摘要: 根据GenBank报道的双子叶植物线粒体atp6和atp9基因编码区保守序列设计简并引物, 通过PCR技术从苎麻细胞质雄性不育系、保持系和恢复系(简称“三系”) mtDNA中扩增目的基因片段, 发现所得序列开放阅读框虽不完整, 但与GenBank报道的其他植物线粒体atp6和atp9基因同源性分别高于94%和85%。采用DNA Walking步移法分别从3′端和5′端扩增两个基因片段的未知侧翼序列, 分离出完整的苎麻线粒体atp6和atp9基因, 包含了完整的开放阅读框。其中“三系”的atp6基因在mtDNA水平、转录和翻译调控水平、蛋白质水平上均无差异。不育系atp9基因在编码区3′端与保持系和恢复系相比存在若干个碱基的差异和缺失; RT-PCR分析还表明, 不育系atp9基因在现蕾期和盛花期的表达量很高。推测不育系atp9基因的结构变异和/或异常表达与苎麻细胞质雄性不育(CMS)的关系密切。

Abstract: The atp6 and apt9 gene fragments associated with cytoplasmic male sterility (CMS) were cloned from the mito-chondrial DNA of a ramie (Boehmeria nivea (L.) Gaud.) cytoplasmic male sterile line and its maintainer and restorer lines using PCR and degenerated primer strategy. The primers were designed according to the reserved sequences in the encoding region of mitochondrial genes atp6 and atp9 of some dicotyledons from GenBank. These fragments did not have complete encoding region but showed the homology of 94% and 85% with atp6 and atp9 genes from the referred dicotyledons in GenBank. The complete atp6 and atp9 genes including the complete open reading frames were cloned by means of ampli-fying the 3′ and 5′end unknown sequences of these gene fragments using DNA Walking method. The atp6 gene showed no difference among ramie male sterile line, maintainer and restorer lines at mtDNA sequence, transcription and translation control and protein level. However, compared to the maintainer and restorer lines, the atp9 gene of the male sterile line was different and deletion in several bases at the 3′ end of the encoding region. An abnormally high expression of atp9 gene in the male sterile line at the budding stage and full-bloom stage was analyzed by RT-PCR analysis. These results indicated that the variation in DNA sequence and/or abnormality in expression of atp9 gene in the male sterile line maybe closely related to ramie CMS.