油菜细胞质PCR鉴定方法初步研究
毕业论文关键词：油菜；细胞质；雄性不育
A Preliminary Study on Indentification of Cytoplasmic Male Sterility by PCR Method in Brassica napus
Abstract：In identification of Brassica hybrids, it is necessary to identify the cytoplasmic origin of Brassica napus parents. Determination of the mitochondrial genome of Brassica female parent is the best way to identify the cytoplasm origin, but it is complex, costs very much, and difficult to manipulate. This study used PCR method to identify the cytoplasm. By extracting the Brassica DNA, primers for the specific sequence of different cytoplasmic mtDNA were designed to amplify the DNA fragment by PCR method, and the cytoplasmic type may be identified by PCR products recogintion. Based on this principle, our experimental results showed that the cytoplasm origin may be identified by use of mitochondrial DNA. However, cytoplasm origin could not be detected by total DNA which includes the nuclear, chloroplastic and mitochondrial DNAs. This result might be explained by the mitochondrial evolution process. Therefore, we may conclude that cytoplasmic identification by PCR need to use the mitochondrial DNA.
Key words: Brassica napus；Cytoplasm；Cytoplamic male sterility
目 录
摘要1
关键词1
Abstract1
Key words1
引言2
1 材料与方法2
1.1 实验材料2
1.2 实验方法3
1.2.1 油菜基因组DNA的提取3
1.2.2 油菜线粒体DNA的提取3
1.2.3 油菜特异性片段的PCR扩增3
2 结果与分析4
2.1 油菜基因组DNA提取物电泳结果分析4
2.2 油菜线粒体DNA提取物电泳结果分析5
3 讨论5
致谢6
参考文献7
附录8
油菜细胞质PCR鉴定方法初步研究
引言
油菜杂交种的细胞质存在多种类型。日本学者ogura[1]在萝卜中发现的一种天然雄性不育类型（ogura CMS）。Thompson [2]以波兰春油菜品种Bronowski作为父本与冬油菜品种RD58作为母本杂交的F2代中，发现细胞质雄性不育，进一步培育成Tcms。Shiga和Baba[3]在以日本甘蓝型油菜品种Chisayanatane为母本，Hokuriku 23为父本的杂交后代中发现细胞质雄性不育，进一步培育成Scms。后研究发现[4]，TCMS与SCMS属同一类型，合称nap型。傅廷栋等[5]于1972年春在甘蓝型油菜品种波里马中，发现了polCMS。1976年湖南省农科院[6]利用这一波里马不育材料首先实现pol CMS三系配套。此后，中国学者发现了多种细胞质雄性不育材料，如MI CMS[7]、ABI CMS[8]、153A CMS以及NCa CMS[9]等。
油菜细胞质的差别与细胞质中线粒体和叶绿体DNA序列差异有关。决定细胞质雄性不育的基因基本上都存在与线粒体基因组上。线粒体基因组研究取得了重要进展。目前，线粒体cam、ole、nap、pol、jun、car的基因组也已测序完成[10]，其线粒体基因组大小分别为219747 bp，360271 bp，38662 bp 、38742 bp 、219766 bp，232241 bp。所含有的功能基因基本相同，但是所含的不育基因是不同的，而且基因组结构有所不同，各自含有一些未知功能蛋白基因。nap和pol线粒体类型中发现的雄性不育基因分别为orf222和orf224 [11]。