自交不亲和基因座的 313 kb 基因组区域的分子特征

• 发表时间: 2004
• 期刊: Breeding Science Vol.54
• 作者: 掛田克行;K.Kakeda;R.N.Tomita;K.Kakeda;K.Kakeda;R.N.Tomita
• 通讯作者: R.N.Tomita

オオムギ野生種の自家不和合性遺伝子座周辺における組換え抑制

抑制野生大麦自交不亲和基因座周围的重组

• 发表时间: 2004
• 期刊: 育種学研究 6(別冊1)
• 作者: 掛田克行

Agrobacterium-mediated transformation of a wild diploid species of sweet potato

农杆菌介导的甘薯野生二倍体物种的转化

• 发表时间: 2003
• 期刊: Breeding Research Vol.5(Suppl.1)
• 作者: 掛田克行;K.Kakeda;R.N.Tomita;K.Kakeda;K.Kakeda;R.N.Tomita;K.Kakeda;掛田克行;K.Kakeda;K.Kakeda
• 通讯作者: K.Kakeda