烟草线粒体基因atp6的SNP及其与CMS的相关性

为更好地在烟草育种中利用雄性不育，对烟草胞质雄性不育(CMS)的分子机理进行了研究。atp6基因是影响植物CMS的一个重要候选基因。本研究以7个烟草CMS系及其相应的保持系为材料，利用PCR特异引物扩增其线粒体基因atp6，通过直接测序和比对，发现atp6中A-59C、T-92C、T-185G、T-253C、T-418C、T-768C 6个核苷酸位点存在碱基变异，其中第59位、92位、185位和418位碱基的变化导致了相应位点编码氨基酸残基的改变，另两个位点(第253位和768位)为同义突变。对atp6基因中第59位A→C的突变进行了大量个体(共222个烟草植株个体)的PCR-RFLP检测及分析，结果表明，所有保持系单株的线粒体atp6基因片段都可以被Bst1107 I酶切，酶切后出现两种条带；而96%以上的雄性不育系单株的线粒体atp6基因片段部分能被Bst1107 I酶切，部分由于第59位A→C突变不能被Bst1107 I酶切，酶切后出现3种条带。说明atp6基因第59位的SNP位点与烟草CMS特性存在显著的相关性。

SNP in Tobacco Mitochondrial Gene atp6 and Its Correlation with CMS

The cytoplasmic male sterility (CMS) of higher plants has great significance for the use of heterosis in crop production and the researches of cytoplasmic heredity and the interaction between cytoplasm and nucleolus. The mitochondrial genome is considered as the carrier of sterility gene of cytoplasm, its mutation and recombination are closely related to CMS. ATP synthase participates in the reactions of oxidative phosphorylation and photophosphorylation and provides energy for the plant growth and development. The atp6 gene is one of the subunits of ATP synthase in mitochondria. The base mutation of DNA sequence of atp6 gene results in the abnormal synthesis of amino acids, causing the dysfunction of ATP synthase and the shortage of energy in plant cells. So the normal physiological function of cells is difficulty to be maintained, which results in the male sterility in higher plant.Single nucleotide polymorphism (SNP) is a frequent sort of genetic variation in organism genome DNA and this genetic variation is generally the direct reason of the changes of biologic traits. To better use the male-sterile lines to breed tobacco varieties, we investigated the molecule mechanism of tobacco CMS. The atp6 is a significant candidate gene carried by chondriosome related to CMS in higher plants. In this study, using specific primers designed, the mitochondrial atp6 gene was distinctively amplified by PCR from seven tobacco (Nicotiana tabacum) CMS lines (named as MS Yunyan 85, MS Zhongyan 90, MS K346, MS K326, MS G28, MS Nordel, and MS Jingyehuang) and their corresponding maintainer lines. Six nucleotide variations at A-59C, T-92C, T-185G, T-253C, T-418C, and T-768C in mitochondrial atp6 gene of CMS were detected by sequencing directly and comparing the sequences of atp6 gene. Four nucleotide variations at 59, 92, 185 and 418 resulted in the changes of amino acids coded by the changed nucleotide.The rest two nucleotide variations at 253 and 768 didn’t cause the changes of amino acids, which was a synonymy mutation. The 59 A→C mutation of atp6 gene was detected and analysed by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) with a total of 222 individual tobacco plants, indicating that all the mitochondrial atp6 gene fragments of maintainer lines plants could be entirely digested by Bst1107 I and showed 2 bands in the electrophoresis pattern of atp6 gene, while these of 96% CMS plants displayed 3 bands in the electrophoresis pattern of atp6 gene could be partly digested by Bst1107 I due to the mutation of A→C at 59. So the A-59C SNP site in atp6 gene exhibited a significant correlation with the tobacco CMS.