A new protocol for the differential display of mtRNA was developed to compare the difference in the mtRNAs of rice (Oryza sativa L.) among cytoplasm male sterility (CMS) line, maintainer line and F1 hybrids. This new procedure was based on the ligation function of T4 RNA ligase and the principle of AFLP technique. By comparing the display patterns of the mtRNAs amplified selectively with four selective primer pairs six differential fragments have been obtained, among which fragment DTA was specific in CMS line, fragment DAB was shared by CMS line and maintainer line, and fragment DBF1,DBF2,DBF3 and DBF4 were shared by maintainer line and F1 hybrid. The results showed the difference of mtRNA between CMS line (Yuetai A) and F1 hybrids (Taiyou 2) was more obvious than the difference of mtRNA between the maintainer line (Yuetai B) and F1 hybrids (Taiyou 2). The fact that the transcription of fragment DTA was proved to be differential among CMS line, maintainer line and F1 hybrid by Northern hybridization inferring that the fragment DTA may be relative to the HL CMS. Fragment DTA was composed of 259 base pairs, though no sequence homology and no open reading frame was found in the sequence. It could be used as a probe to clone the whole length gene from the cDNA library.
水稻红莲型细胞质雄性不育系与保持系mtRNA差异显示和差别片段的分析
凌杏元1,2 周培疆1 朱英国1*
（1. 武汉大学生命科学学院遗传学研究所，武汉430072；2. 安徽农业大学生物技术研究中心，合肥230036）
摘要：将T4 RNA连接酶和AFLP技术特点相结合构建了适于mtRNA的差异显示方法 ,并比较了水稻 (OryzasativaL .)红莲型细胞质雄性不育系、保持系和杂种一代mtRNA的差异。在 4组引物对的选择扩增产物中共找到 6个差异片段 ,其中差异条带DTA为不育系仅有 ,条带DAB为不育系和保持系特有 ,而条带DBF1、DBF2 、DBF3 、DBF4 为保持系和F1杂种共有。这表明水稻红莲型不育系粤泰A与杂种一代泰优 2号mtRNA的差异大于保持系粤泰B和杂种一代泰优 2号mtRNA的差异。Northern杂交证实条带DTA在不育系、保持系和杂种一代的转录确有差异 ,表明它与红莲型细胞质雄性不育有关。条带DTA全长 2 5 9bp ,尽管未发现有同源序列和新的开放阅读框 ,但它可作为探针从cDNA文库中筛选全基因序列 ,进而寻找与细胞质雄性不育有关的开放阅读框架。
关键词： 水稻；细胞质雄性不育；线粒体RNA；差异显示
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